Biochemical Characterization of FIKK Kinase from

Cryptosporidium parvum and Discovery of Potent

Inhibitors.

by

Khan Tanjid Osman

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Molecular Genetics University of Toronto

© Copyright by Khan Tanjid Osman 2016

ii

Biochemical Characterization of FIKK Kinase from

Cryptosporidium parvum and Discovery of Potent Inhibitors.

Khan Tanjid Osman

Doctor of Philosophy

Molecular Genetics

University of Toronto

2016

Abstract

Cryptosporidium parasites cause serious human and animal diseases and affect millions of

children worldwide. Drug discovery attempts against the parasites are insufficient and new drug

targets are necessary. C. parvum harbors some unique protein kinases including one called FIKK

kinase. FIKKs are parasite-specific protein kinases with distinctive sequence motifs and

restricted to phylum Apicomplexa. The biochemistry and biology of the evolutionarily conserved

members of the FIKK family have not been elucidated before this project. I explored the

biochemical nature of the most conserved FIKK members in C. parvum and malaria causing P.

falciparum, known as CpFIKK and PfFIKK8, respectively. I have identified the soluble domain

boundary of the proteins and their substrate preferences, and characterized their activity in vitro.

FIKKs need a ~40 residue extension to the predicted kinase domain to be soluble. They prefer

Ser as phosphoacceptor residue flanked by Arg at the -3 and +3 positions in the substrate.

Because their biological roles have not been completely elucidated, potent, selective and cell

iii

permeable inhibitors would be useful to understand the biological roles of FIKKs in parasites.

Here, I report the first Cryptosporidium FIKK (CpFIKK) inhibitor and its selectivity profile. I

systematically explored the structure activity relationship for CpFIKK inhibition and for

selectivity against CpCDPK1. I identified 4b as a potent (IC50 = 0.2 nM) inhibitor of CpFIKK

catalytic activity, and confirmed CpFIKK binding using a thermal melt assay. Minor variations

of inhibitor structure led to significant change in selectivity profiles against CpCDPK1 and

identified CpCDPK1 selective as well as dually acting C. parvum FIKK-CDPK1 inhibitors from

the same structural class of compounds. I evaluated these CpFIKK inhibitors for inhibition of

parasite growth in vitro. The observed effect in parasite growth did not correlate with CpFIKK

inhibition.

iv

Dedication

To my parents

Dr. Khan Towhid Osman and Ms. Taslima Begum

From whom I inherited my set of kinase genes

v

Acknowledgments

First and foremost, I like to thank my advisor Dr. Aled M. Edwards. It has been a privilege to

work under his supervision. His tremendous support, endless patience, sincerity and guidance

were crucial for the fulfillment of my project. His views on science, enthusiasm and wonderful

ideas motivated me to take risk and challenge myself, helped me to design experiments and

forced me to think outside the box. I appreciate his contributions of time, advice and funding to

make my research productive, fun and stimulating. I count myself extremely fortunate to be

supervised by such an outstanding science leader and one of the frontline advocates of open

access science.

Most of my research work was conducted in the Structural Parasitology laboratory within the

Structural Genomics Consortium (SGC), Toronto (thesgc.org). I would like to thank the principal

investigator of the group and my co-supervisor Dr. Raymond Hui for his excellent support.

Working in a super-productive lab under his leadership helped my research tremendously. I am

greatly thankful for the time, patience, effort and ideas he contributed for my project. I would

also like to extend my sincere gratitude to the supervisory committee members Drs. Frank

Sicheri and Scott Gray-Owen for their suggestions, ideas and time that led this research towards

right direction. I am specially grateful to Dr. Vijayaratnam Santhakumar from ChemNet, SGC,

for leading the inhibitor compound discovery project.

Lab members of Structural Parasitology helped me immensely in numerous ways. Special thanks

to Diego Lovato, Linda Lin, Mehrnaz Amani, Ashley Hutchinson, Majida El Bakkhouri, Tania

Hills, David Hou, Wei Qiu, Maria Mangos, Dunquan Jiang and Verena Brand for their

tremendous support. I am grateful to the members from other groups of SGC for their assistance,

specially to Peter Loppnau for cloning, Ashley Hutchinson for protein expression, Guillermo

Senisterra and Abdellah Allali-Hassani for biophysical and biochemical assays, and Mani

Ravichandran for crystallography. I would like to extend my gratitude to Greg Brothers, Merilyn

Pereira and Rebecca Clare of SGC for their kind support in administrative matters.

This work was aided by several collaborations and I would like to thank all of the awesome

collaborators: laboratories of Dr. Benjamin Turk from Yale University, Dr. Mark Lautens from

University of Toronto, Dr. Dana Mordue from New York Medical College, Christopher Huston

vi

from University of Vermont, Dr. Serge Muyldermans from VIB Structural Biology Research

Center, Dr. Bill Zuercher from University of North Carolina. Particularly, I would like to thank

Hua Jane Lou from Turk lab for peptide library screening, to Rajiv S. Jumani from Huston lab

for performing Cryptosporidium parvum inhibition assays, to Juntao Ye from Lautens lab for

synthesizing inhibitor compounds and to Odaelys Walwyn from Mordue lab for performing

Toxoplasma gondii inhibition assays. During the period of my PhD program, I have visited many

labs to learn different techniques on cell biology and genetics experiments and I want to express

my sincere gratitude to Dr. John Parkinson from SickKids, University of Toronto (for T. gondii

cellular and inhibition assays), Dr. David Sibley from Washington University in St. Louis (for T.

gondii genetic manipulation techniques) and Dr. Momar Ndao (for C. parvum cellular and

inhibition assays) for their kind permission to work in their labs.

This research was funded by different academic and industry partners through the SGC. The

SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma

AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for

Innovation, Genome Canada through Ontario Genomics Institute, Innovative Medicines

Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co., Novartis Pharma

AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research

Foundation-FAPESP, Takeda, and the Wellcome Trust. I would like to thank all of the funders

for providing me with this opportunity.

Last but not least, I want to thank my family members, without whom this work would not be

possible. My parents encouraged and motivated me to pursue the PhD degree. Finally, I would

like to thank my wonderful wife Ms. Sadiah Mussarrat for her support, love and care. Her

comfort, guidance and assistance were indispensable for me throughout this work.

vii

Table of Contents

Contents

Dedication ...................................................................................................................................... iv

Acknowledgments............................................................................................................................v

Table of Contents .......................................................................................................................... vii

List of Tables ...................................................................................................................................x

List of Figures ............................................................................................................................... xii

Chapter 1 ..........................................................................................................................................1

Introduction .................................................................................................................................1 1

1.1 Cryptosporidium causes serious human and animal diseases ..............................................1

1.2 Epidemiology, taxonomy and life cycle of C. parvum ........................................................2

1.3 Drug discovery efforts against Cryptosporidium and other apicomplexans ........................5

1.4 Targeting protein kinases in parasites for new drug discovery: learning from the past ......7

1.4.1 Ligand-substrate interaction in protein kinases .......................................................8

1.4.2 Known inhibitors of parasite kinases .....................................................................10

1.5 The Cryptosporidium kinome: a hub of unique kinases ....................................................11

1.6 The FIKK kinase family ....................................................................................................13

1.6.1 Number of FIKK members in apicomplexan genomes .........................................13

1.6.2 Domain architectures and known biological functions of FIKKs .........................14

1.6.3 Lone FIKKs are orthologous to FIKK8 from P. falciparum .................................19

1.6.4 FIKKs contain putatively active kinase domains...................................................19

1.6.5 The FIKK kinase domains share some features of atypical protein kinases ..........21

1.6.6 Evidence for catalytically active FIKKs ................................................................23

1.7 Conclusion .........................................................................................................................25

viii

Chapter 2 ........................................................................................................................................26

Biochemical characterization of FIKKs from C. parvum (CpFIKK) and P. falciparum 2

(PfFIKK8) .................................................................................................................................26

2.1 Soluble FIKK constructs include an extension region .......................................................26

2.2 CpFIKK and PfFIKK8 are active kinases ..........................................................................39

2.2.1 Purified FIKKs bind ATP ......................................................................................30

2.2.2 CpFIKK and PfFIKK8 autophosphorylate ............................................................31

2.2.3 CpFIKK and PfFIKK8 phosphorylate exogenous substrates ................................32

2.3 CpFIKK and PfFIKK8 target serine with flanking arginines ............................................39

2.3.1 Positional-scanning peptide array ..........................................................................33

2.3.2 Characterization of enzyme parameters using optimized substrates .....................37

2.4 Materials and methods .......................................................................................................39

2.4.1 Cloning and expression ..........................................................................................39

2.4.2 Differential static light scattering (DSLS) .............................................................40

2.4.3 In vitro phosphorylation .........................................................................................41

2.4.4 Kinase substrate peptide assay ...............................................................................41

2.4.5 Enzymatic assay .....................................................................................................42

Chapter 3 ........................................................................................................................................43

Identification of potent inhibitors of CpFIKK ..........................................................................43 3

3.1 Inhibitor screening yielded multiple naphthyridine-based CpFIKK-inhibitors .................44

3.2 Selectivity of 4a within C. parvum kinome .......................................................................45

3.3 Structure activity relationship of CpFIKK inhibitors ........................................................46

3.4 Orthogonal confirmation of CpFIKK inhibition ................................................................52

3.5 Selectivity profile of CpFIKK inhibitor 4b against human kinases ...................................54

3.6 Structural understanding of inhibitor binding ....................................................................59

3.7 Materials and methods .......................................................................................................63

ix

3.7.1 Enzymatic inhibition assay ....................................................................................63

3.7.2 Screening with human kinases ...............................................................................64

3.7.3 X-ray crystallography ............................................................................................64

Chapter 4 ........................................................................................................................................66

Phenotypic effects of CpFIKK inhibitors on parasites .............................................................66 4

4.1 Evaluation of CpFIKK inhibitors in C. parvum growth inhibition ....................................66

4.2 Chemical genetics experiments with FIKK knock out (KO) strains of T. gondii..............69

4.3 Materials and methods .......................................................................................................71

4.3.1 C. parvum growth inhibition assay ........................................................................71

4.3.2 T. gondii growth inhibition and cystogenesis inhibition assay ..............................72

Chapter 5 ........................................................................................................................................73

Discussion .................................................................................................................................73 5

5.1 The role of N-terminal extension (NTE) ...........................................................................74

5.2 FIKK kinases are unique in substrate preference ..............................................................75

5.3 CpFIKK/CpCDPK1-inhibitors: what makes them selective? ............................................76

5.3.1 The Hydrophobic region I selectivity: role of gatekeeper .....................................77

5.3.2 Selectivity between CpFIKK and PfFIKK8 ..........................................................79

5.3.3 A common inhibitor for both CpFIKK and PfFIKK8 provides insights to

design CpFIKK-selective inhibitors. .....................................................................80

5.4 Parasite killing by compound 4b is probably due to CDPK1 inhibition............................81

5.5 Utility of a chemical tool to understand the biological role of CpFIKK in parasites ........82

References ......................................................................................................................................85

x

List of Tables

Table 1.1 Family, genus, species and strains from the phylum Apicomplexa and the number of

FIKK members in their genomes. ................................................................................................. 14

Table 1.2 FIKK proteins from P. falciparum and P. reichenowi. ................................................ 16

Table 1.3 Domain boundaries of the kinase domains (KD) of the FIKK proteins from different

apicomplexans............................................................................................................................... 20

Table 1.4 Key catalytic motifs of FIKK proteins from different organisms. ............................... 24

Table 2.1 Cloning and expression profile of constructs of PfFIKK8, CpFIKK and TgFIKK. ..... 27

Table 2.2 Normalized average intensity values of enzyme screening spots for PfFIKK8l in Fig.

2.5 A .............................................................................................................................................. 35

Table 2.3 Normalized average intensity values of enzyme screening spots for CpFIKKd in Fig.

2.5 B .............................................................................................................................................. 36

Table 2.4 List of consensus peptides tested and the kinetic parameters obtained, including the

Michaelis constant (Km), turnover (kcat) and catalytic efficiency (kcat/Km). ............................ 39

Table 2.5 Kinetic parameters of purified FIKK proteins in using ATP. ....................................... 39

Table 3.1 Cryptosporidium kinases tested for selectivity. The construct boundaries of the kinases

are included. .................................................................................................................................. 45

Table 3.2 CpFIKK kinase activity of compounds 4a-i and selectivity against CpCDPK1. ......... 48

Table 3.3 CpFIKK kinase activity of compounds 4m-q and selectivity against CpCDPK1. ....... 50

Table 3.4 CpFIKK kinase activity of compounds 4r, 5a, and 6 and selectivity against CpCDPK1.

....................................................................................................................................................... 52

Table 3.5 Thermal stabilization of CpFIKK by inhibitors. ........................................................... 53

Table 3.6 Percentage of activity of human kinases in the presence of 500 nM 4b. ...................... 56

xi

Table 3.7 Probable inhibitor interacting residues in TgCDPK1, CpCDPK1 and CpFIKK .......... 60

Table 3.8 Data collection, phasing, and refinement statistics for the TgCDPK1 co-crystal

structures. ...................................................................................................................................... 65

Table 4.1 Parasite invasion inhibition profiles of CpFIKK inhibitors. ......................................... 68

Table 5.1 Substrate preference of different protein kinases. ........................................................ 76

Table 5.2 Highest inhibited protein kinases from human kinome by compound 4b at 500 nM and

the gatekeeper residues. ................................................................................................................ 79

xii

List of Figures

Figure 1.1 C. parvum life cycle. ..................................................................................................... 4

Figure 1.2 Representation of the bilobate structure of a typical protein kinase. ............................ 8

Figure 1.3 Representation of the ATP binding pocket of a typical protein kinase. ........................ 9

Figure 1.4 Evolutionary relationships between the C. parvum kinases. ....................................... 12

Figure 1.5 Domain construction of the FIKK proteins. ................................................................ 15

Figure 1.6 Phylogenetic tree of the FIKKs from different apicomplexans. .................................. 18

Figure 1.7 Sequence alignment of the kinase domains of the orthologous FIKKs from different

apicomplexans............................................................................................................................... 22

Figure 2.1 Test expression gel showing that constructs PfFIKK8h to PfFIKK8o were soluble. . 28

Figure 2.2 Purity of the purified proteins...................................................................................... 29

Figure 2.3 Thermal melting curves of CpFIKK and ΔTm values for both kinases with different

ligands. .......................................................................................................................................... 31

Figure 2.4 Maps of phosphorylated residues in (A) PfFIKK8l and (B) CpFIKKd (C) MBP. ..... 32

Figure 2.5 Peptide substrate preference array of PfFIKK8 and CpFIKK identifies a motif. ....... 34

Figure 2.6 Enzymatic activities of recombinant FIKKs (A) PfFIKK8o (B) PfFIKK8l and

CpFIKKd (C). ............................................................................................................................... 37

Figure 2.7 Enzyme kinetics of FIKKs with ATP. ......................................................................... 38

Figure 3.1 Molecular structures of compounds 4a and 4j. PKIS IDs and IC50 values against

CpFIKK are shown. ...................................................................................................................... 44

Figure 3.2 The naphthyridine derivative with HsALK5 active site. ............................................. 47

Figure 3.3 The enzymatic inhibition of CpFIKK and CpCDPK1 by 4b. ..................................... 49

xiii

Figure 3.4 CpFIKK stabilization by 4b in differential static light scattering (DSLS or thermal

melt) assay. ................................................................................................................................... 53

Figure 3.5 Selectivity of 4b against 140 human kinases. .............................................................. 55

Figure 3.6 Structure analysis of parasite kinases with compound 4b. .......................................... 60

Figure 3.7 Sequence alignment of the kinase domains of TgCDPK1, CpCDPK1 and CpFIKK. 62

Figure 3.8 Thermal melt assay with CpFIKK-gatekeeper-residue-mutant CpFIKKΔS516M. .......... 63

Figure 4.1 Inhibition of C. parvum growth in vitro. ..................................................................... 69

Figure 4.2 In vitro growth inhibition results of wild type (WT) and FIKK knocked out (KO)

strains of T. gondii with compound 4b. ........................................................................................ 70

Figure 5.1 Sequence alignment of the N-terminal extension of different apicomplexan FIKKs. 75

Figure 5.2 Representation of the active site of TgCDPK1 occupied by compound 4b. ............... 78

Figure 5.3 A) Active site of the co-crystal structure of CGP060476-TgCDPK1 (top left) and

superposed structures of CGP060476 and 4b-bound TgCDPK1 (bottom left). B) Proposed

compound structures to occupy the Hydrophobic region II of CpFIKK. ..................................... 80

1

Chapter 1

Introduction 1

1.1 Cryptosporidium causes serious human and animal diseases

Cryptosporidium parasites belong to the phylum of unicellular parasites Apicomplexa. The

phylum contains some medically important human parasites that cause serious human diseases.

These include malaria-causing Plasmodium falciparum, a global opportunistic parasite

Toxoplasma gondii, and Cryptosporidium parvum (and C. hominis), which causes severe

diarrhoea in infants and AIDS patients.

Cryptosporidium is an enteric parasite of human, cattle and poultry. Children under the age of

five in under-developed regions of the world are most vulnerable, with hundreds of thousands

dying every year and millions infected and often re-infected (Ochoa, Salazar-Lindo, and Cleary

2004; Checkley et al. 2015; Striepen 2013; Kotloff et al. 2013). Developmental problems among

this age group of children are also associated with Cryptosporidium infection (Guerrant et al.

1999). It is also a significant veterinary concern affecting ruminants and poultry. There are also

periodic outbreaks of cryptosporidiosis in North America and Europe. With the advancement of

very effective vaccine productions against rotaviruses, Cryptosporidium is soon expected to be

the most important diarrheal agent in world.

Cryptosporidiosis is a disease of watery diarrhea, sometimes with prolonged and chronic

infection (Bouzid et al. 2013; Current and Garcia 1991) and sometimes accompanied by

stomach-ache, nausea, vomiting and fever. Non-specific symptoms, such as myalgia, weakness,

malaise, headache and anorexia can occur occasionally (Current and Garcia 1991). A

symptomatic but self-limiting disease occurs in immunocompetent individuals. However,

immunocompromised individuals, such as HIV infected patients, show symptoms of acute and

prolonged diarrhea, which can be deadly if HIV infection is not treated (Hunter and Nichols

2002; Manabe et al. 1998). HIV infected individuals can also develop extra-intestinal infections,

such as in gall-bladder, biliary tract, pulmonary system and pancreas (Hunter and Nichols 2002).

2

1.2 Epidemiology, taxonomy and life cycle of C. parvum

Cryptospodium transmission occurs by the faecal-oral route. Ingestion of contaminated food or

water is the most common cause for human infections or outbreaks. Cryptosporidium was the

major etiological agent of waterborne parasitic outbreaks worldwide between 2004 and 2010,

responsible for 60.3% of them (Baldursson and Karanis 2011). However, human-to-human

contact or animal-to-human contact can also cause transmission (Xiao 2010).

Being in the apicomplexan phylum, Cryptosporidium shares some features with parasites from

Eucoccidioirida, which contains parasites Toxoplasma, Cyclospora, Isospora and Sarcocystis.

These include environmental cyst production, similar Type I fatty acid metabolic pathways and

polyketide synthetic enzymes and having a number of extracellular proteins sharing similar

domain architecture (Templeton et al. 2010). However, despite the similarities, Cryptosporidium

differs significantly from the eucoccidian parasites and its biology and physiology resembles the

more primitive gregarine parasites (Carreno, Martin, and Barta 1999; Leander, Clopton, and

Keeling 2003). Unlike most apicomplexans, Cryptosporidium has lost the apicoplast organelle as

well as genomes for plastid and mitochondria (Zhu, Keithly, and Philippe 2000; Xu 2004;

Abrahamsen et al. 2004). There are multiple Cryptosporidium-specific and gregarine-like

features that also separate this parasite from eucoccidians. For example, Cryptosporidium

parasites occupy an intracellular, but extracytoplasmic location in the infected host cell;

possesses two morpho-functional types of oocysts: thin and thick walled, which are very small

(5.0 – 4.5 µm for C. parvum) and lack sporocyst, micropyle and polar granules (Petry 2004);

nutrient uptake from host cells is facilitated by multi-membranous feeder organelles formed in

the host-parasite attachment site and at the base of the parasitophorous vacuole (PV); parasites

are not sensitive towards anti-coccidal agents tested (Cabada and White 2010); antibodies against

gregarine parasites cross react with Cryptosporidium parasites (Bull et al. 1998); and

Cryptosporidium has gregarine like life-cycle stages, for example gamont like extracellular

stages (Borowski et al. 2010; Karanis and Aldeyarbi 2011; Hijjawi et al. 2002; Koh et al. 2013).

These features, supplemented with molecular studies, suggest that Cryptosporidium parasites are

more like gregarine parasites than eucoccidian (Carreno, Martin, and Barta 1999; Leander,

Clopton, and Keeling 2003). Phylogenetic association with gregarine parasites have been

elucidated too by recent whole genome analysis (Templeton et al. 2010). Future and more

detailed comparative genomic studies and further characterization will provide a clearer

3

understanding about the phylogeny of the parasite and new ideas about investigating the

epidemiology, pathogenicity, treatment and control of Cryptosporidium parasites.

There have been 27 species of Cryptosporidium identified so far (U. Ryan and Hijjawi 2015; U.

N. A. Ryan, Fayer, and Xiao 2014). 19 species infect mammals, 3 infect birds, 2 infect reptiles, 2

in fishes, and 1 has been found in amphibians. C. hominis is highly specific for human, whereas

C. parvum has a broader range and most of them are transmitted zootonically, except for the

subtype IIc family, which is transmitted anthroponotically (Xiao 2010; U. N. A. Ryan, Fayer,

and Xiao 2014).

Cryptosporidium exhibits a monoxenous (development restricted to a single host system), but

complex life cycle with multiple developmental stages involving both sexual and asexual life

cycles, which are completed within the gastrointestinal tract of hosts (Figure 1.1). The life cycle

can be divided into six major developmental phases: exystation, merogony, gametogony,

fertilization, oocyst wall formation and sporogony (Current and Garcia 1991).

The life cycle begins in a susceptible host with the ingestion of the sporulated oocysts. Upon

ingestion the exystation occur, which is thought to be triggered by multiple factors, such as

reducing conditions, temperature, carbon dioxide, pancreatic enzymes and bile salts (Blagburn et

al. 1987; Fayer and Leek 1984; Robertson, Campbell, and Smith 1993). Exystation helps four

sporozoites to come out of a single oocyst, and the sporozoites glide over the intestinal epithelial

cells (Okhuysen and Chappell 2002; X. M. Chen et al. 2004; Wetzel et al. 2005). The sporozoites

secrete apical complex components to allow them to invade the host cells. Throughout the whole

life cycle of Cryptosporidium the parasite preferentially infects the host cells at the apical site.

The apical complex is made up of micronemes, a single rhoptry and dense granules (Tetley et al.

1998). With the help of these components the sporozoites invade the host cells and the host cell

membrane encloses the parasites, forming the parasitophorous vacuole (PV) (Petersen et al.

1992; Boulter-Bitzer, Lee, and Trevors 2007; Tomley and Soldati 2001). Each sporozoite

transforms into a spherical trophozoite and undergoes merogony to form type I mermont

containing eight merozoites. After the merozoites are released, they attach to more epithelial

cells and either remain as type I mermonts or convert to type II mermonts. Type II mermonts

contain four merozoites each and after their release, the merozoites attach to epithelial cells, but

instead of producing more mermonts, these merozoites undergo gametogony. One merozoite

4

produces either a microgamont or a macrogamont. Each microgamont undergoes mitotic division

and emerge into at least 16 additional microgametes. Similarly, macrogamonts differentiate into

macrogametocytes, which are located and fertilized by the microgametes after being released

from the PV. The resulting diploid zygote undertakes two rounds of asexual sporogony to

produce an oocyst with four haploid sporozoites inside (Smith and Rose 1998). As mentioned

earlier, the oocysts can be of two types, thin walled and thick walled (Current and Reese 1986).

The thin walled oocysts can autoinfect the same host once they are liberated from the epithelium

and exyst (Current and Reese 1986). The thick walled oocysts are excreted from the lumen to the

outside of the body through feces and spread in the environment can infect another host. The

persistence and chronic infection occurs due to both of the autoinfection and recycling of the

type I mermonts (Smith and Rose 1998).

Figure 1.1 C. parvum life cycle. (Copied from Bouzid et al. 2013, Cryptosporidium

pathogeneciry and virulence. Courtesy of Clinical Microbiology Reviews, ASM.)

5

1.3 Drug discovery efforts against Cryptosporidium and other apicomplexans

Anti-cryptosporidium drug discovery has been hampered by many factors. Continuous culturing

and genetic manipulations had been extremely difficult until very recently (Vinayak et al. 2015).

Rodent models are considered inadequate too. Because of the limited tools, understanding the

biology of the parasite at the molecular level has been challenging, and identifying targets,

proper testing and validating new drugs remain very difficult.

Sometimes, well studied organisms can be exploited to understanding the biology and inhibition

of another less understood organism from similar evolutionary family or group. Two other

organisms apicomplexans, P. falciparum and T. gondii have been studied explicitly and are being

used as models for Cryptosporidium. The problem with this approach is that apicomplexans,

despite being in the same family, exhibit very different life cycles and host and environmental

preferences. Cryptosporidium metabolism is particularly different from that of P. falciparum and

T. gondii: Cryptosporidium lost its plastid-derived apicoplast and lacks the citric acid cycles and

cytochrome systems in the remnant mitochondrion. Cryptosporidium parasites also lack some

enzymes for de novo synthesis of amino acids, nucleotide and sugars (Abrahamsen, 2004; Xu,

2004). The loss of multiple important genes from the metabolic pathways suggests that the

parasite relies heavily on the scavenging nutrients and other factors from the host and a unique

group of genes may be involved in these pathways, rather than de novo synthesis, that are not

found in human and animals. Therefore, many classic drugs that are effective against many of

the other apicomplexans are not effective against Cryptosporidium, which limits the drug

discovery attempts. There is a need for identifying new drug targets (Miyamoto and Eckmann

2015).

Different cellular pathways including lipid metabolism, energy metabolism and signaling

pathways are essential for survival or completing life cycle or transmission in these parasitic

organisms. Proteins playing roles in these pathways may serve as new drug targets against

Cryptsporidium, especially which are unique to the parasite. For example, a recent report has

identified an essential enzyme called long chain fatty acyl-coenzyme A synthetase (LC-ACS) as

potential new target of ACS inhibitor triacsin A and showed efficacy in mice (Guo et al. 2014).

6

The most extensively studied pathway for drug discovery in Cryptosporidium is purine salvage.

The parasites are not able to synthesize de novo purine nucleotides and are completely dependent

on salvaging guanine nucleotides from the host via a pathway involving inosine 5’-

monophosphate dehydrogenase (IMPDH) (Umejiego et al. 2004). The IMPDH gene has been

laterally transferred from bacteria and is structurally distinct from mammalian IMPDH making it

potentially an important drug target (Striepen et al. 2004). A number of structurally diverse

inhibitors of CpIMPDH inhibitors have been identified by high-throughput screening (Striepen et

al. 2004) and some showed activity in mouse infection model as well (Gorla et al. 2014). Some

peptide inhibitors of CpIMPDH have also shown efficacy in enzymatic inhibition, however, the

in vivo activity needs to be verified (Jefferies et al. 2015). Nevertheless, IMPDH in

Cryptosporidium has been found to be potential drug targets and needs to be further explored for

drug development.

Some other molecular targets tested are calcium dependent protein kinases, such as CDPK1

(Castellanos-Gonzalez et al. 2013; Kuhlenschmidt et al. 2016; Artz et al. 2011) and cysteine

proteases (Perez-Cordon et al. 2011). Cysteine proteases are associated with host cell invasion.

The other essential function of these proteins in the completion of the life cycle is helping in

exystation. Cysteine protease inhibitors show promising results both in vivo and in vitro studies.

However, the selectivity by these compounds for the 20 or more cysteine papain-like proteases

remains to be identified (Ndao et al. 2013).

Current cryptosporidiosis treatment is limited to only a single approved drug, called

nitazoxanide, by the Food and Drug Administration (FDA). Nitazoxanide is effective against

other parasitic and viral infections too. The efficacy of this drug against cryptosporidiosis in

HIV-negative individuals varies between 50-90% (Amadi et al. 2002). The drug is well tolerated

too. However, for the immunocompromised individuals like HIV-infected individuals and

malnourished children, where the pathogenesis is severe, the drug is inactive (Amadi et al. 2002)

and administration is not FDA approved for this group of patients.

Other anti-cryptosporidium interventions have been developed and tested clinically, but have

lower efficacy than nitazoxanide and have not been approved by FDA for cryptosporidiosis

treatment. For example, paromomycin shows some level of efficacy against the disease in

immunocompetent individuals (Perkins, Wu, and Le Blancq 1998). However, the clinical results

7

in HIV-infected individuals are highly variable (Hewitt et al. 2000). Some immunotherapy has

been developed to restore immunocompetency in AIDS patients (Mead 2014). However,

nitazoxanide treatment remained the most effective therapeutic choice against cryptosporidiosis

in immunocompetent individuals while no effective treatments are available against

immunodeficient individuals (Miyamoto and Eckmann 2015).

As mentioned before, Cryptosporidium is very different from some of the better studied parasites

P. falciparum and T. gondii. However, there are some efforts taken to repurpose some of the

known parasitic drugs. These attempts include refurbishing FDA approved drugs and chemical

compounds from the malaria box by Medicines for Malaria Venture (MMV) that have been

tested against the C. parvum in vitro and multiple compounds have been identified having IC50s

<10 µM (Bessoff et al. 2014). However, none of these compounds have been clinically tested yet

for cryptosporidiosis.

1.4 Targeting protein kinases in parasites for new drug discovery: learning from the past

In humans, phosphorylation of proteins by kinases plays role in regulation of multiple essential

cellular processes including transcription, translation, protein synthesis, cell cycle and apoptosis.

Life cycles of apicomplexan parasites are composed of multiple stages having morphologically

and phenotypically different forms in each stage, suggesting ample opportunities for

pharmacological inhibition. Protein kinases may be suitable targets in parasites because protein

kinases are known to trigger development of the stages (Kato, Sugi, and Iwanaga 2012).

However, suitability of kinases as drug targets for Cryptosporidium is not clear because our

understanding of the biology of Cryptosporidium at the molecular level is still limited. That

said, protein kinases are better studied in Plasmodium and Toxoplasma, and in these organisms

protein kinase inhibitors demonstrate anti-parasitic activity. Therefore I hypothesize protein

kinases will participate in essential events shared across apicomplexans and homologous protein

kinases will have similar functions and be pharmacologic targets in Cryptosporidium

8

1.4.1 Ligand-substrate interaction in protein kinases

Protein kinases catalyze phosphorylation reaction by hydrolyzing ATP in the presence of Mg2+

and transferring a phosphate group from ATP to an amino acid of a substrate protein or peptide.

The kinase domain is highly conserved among eukaryotes. Its sequence can be divided into 12

subdomains that fold to produce a bilobate 3D structure (Figure 1.2). The catalytic pocket

situates between two lobes, where ATP binds too. The ATP-binding segment in the pocket is

called the ‘hinge’ and it connects the two lobes. The adenine ring of the ATP makes hydrogen

bonds with residues from the hinge region and this anchors ATP in the pocket during the

reaction. Ribose and the triphosphate interact with a hydrophilic channel and span up to the

substrate-binding site. There is a conserved activation loop in every kinase; this is marked by

conserved DFG and APE motifs at the beginning and the end of the loop, respectively. The

activation loop can adopt different conformational states, such as a catalytically competent

conformer (Figure 1.2 A) or an inactive conformer in which the active site is blocked (Figure

1.2B).

A. Active conformation, B. Inactive conformation. The activation loop movement helps the

protein to toggle between active and inactive conformations.

A

Figure 1.2 Representation of the bilobate structure of a typical protein kinase.

B

9

Hinge and ATP are shown in blue and red lines, respectively. Hydrogen bonding between the

hinge and the ATP are shown by dotted lines. Regions that are targeted by different types of

kinase inhibitors are shaded in different colors.

Most common kinase inhibitors bind in the hinge region at the active site by mimicking the

hydrogen bonding with adenine of ATP. These are classified as type I inhibitors. Compounds

exploiting the ribose binding site or the triphosphate binding regions are not very common. More

recently developed inhibitors, types II-V, are not ATP-competitive. They act by inducing a

conformational change in the target enzyme in a way that the kinases no longer can function.

Type II inhibitors not only bind at the ATP-binding pocket, but also occupy the hydrophobic,

allosteric pocket exposed by the movement of the DFG motif at an ‘out’ position. Type III

inhibitors occupy the catalytic pocket but do not interact with the ‘hinge’ region and occupy the

Figure 1.3 Representation of the ATP binding pocket of a typical protein kinase.

10

DFG-out hydrophobic pocket. Type IV are truly allosteric inhibitors binding away from the

catalytic site and type V inhibitors are bivalent or bisubstrate inhibitor compounds and interacts

with the kinase irreversibly. Figure 1.3 shows the representations of the chemical space and

inhibitor binding modes of different kinase inhibitors.

1.4.2 Known inhibitors of parasite kinases

Protein kinases have been studied for many years as drug targets and there are currently more

than 25 approved drugs that target human kinases (Zhang, Yang, and Gray 2008). Rich

biochemical and structural tools are available to study kinases. These tools have been applied to

understand the parasite kinases and to target them with small molecules. In Plasmodium, the best

studied parasite, protein kinases play roles in different life cycle stages. For example, PfPK2,

PfPKB, PfCDPK1 and PfPKA function in erythrocyte invasion (Kumar et al. 2004; Kato et al.

2008; Merckx et al. 2008; Bansal et al. 2013), PfCDPK1, PfGSK3, PfPK7 and PfPKA play roles

during replication at the erythrocyte stages (Bansal et al. 2013; Droucheau et al. 2004; Dorin et

al. 2005; Merckx et al. 2008) and PfCDPK5 plays roles in egress (Dvorin et al. 2011). PbCDPK6

is critical in invasion during liver stage (Coppi et al. 2007), PbCDPK4, PfMAP2 and PfPKG are

functional during gametogenesis (Billker et al. 2004; McRobert et al. 2008; Rangarajan et al.

2005) and PbCDPK3 function during ookinete migration in the mosquito (Ishino et al. 2006). In

T. gondii, TgCDPK1 and TgPKG1 work in invasion (Lourido et al. 2010; Donald et al. 2002),

TgCDPK1 functions in egress (Lourido et al. 2010), TgNEK1, TgCK1, TgTPK2 and TgPKA are

critical for cell proliferation (Kurokawa et al. 2011; C.-T. Chen and Gubbels 2013; Khan et al.

2002; Donald et al. 2005), TgMAPK1, TgPKA and TgEIF2K play roles in stress response and

stage conversions (Cao et al. 2015; Kurokawa et al. 2011; Sullivan et al. 2004) and T. gondii

specific protein kinases ROP18, -16 and -38 are important in host immune system manipulation

(Talevich and Kannan 2013; Fox et al. 2016). Recently, TgFIKK was found to play role in tissue

cyst formation in T. gondii (Skariah et al. 2016). Functions of the very few members of the C.

parvum proteome are known. One of the most studied kinases, CpCDPK1, has been found to be

essential for parasite growth in vitro (Larson et al. 2012; Castellanos-Gonzalez et al. 2013).

Small molecule inhibitors targeting some of the protein kinases from Plasmodium and

Toxoplasma parasites have produced phenotypic changes in the parasites, i.e. reduction of

11

growth, blockage in stage conversions and inhibition of infection processes (Eaton, Weiss, and

Kim 2006; Wiersma et al. 2004; Donald et al. 2006; Sicard et al. 2011; Carruthers, Giddings, and

Sibley 1999; Wei et al. 2002). For example, non-selective inhibitors of PKG (e.g. Compound 1)

(Wiersma et al. 2004), PKA (e.g. H89) (Syin et al. 2001), CDK (e.g. Purvalanol A) (Donald et al.

2005) and MAPK (e.g. U0126) (Sicard et al. 2011) reduce growth of both parasites. Inhibitors

targeting small gatekeepers in the kinase active site (e.g. bumped kinase inhibitor or BKIs)

inhibit orthologous CDPK kinases CpCDPK1 (S. M. Johnson et al. 2012; Larson et al. 2012),

PfCDPK4 (Ojo et al. 2014) and TgCDPK1 (S. M. Johnson et al. 2012; Wernimont et al. 2010),

and block host infection and parasite growth. PKA (Eaton, Weiss, and Kim 2006) and PKG

(Ramdani et al. 2015) inhibitors inhibit gametogenesis in Plasmodium. These studies suggest that

parasite kinases can be potential drug targets to block growth and transmission of parasites and

should be explored more extensively to identify potential drug targets.

1.5 The Cryptosporidium kinome: a hub of unique kinases

The kinome of C. parvum (CpKinome) is smaller but more diverse than those in T. gondii and P.

falciparum. At least 73 protein kinases have been identified in C. parvum; these belong to sub-

families AGC, CaMK, CK1, CMGC, TKL, Atypical and OPKs (other protein kinases) (Artz et

al. 2011). No STE and tyrosine kinases have been identified. A quarter of the kinases identified

have no known orthologues in other eukaryotic kinomes.

Two sets of kinases in CpKinome have orthologues within apicomplexans and other orders, but

not in mammals. Being evolutionary conserved among apicomplexans, these kinases can be

considered potentially important for the parasites. The first group is called the calcium dependent

protein kinases (CDPK), which are regulated by Ca2+

in the cellular environment (Billker,

Lourido, and Sibley 2009). There are eight CDPKs in the C. parvum kinome and each has

multiple EF-hands that bind Ca2+

to become active. CpCDPK1 orthologues in P. falciparum and

T. gondii are essential for parasite infection; they play role in invasion of host cells and egress.

CpCDPK1 has been found to be essential in parasite growth in vitro by inhibition studies (Larson

et al. 2012; Castellanos-Gonzalez et al. 2013).

12

A second group of unique kinases are the FIKKs. These kinases are unique in sequence and are

restricted toapicomplexan parasites, but are not well studied. I decided to focus on this relatively

less well-understood kinase sub-family to understand their function and inhibition and explore if

they are drug targets. The next sections discuss FIKK kinases from different parasites.

The families are colour-coded as follows: AGC (yellow), CK1 (orange), Atypical (pink), CaMK

(blue), CMGC (green), TKL (red), and OPK (grey). CpFIKK belong to the OPK family.

Figure 1.4 Evolutionary relationships between the C. parvum kinases. Maximum

likelihood tree from a rapid bootstrap analysis describing the classification of the 73 C.

parvum protein kinases. (Copied from Artz et al., 2011, The Cryptosporidium parvum

kinome. Courtesy of BioMed Central Genomics.)

13

1.6 The FIKK kinase family

1.6.1 Number of FIKK members in apicomplexan genomes

Protein kinases comprise one of the largest protein families in eukaryotic genomes. The size of

the kinome varies widely throughout the apicomplexan clades, e.g. 90, 159 and 73 membered

kinomes are present in P. falciparum, T. gondii and C. parvum, respectively. Not all

apicomplexans contain obvious FIKK orthologues. FIKK kinases have been identified in

genomes of some apicomplexan families - Cryptosporidiiae, Plasmodiiae, Sarcocytidae and

Eimeriidae (Table 1.1), but not in genomes from piroplasms containing Babesiidae and

Theileriidae families, and in Gregarinidae. Some of FIKK-containing species are potent human

pathogens.

The number of FIKK genes varies between species and even among species from the same

genus. Three closely related Plasmodium species, P. falciparum, P. reichenowi and P. gaboni

genomes contain more than 20 FIKK members each (Otto et al. 2014; Schneider and Mercereau-

Puijalon 2005). (Sundararaman et al. 2016). P. falciparum and P. reichenowi contain 21

members each and P. gaboni contains 22 member. The only outlier or non-orthologous (22nd

)

FIKK in P. gaboni is found in chromosome 9 and called PfFIKK9.15. Remarkably, all three

species, having the expanded FIKK family are from the Laverania subgenus (Sundararaman et

al. 2016). All the other FIKK-containing parasites possess a single member. The reason for the

expansion in two Plasmodium species is not clear. Predictions include their role in virulence

related host cell modifications during human and chimp malaria. The answer though is likely

more complex; according to current annotation, multiple Plasmodium species contain no FIKK

members (Table 1.1).

The FIKK genes are distributed throughout the chromosomes of P. falciparum and P. reichenowi

genomes. 12 out of 14 chromosomes from these parasites have at least a single FIKK gene (no

FIKK genes have been found in chromosomes 2 and 6). The location of FIKK genes within the

single FIKK-containing Plasmodium genomes tends to be influenced by the evolutionary

relationship between species. For example, the single gene in evolutionarily close species P.

berghei, P. yoleii and P. chaubadi contain the FIKK gene in the 12th

chromosome, whereas, P.

vivax and P. knowlesi contain the gene in the 1st chromosome. A single FIKK gene has been

found in other, but not all, apicomplexan parasites. Table 1.1 presents the number of FIKK

14

members in different parasites. CpFIKK gene is found at the chromosome 5 of the C. parvum

genome.

The data were collected from Eukaryotic pathogen database resources (eupathdb.org).

1.6.2 Domain architectures and known biological functions of FIKKs

FIKKs are conserved in their domain architecture. All the FIKKs contain a C-terminally

conserved kinase domain and a non-conserved N-terminal region (Figure 1.5). Paralogous FIKK

sequences, except the FIKK8, from P. falciparum and P. reichenowi (PfFIKK8 and PrFIKK8)

Table 1.1 Family, genus, species and strains from the phylum Apicomplexa and the

number of FIKK members in their genomes.

15

contain two additional domains or motifs at the very N-terminal region. The additional domains

are either a transmembrane domain (TMD) or a signal peptide (SP) and are situated very close to

the N-terminal. The predicted TMD or SP lengths varied from 17 to 42 residues (Table 1.2). 2

FIKKs (PfFIKK5, PfFIKK7.2,) do not contain any transmembrane region but retained the,signal

peptide.

All FIKK sequences from P. falciparum (PfFIKK) and P. reichenowi (PrFIKK), except FIKK8s,

contain an export signal, known as the PEXEL motif. This penta-residue motif is found in many

Plasmodium proteins and helps parasite proteins to be exported from the parasitophorous vacuole

to the different compartments of the infected erythrocyte cells. PEXEL motifs have been found

downstream of the TMD or SP. CpFIKK, like Pf- and PrFIKK8s, does not contain any signal

peptide or transmembrane regions.

Figure 1.5 Domain construction of the FIKK proteins.

16

Total length and boundaries of the kinase domains (KD) and other motifs are mentioned here.

The domain boundaries were predicted with Prosite and the signal and transmembrane

sequences were predicted by Pfam.

Table 1.2 FIKK proteins from P. falciparum and P. reichenowi.

17

Substrate selectivity of protein kinases is often achieved by localization the protein kinases to

distinct subcellular compartments or structures. In Plasmodium species, as many as 400 proteins,

including many of the FIKK paralogues, are secreted and have been assigned to the remodeling

process of the host cells (Cooke et al. 2004; Maier et al. 2009; Marti et al. 2005). Some of the

secreted FIKK members, containing the PEXEL motif, are localized to a distinct cellular

compartment called the Maurer’s cleft (PfFIKK4.1, PfFIKK9.3, PfFIKK9.6 and PfFIKK12)

and/or in infected erythrocyte (IE) membranes (PfFIKK12). PfFIKK4.1 has been found to

phosphorylate an erythrocyte cytoskeleton protein, dematin, and takes part in the remodeling

process (Brandt and Bailey 2013). A more recent study has shown that PfFIKK4.2 is exported to

another distinct cellular structure, called the K-dots, in infected erythrocytes (Kats et al, 2014).

Two other Pf- and PrFIKKs, FIKK5 and FIKK7.2, do not have a TMD or SP and can be

predicted not to anchor to any membranes, but possess PEXEL motifs. Therefore, they may be

secreted protein, but not anchored. PrFIKK7.2 has a stop codon on the PEXEL motif and is

predicted as non-exported. In addition to this, knock-out studies on three PfFIKKs suggests that,

although not essential for parasite survival, PfFIKK4.1, PfFIKK7.1 and PfFIKK12 play roles in

remodeling the infected erythrocyte membrane. This role might be achieved by the FIKKs to be

directly present in the IE membrane. Finally, although the presence of these sequence motifs

suggests a secreted function, this is not universally true. For example, despite containing a

PEXEL motif (RNLSE) and TMD, one of the PfFIKKs, PfFIKK9.2 was localized inside the

parasite in infected erythrocytes (Table 1.2).

FIKK8s (Pf and Pr) and the sole FIKKs from other organisms lack the PEXEL motif in the

sequence. They also do not contain any TMD or SP. Accordingly, FIKK8s and sole FIKKs are

predicted to be non-exported kinases. Indeed, a co-localization study by Skariah et al. (2016) has

discovered that FIKK from T. gondii, TgFIKK, is located in the posterior part of T. gondii

tachyzoites in the infected host cells and co-localized with the proteins from the basal complex.

TgFIKK is not essential in the lytic cycle in vitro and for acute infection in vivo (Skariah et al.

2016).

18

CpFIKK orthologues clustered together. The tree was created with ClustaW2-Phylogeny tool

using multiple sequence alignment of the full sequences of FIKKs and was rendered with

TreeDyn server. A neighbor-joining algorithm was employed having distance matrix enabled.

Figure 1.6 Phylogenetic tree of the FIKKs from different apicomplexans.

CpFIKK and

orthologues

19

1.6.3 Lone FIKKs are orthologous to FIKK8 from P. falciparum

Pf- and PrFIKK8 and the sole FIKKs from other apicomplexan organisms, e.g. CpFIKK, lack all

of the three N-terminal components found in the other Pf- and PrFIKKs. Sequence analysis of

the kinase domains of FIKK8s and orthologous FIKKs shows that they have higher sequence

similarities than rest of PfFIKKs. Moreover, on average, FIKK8s and sole-FIKKs have longer

protein sequences than other FIKKs. To further investigate the evolutionary relationship of the

FIKKs, I created a phylogenetic tree with the full sequences of the FIKKs from different

organisms (Figure 1.6). FIKK8s and the sole FIKKs from different apicomplexans were found to

group together suggesting that FIKK8s from Pf- and PrFIKK family are homologues of the sole-

FIKKs in different apicomplexan genomes. FIKK8s may also be the ancestral FIKK member in

the P. falciparum and P. reichenowi genomes too, where the FIKK family has been expanded in

to 21 members.

1.6.4 FIKKs contain putatively active kinase domains

Typical eukaryotic PKs contain a conserved catalytic core that is important for activity.

Sequence comparison of FIKKs with a canonical protein kinase, human protein kinase A

(HsPKA), showed that most of the catalytically important residues are conserved among FIKKs,

with some notable exceptions (Table 1.4). A characteristic feature of an active protein kinase is

the presence of the catalytic triad in the sequence, which includes the three invariant residues

D166 (catalytic activity), N171 and D184 (Mg2+

-binding) (residue numbers correspond to

HsPKA). All the known FIKK kinases retain the catalytic triad. The invariant K72 (salt-bridge

and ATP binding) and E91 (in HsPKA) of the canonical ePKs are conserved throughout the

FIKK family. The most conserved other catalytically important residues are also conserved

among FIKK kinase domains (Table 3). These conservations initially suggest putatively active

kinase members are present in the FIKK family.

Two key regions play critical roles during catalysis reaction by kinases - the catalytic loop and

the activation segment. Subdomain VIb contains the catalytic loop, which has a conserved motif

HRDLKxxN (Y instead of H in HsPKA). FIKK8s, CpFIKK and other orthologues contain an

invariant HLDLTPEN motif in the catalytic loop. The invariant D of this motif acts as the

catalytic base that accepts the hydrogen removed from the hydroxyl group being phosphorylated.

20

The arginine in this motif is often associated with autophosphorylation of the kinase at the

activation loop. FIKKs lack this residue and in most cases this residue is replaced by a Leu. The

activation segment starts with a conserved DFG motif and ends with APE motif. In FIKKs, the

2nd

and 3rd

positions of DFG motif are not conserved, and have D(L/F)(S/A/G) sequences. The

invariant D chelates Mg2+

that bridges gamma and beta phosphates of ATP and positions the

gamma phosphate for transfer to substrate.

Protein kinases toggle between active and inactive conformations by forming and deforming two

hydrophobic spines - the catalytic spine (C-spine) and the regulatory spine (R-spine). During

activation, the spines are formed as a result of binding of ATP. The C-spine is composed of the

following residues A70, V57, L173, I174, M128, M231 and L227 in HsPKA and the ATP

molecule is positioned between V57 and L173 during activation. The R-spine is composed of

Table 1.3 Domain boundaries of the kinase domains (KD) of the FIKK proteins

from different apicomplexans.

21

four residues L106, L95, F185, Y164 in HsPKA. Construction of both spines is required for a

kinase to obtain an active conformation. Unfortunately, no structure of any member of the FIKK

family has been solved yet and therefore, pinpointing the spine components in FIKKs is

particularly difficult, given the fact that some regions of the FIKK kinase domain sequences do

not align well with any other ePKs. The closest structural homologue of CpFIKK kinase domain

in protein data bank (PDB.org) is human MNK1 kinase having less than 19% sequence identity

(PDB ID 2HW6). As this sequence identity, it would be too speculative to predict the spine

residues from sequence alignments and homology modelling.

About 10% of human kinases are predicted to be pseudokinases (Manning et al. 2002), enzymes

that share sequence similarity with protein kinases but lack some or all of the residues important

for catalysis. The fraction increases in some parasite kinomes; the Toxoplasma kinome, for

example, is predicted to contain ~30% pseudokinases (Peixoto et al. 2010). Pseudokinases

usually lack multiple catalytically important residues and components of the spines and are

unable to form the hydrophobic spines. Increasing evidence suggests that pseudokinases have

potential noncatalytic functions in signaling pathways. For example, ROP2 from T. gondii, a

pseudokinase that has an incomplete C-spine, has been found to play critical role in parasite

virulence (Taylor et al. 2013).

1.6.5 The FIKK kinase domains share some features of atypical protein kinases

The overall sequence identity between FIKKs and other eukaryotic protein kinases is very low.

FIKKs may this be considered a diverged kinase sub-family, and be classified as atypical protein

kinases.

Atypical protein kinases are catalytically active and share structural features and catalytic core

with typical protein kinases, but can vary in some of the conserved motifs comprising classical

protein kinases domains. For example, Haspin, an active atypical protein kinase, contains a non-

canonical DYT motif instead of DFG and the APE motif is completely absent.

Here, I examined FIKK sequences to determine if FIKKs have atypical kinase like features. The

first noticeable difference in FIKKs is that their kinase domains do not have the canonical

22

GxGxxG motif in the first subdomain. Instead, FIKK8s and orthologues have a conserved

GxSxxS motif (Figure 1.6). Sequences in other FIKKs vary in this region, but the first Gly is

present in most of the members. Indeed, the first Gly position is the most conserved in the motif

throughout the protein kinome. This said, although highly conserved, the canonical GxGxxG

motif is not essential for a protein kinase to be active.

Conserved motifs and the sub-domain boundaries are indicated on the top of the alignment.

Activation segment (DFG to APE motifs) is shown by red bar. The subdomain boundaries are

determined by following Hanks and Hunter (1995).

Non-canonical DLG or DYG motifs are found in many other protein kinases, including human

and apicomplexan kinases, e.g. HsNek7 (DLG), HsNek9 (DYG) and PfCDK (DLG) etc.

Substitution of Ala by Pro in the APE motif is not very rare either. Examples include human

Janus kinase 2 (HsJak2) and Bruton’s tyrosine kinase (HsBtk). In some proteins, the Ala is

Figure 1.7 Sequence alignment of the kinase domains of the orthologous FIKKs from

different apicomplexans.

23

replaced by Ser, e.g. in interleukin-2-inducible T-cell kinase (Itk). Interestingly, beside FIKKs,

another parasite specific kinase family, ROPKs, also contain the PPE motif instead of APE in the

activation segment. Thus, although the FIKK sequences diverge from the canonical motifs, each

FIKK variant can be found in at least one other member of the eukaryotic protein kinome.

In addition to the above features, some of the FIKK-specific conserved motifs and insert

sequences are unique to the FIKK family. Non-canonical insert sequences have been found

mostly in the subdomain II, III, IV and VIII regions of different FIKKs sequences. The activation

segment of Plasmodium FIKKs contains a 16-residue insert, and the FIKKs from

Cryptosporidium and Toxoplasma species have an even longer insert in this region. Sequence

alignment of the kinase domains of the orthologous FIKKs is shown in Figure 1.7.

1.6.6 Evidence for catalytically active FIKKs

Sequence analysis can be used to predict whether a protein kinase is catalytically active, but

experimental evidence is still essential for confirmation. Some unique sub-families of kinases

present in Apicomplexans are predicted to be pseudokinases and some, for example ROPKs in

Toxoplasma parasites, have many predicted pseudokinases (Talevich and Kannan 2013). Some

of the pseudokinases appear to be functionally important for parasite biology, despite lack of

catalytic ability.

All the FIKK sequences found in the parasite genome databases have complete catalytic triads

(D166, N171 and D184 in HsPKA) and on this basis are predicted to be catalytically active.

However, other features in the sequences in some of the FIKKs are more consistent with

pseudokinases. For example, four FIKK sequences, PfFIKK7.2, PrFIKK7.2, PrFIKK9.5 and

PrFIKK9.2 contain ‘in sequence’ stop codon(s). Although these may result from genome

sequencing errors or database deposition failures, they are for now considered pseudokinases.

Another FIKK8 orthologue, EtFIKK, lacks the first, second and last subdomains of the canonical

kinase domain. Two other sequences - PfFIKK4.2 and PrFIKK4.2, contain long inserts of

repeated sequences in the kinase domain, although an insert-excluded recombinant version of the

PfFIKK4.2 showed in vitro kinase activity by one study (Kats et al. 2014).

24

The FIKK family members have proven difficult to express and purify, and thus not many have

been shown to be catalytically active. Exported FIKK members, including PfFIKK4.1,

PfFIKK4.2, PfFIKK12 and PfFIKK4.1, have been found to be active (Nunes et al. 2010; Nunes

et al. 2007; Kats et al. 2014; Brandt and Bailey 2013). PfFIKK4.1 was able to phosphorylate

human dematin , and PfFIKK4.2 and PfFIKK12 phosphorylated myelin basic protein (MBP), a

generic kinase substrate (Kats et al. 2014; Nunes et al. 2007). I have also shown recently that the

putatively non-exported FIKKs, PfFIKK8 and CpFIKK show both auto-phosphorylation and

kinase activity on specific substrates. Thus, we are now accruing increasing evidence that the

FIKKs comprise a family of catalytically active kinases.

Table 1.4 Key catalytic motifs of FIKK proteins from different organisms.

25

1.7 Conclusion

In broad terms, I set out to explore FIKKs as drug targets. The specific objectives of my thesis

are to - a) better understand the biochemistry of orthologous FIKK proteins from C. parvum

(CpFIKK) and P. falciparum (PfFIKK8), b) identify inhibitors against CpFIKK as chemical

probes or early drug leads, in order to provide confidence in the concept that FIKK8 might be a

drug target, c) investigate if the chemical inhibitors have biological relevance. To achieve these

goals, I carried out the following experiments during my PhD research project. First, I

developed methods to express and purify the FIKK kinase domains from C. parvum and P.

falciparum. In so doing I identified a new domain, found adjacent to the kinase domain, and

showed that this domain was essential for expression of the FIKK kinase domain. Second, using

biochemical and screening approaches, I characterized the enzymatic properties of the enzyme,

as well as its substrate preferences. Third, I developed a screening assay and I identified small

molecule inhibitors of CpFIKK enzyme, and confirmed inhibition using orthogonal assays.

Fourth, I performed structure-activity relationship assay with compounds targeting CpFIKK and

checked selectivity of potent compounds against panels of CpKinases and human kinases. Fifth,

I investigated if potent CpFIKK inhibitors have any effect on the cellular growth of the parasites.

My studies will help us understanding the biochemistry, biology and inhibition of FIKK kinases

from apicomplexan parasites.

26

Chapter 2 Part of this chapter has been published in the Journal of

Molecular and Biochemical Parasitology, 2015 Jun;201(2):85-9.

doi: 10.1016/j.molbiopara.2015.06.002.

Biochemical characterization of FIKKs from C. 2

parvum (CpFIKK) and P. falciparum (PfFIKK8)

Protein kinases play essential roles in many important cellular pathways. Their catalytic

activities are regulated in many different ways (Endicott, Noble, and Johnson 2012; Huse and

Kuriyan 2002; L. N. Johnson, Noble, and Owen 1996; Nolen, Taylor, and Ghosh 2004) and

substrate specificity is achieved mostly through the recognition of the peptide sequence around

the phospho-acceptor residues but also via their cellular localization. The rate of the reactions,

ligand binding affinity, substrate preference and regulation of the catalysis differ from kinase to

kinase based on their sequences and structures. As yet it is not possible to predict all the catalytic

functions of these enzymes and thus it is essential to biochemically characterize a novel kinase to

understand its behaviour.

My goal in this chapter is to understand the biochemical features of FIKK kinases from P.

falciparum and C. parvum. I used high-throughput techniques to clone, express and purify

FIKKs from the two parasites and developed enzyme assays to characterize them and determine

substrate preference. This is the first biochemical characterization of the evolutionarily

conserved FIKK proteins from C. parvum (CpFIKK) and P. falciparum (PfFIKK8).

2.1 Soluble FIKK constructs include an extension region

To improve the chance of obtaining an expressible, soluble and stable protein construct, I

initially cloned at least 6 constructs of different lengths into bacterial expression vectors, usually

keeping the predicted catalytic domains intact. To design the lengths of the constructs, I used

bioinformatic prediction tools that predicted catalytic domain lengths (Prosite and Pfam),

secondary structures (psipred), and disordered (psipred), hydrophobic (TMpred) and

transmembrane (TMpred) regions.

27

21 PfFIKK8, 6 CpFIKK and 9 TgFIKK constructs were cloned and tested for expression. The

well-expressed constructs (>1 mg soluble and stable protein per litre of culture) are marked by

“Y” in the right column. Among the expressing constructs, PfFIKK8l (N1025-L1457), PfFIKK8o

(M1049-L1457) and CpFIKKd (K344-I796) were selected for the biochemical analysis.

Table 2.1 Cloning and expression profile of constructs of PfFIKK8, CpFIKK and TgFIKK.

28

The catalytic domains are located at the C-termini of the FIKK sequences, and therefore I mostly

varied the lengths at the N-terminal regions of the kinase domains (KD). I tried to design

constructs avoiding the disordered and hydrophobic regions in the N-terminal regions and

keeping the catalytic domain intact.

I initially cloned 6-8 constructs for each of PfFIKK8, TgFIKK and CpFIKK (Table 2.1) using

an Escherichia coli system successfully used previously for parasite PKs (Vedadi et al. 2007),

and that appended an N-terminal hexa-His tag to help to purify the proteins. None of the TgFIKK

constructs expressed a soluble version. Many constructs of PfFIKK8 and CpFIKK expressed, but

only those including an extension at the N-terminus of the predicted kinase domain (KD) yielded

soluble and stable protein samples (Table 2.1). To understand the expression pattern better, I

screened an additional 21 construct of PfFIKK8, more rigorously exploring the requirement for

the extension region (P915 to K1056). I found that the inclusion of 38 or more residues at the N-

terminal extension (NTE) was essential for the expression of the protein (Table 2.1, Figure 2.1).

The inclusion of an extra N-terminal region was needed to express CpFIKK. For PfFIKK8 the

minimal well expressed construct encoded 409 amino acid residues (M1049-L1457). The NTE

contains both polar and non-polar residues as well as multiple potential phospho-acceptor

residues (S/Y) (Figure 2.1). To obtain protein for biochemical studies, I purified two constructs

(recombinant proteins) from PfFIKK8 and one of CpFIKK

(PfFIKK8l, PfFIKK8o and CpFIKKd in Table 2.1) by Ni-affinity and size exclusion

chromatography. The purity is shown in the Figure 2.2. The proteins were concentrated to >5

mg/mL. These were used to assess their auto-phosphorylation activity and determine their ability

to phosphorylate a set of common peptide substrates.

The shortest soluble construct was PfFIKK8o, containing an N-terminal extension starting at

S1049. The predicted kinase domain based on alignment with known kinases without an N-

terminal extension (i.e. a few amino acids upstream of the glycine-rich region) starts around

PfFIKK8s at D1087.

Figure 2.1 (A) Test expression gel showing that constructs PfFIKK8h to PfFIKK8o (see

Table 2.1) were soluble. (B)

29

10 µg of protein of each sample was resolved by SDS-PAGE and stained with Coomassie Blue.

Protein molecular weight markers show the relative size of the proteins.

95 kDa

70 kDa

62 kDa

51 kDa

42 kDa

29 kDa

22 kDa

PfFIKK8

Figure 2.2 Purity of the purified proteins.

30

2.2 CpFIKK and PfFIKK8 are active kinases

Almost 10% of the human kinases are predicted to be catalytically inactive or pseudokinases

(Manning et al. 2002). Toxoplasma has the highest number of pseudokinases (about 30% of the

genome) of all known genomes (Peixoto et al. 2010). Therefore, there is always a chance that a

newly characterized kinase from an apicomplexan genome turns out to be catalytically dead.

Pseudokinases harbour mutations in the positions that are critically important residues for

catalysis (Manning et al. 2002). However, despite the mutations, some of the pseudokinases (e.g.

WNK kinases) that were thought to be catalytically incompetent find ways to catalyze the

phosphorylation reaction (Xu et al. 2000; Taylor and Kornev 2011). Therefore, prediction of

catalytically active kinases from the sequence is not very straightforward and this is especially

difficult if the kinase family is evolutionary distant from the known and well-studied families

and sequence similarity is poor. Because of these considerations, although the sequence

prediction suggests that PfFIKK8 and CpFIKK are active kinases (Chapter 1), it was still very

important to show this.

2.2.1 Purified FIKKs bind ATP

Protein kinases catalyze the transfer of the γ-phosphate from a Mg2+

-ATP complex to their

substrates. Although most of the catalytically important residues are predicted to be present in

FIKK kinases, FIKKs lack the canonical GxGxxG ATP-binding motif, raising the possibility that

the kinases do not interact with ATP. To test whether the purified FIKKs bind ATP, I employed

differential static light scattering (DSLS), which is a thermal shift assay (Vedadi et al. 2010;

Senisterra et al. 2006). I found that the stability of the proteins increased when bound to ATP and

ADP, where ADP-binding showed higher stability (Figure 2.3). This suggests that the ATP-

binding pocket of the kinase is functional.

However, ATP-binding alone does not provide evidences of an active kinase and despite having

ATP-binding ability, some pseudokinases are not catalytically active or perform phosphorylation

reaction (Murphy et al. 2014). Therefore, even though PfFIKK8 and CpFIKK proteins bind ATP,

there is a need to prove that the recombinant kinases are catalytically active. We employed

phosphorylation reaction assays for this proof.

31

Purified CpFIKK denatured in the presence or absence of ligands and the melting temperature

was measured, as described in the Materials and Methods. The transition points or melting

temperatures (Tm) were computed (inset). The ATP containing sample (red) showed a 2oC

temperature shift (ΔTm) from the control sample (no ligand). ADP produced the highest ΔTm

and non-hydrolysable ATP analogue, AMPPNP, showed almost similar ΔTm as that of ATP.

2.2.2 CpFIKK and PfFIKK8 autophosphorylate

Protein kinases display a variety of regulatory mechanisms (L. N. Johnson, Noble, and Owen

1996; Huse and Kuriyan 2002; Nolen, Taylor, and Ghosh 2004). A common method of

activating a protein kinase is through auto-phosphorylation, where the kinase adds a phosphate

group to itself (cis) or to another protomer (trans). Autophosphorylation activity can be an initial

indication of a whether a protein kinase does or does not have catalytic activity. In the absence of

knowing a suitable substrates, I tested if the purified FIKKs autophosphorylate.

To test for autophosphorylation, purified PfFIKK8 (PfFIKK8l) and CpFIKK

(CpFIKKd) samples were incubated with ATP and MgCl2 (2 mM each) for overnight at room

Figure 2.3 Thermal melting curves of CpFIKK and ΔTm values for both kinases with

different ligands.

32

temperature. The samples were then trypsinized and the peptides resolved using LC–MS–MS. I

used the maps of the resulting peptides (Figure 2.4 A and B) to identify 6 phospho-serines (pS)

in the Plasmodium sample, 5 phospho-serines and 4 phospho-threonines (pT) in

the Crytposporidium sample and one phospho-tyrosine (pY) on each. One of the phosphorylated

serines (pS1320) on PfFIKK8 is located in the predicted activation loop. The corresponding

serine on CpFIKK was not detected. Furthermore, some of the phosphorylated residues were

found in the NTE, including a phospho-serine that is conserved in both proteins.

2.2.3 CpFIKK and PfFIKK8 phosphorylate exogenous substrates

Given that the FIKK samples are active, I set out experiments to determine if the FIKKs are

active on exogenous substrates, using bovine casein, bovine histones (H1 and H3), bovine

myelin basic protein (MBP) and Syntide-2 (PLARTLSVAGLPGKK) as generic substrates. I

incubated the kinases in the presence of ATP and MgCl2 (same conditions as

autophosphorylation reactions) and monitored the phosphorylation by LC-MS-MS. Both proteins

(PfFIKK8 and CpFIKK) phosphorylated bovine MBP and Syntide-2. MBP was phosphorylated

on multiple Ser and Thr residues LC-MS-MS (Figure 2.4 C). Since MBP is particularly Arg-

rich, this led to my initial hypothesis that the FIKKs might prefer basic residues in their

substrates.

Both FIKKs were autophosphorylated and MBP was phosphorylated by CpFIKK. Residues with

post-translational modifications are highlighted in green. Yellow regions represent those

identified by MS. In A and B Phospho-serines in the NTE are highlighted in red while those in

the catalytic domain are circled in blue. Conserved phosphoserines are highlighted in solid

squares. Serines that are conserved in both proteins but only phosphorylated in one are

highlighted in squares with a dashed border. The latter includes a serine in the activation loop

(pS1320) on PfFIKK8l. While this serine is conserved in CpFIKKd, it was not covered in the MS

analysis. All the phosphorylated residues are circled blue in MBP (C).

Figure 2.4 Maps of phosphorylated residues in (A) PfFIKK8l and (B) CpFIKKd (C) MBP.

33

2.3 CpFIKK and PfFIKK8 target serine with flanking arginines

The cellular substrates for the FIKKs are unknown, but understanding the substrate sequence

preferences may provide clues to identifying potential biological substrates. Here I carried out

two different experiments to discover the substrate preferences of CpFIKK and PfFIKK8.

2.3.1 Positional-scanning peptide array

To determine the sequence preferences of PfFIKK8l and CpFIKKd in an unbiased manner, I

collaborated with Dr. Benjamin Turk’s laboratory at Yale University to test the proteins using a

positional-scanning peptide array (C. Chen and Turk 2011). Both kinases demonstrated a strong

preference for basic residues (Figrue 2.5), primarily Arg at positions −3 and +3 relative to the

A

B

C

34

phosphorylation site. Arginine in the −4 position was also favoured by both kinases, albeit not as

strongly. In addition, both selected Ser over Thr as the phosphate acceptor by about 2–3-fold

(Tables 2.2 and 2.3).

A), B) Peptide array probing FIKK8 substrate specified by radiolabeled kinase assay. Different

amino acids and their fixed positions are mentioned at the X and Y axis respectively. The spot

intensity at the 0 position shows that FIKK8s prefer Ser over Thr/Tyr. Arg at the +3 and -3

positions were highly preferred by the FIKK8s. C), D) The sequence logo of FIKK8s substrate

preference was built from the normalized average intensities of the spots from the peptide array.

Intensities having higher values than phosphor-acceptor residues were only taken into account.

The height of the amino acids is indicative of the probability of the preference at the single

position.

Figure 2.5 Peptide substrate preference array of PfFIKK8 and CpFIKK identifies a motif.

A B

C D

35

Table 2.2 Normalized average intensity values of enzyme screening spots for PfFIKK8l in

Fig. 2.5 A (average of two runs).

-5 -4 -3 -2 -1 0 +1 +2 +3 +4

P 0.72 0.47 0.28 0.03 2.05 0.03 0.16 0.27 1.19

G 0.59 0.71 0.21 0.82 1.12 0.95 0.27 1.11 1.05

A 0.55 0.62 0.27 1.05 2.35 0.41 0.88 0.85 0.99

C 0.88 0.66 0.14 0.41 1.51 0.57 1.72 0.80 0.58

S 1.91 1.00 0.55 5.49 0.98 1.39 1.33 3.53 1.48 1.44

T 1.74 1.00 0.28 3.10 0.96 0.61 1.05 3.08 0.71 1.09

V 1.27 0.89 0.15 0.66 0.62 0.25 1.14 0.50 0.86

I 1.32 0.98 0.13 0.33 0.48 0.28 0.26 0.51 0.99

L 1.08 0.43 0.13 0.74 0.12 0.69 0.18 0.67 0.91

M 0.95 1.09 0.23 1.49 0.55 2.05 0.24 1.35 1.13

F 0.97 0.86 0.24 0.49 1.32 1.26 1.09 0.98 1.10

Y 1.87 1.12 0.12 0.48 1.65 1.59 1.58 1.01 0.79

W 1.20 1.04 0.18 0.41 1.73 0.61 1.45 1.42 1.21

H 0.85 1.25 1.87 0.62 2.33 2.19 1.98 1.27 1.69

K 1.04 1.47 2.89 0.63 0.34 0.86 0.30 1.31 1.91

R 0.71 2.96 11.68 0.96 0.49 1.88 0.31 4.52 0.84

Q 0.71 1.32 0.31 1.34 0.68 0.41 0.19 0.34 0.92

N 0.58 0.93 0.15 0.28 0.33 3.38 0.49 0.34 0.63

D 0.57 0.52 0.07 0.32 0.12 0.12 0.51 0.41 0.44

E 0.48 0.67 0.12 0.34 0.28 0.10 0.60 0.14 0.24

pT 0.43 0.94 0.14 0.13 0.38 0.05 0.11 0.06 0.14

pY 0.97 1.51 0.18 0.24 1.46 0.19 0.16 0.11 0.14

36

Table 2.3 Normalized average intensity values of enzyme screening spots for CpFIKKd in

Fig. 2.5 B (average of two runs).

-5 -4 -3 -2 -1 0 +1 +2 +3 +4

P 0.57 0.88 0.64 0.10 2.10 0.07 0.09 0.11 1.17

G 1.14 0.95 0.44 0.83 1.69 0.37 0.30 0.67 0.75

A 0.61 0.83 0.60 1.43 1.91 0.64 0.74 0.48 0.97

C 0.75 0.93 0.24 0.50 1.62 0.65 2.14 0.53 0.71

S 2.18 1.37 0.61 4.35 1.52 1.58 1.43 2.48 0.82 1.58

T 1.32 0.89 0.35 2.45 0.89 0.42 0.92 2.24 0.37 0.87

V 0.75 0.96 0.22 0.68 0.68 0.42 1.01 0.22 0.96

I 0.69 1.11 0.23 0.45 0.53 0.70 0.50 0.30 1.23

L 1.24 0.60 0.17 0.82 0.19 1.58 0.25 0.45 0.86

M 1.08 1.02 0.35 1.43 0.62 2.69 0.35 0.74 0.95

F 1.05 0.79 0.29 0.76 1.27 2.50 1.25 0.66 0.97

Y 1.39 0.99 0.36 0.64 1.59 2.44 2.04 0.73 1.07

W 0.71 0.65 0.34 0.48 1.53 1.33 1.35 0.72 0.87

H 0.94 1.08 1.69 0.67 1.92 1.36 1.80 1.46 1.65

K 1.39 1.18 2.84 0.53 0.32 0.37 0.42 1.18 2.04

R 1.59 2.18 9.42 0.69 0.26 0.46 0.82 9.37 0.91

Q 0.93 1.31 0.53 1.35 0.53 0.37 0.66 0.31 1.10

N 0.53 0.86 0.29 0.48 0.41 1.48 0.86 0.53 0.58

D 0.51 0.64 0.16 0.53 0.13 0.07 0.27 0.23 0.37

E 0.64 0.77 0.22 0.83 0.29 0.15 0.44 0.08 0.39

pT 0.87 1.24 0.32 0.23 0.43 0.04 0.21 0.06 0.12

pY 0.98 1.49 0.35 0.55 1.71 0.34 0.49 0.06 0.16

37

2.3.2 Characterization of enzyme parameters using optimized substrates

To evaluate the contributions of the arginines and other flanking residues to phosphorylation

efficiency, we designed an optimized peptide substrate (PO) with the sequence RRRAPSFYRK,

as well as three variants. The variants featured mutations of Arg to Ala at the +3 (PAR) and −3

(PRA), as well as PT, a truncated version of PO. Using an LDH–PK coupled kinase

assay (Kiianitsa, Solinger, and Heyer 2003), I compared the kinetics of both FIKKs using these

substrates. The two PfFIKK8 constructs and CpFIKKd behaved very similarly to each other

with almost all the peptides (Figure 2.6). All the kinetic parameters were within the same order

of magnitude, though CpFIKKd appeared more active than its P. falciparum orthologues. Similar

Michaelis constants (Km) and catalytic efficiency values (kcat) were obtained using PO and PT for

all three FIKK samples. On the other hand, both mutants (PAR and PRA) resulted in

higher Km and lower kcat values, thus, emphasizing the importance of the flanking arginines.

All three FIKK8 constructs are most active against PO and PT (the truncated version of PO).

Replacing Arg in the -3 or +3 position substantially reduced activity in most cases. PfFIKK8o

behaved very similarly as the longer PfFIKK8l.

Figure 2.6 Enzymatic activities of recombinant FIKKs (A) PfFIKK8o (B) PfFIKK8l

and CpFIKKd (C).

38

0 50 100 150 200 250 300

0

200

400

600

800

1000

1200

1400

[ATP] (μM)

Velo

city (

nm

ol/m

in/m

g)

CpFIKKd

PfFIKK8o

PfFIKK8l

Figure 2.7 Enzyme kinetics of FIKKs with ATP.

39

Table 2.4 List of consensus peptides tested and the kinetic parameters obtained, including

the Michaelis constant (Km), turnover (kcat) and catalytic efficiency (kcat/Km).

Peptides Km (μM) kcat (min-1) kcat/Km (μM

-1min

-1)

Name Sequence PfFIKK8l CpFIKKd PfFIKK8l CpFIKKd PfFIKK8l CpFIKKd

PO RRRAPSFYRK 11.0±0.9 7.0±1.5 31.0±0.7 77.0±4.6 2.8 12

PAR RRAAPSFYRK 74.0±6.3 10.0±0.4 38.0±1.8 70.0±0.7 0.5 7

PRA RRRAPSFYAK 150±42 100±4 20.0±5.6 120.0±2.8 0.1 1.2

PT --RAPSFYR- 19.0±0.8 18±1 31.0±0.5 80.0±1.5 1.6 4.5

Table 2.5 Kinetic parameters of purified FIKK proteins in using ATP (PT was used as the

phospho-acceptor substrate).

2.4 Materials and methods

2.4.1 Cloning and expression

The full length DNA constructs of PfFIKK8 and CpFIKK were cloned from cDNA library of P.

falciparum 3D7 (generous donation from the laboratory of Dr. Kevin Kain, Toronto General

Hospital) and C. parvum Iowa I genomic DNA (obtained from MR4) into the pET15-MHL

Km (μM) kcat (min-1) kcat/Km (μM

-1min

-1)

PfFIKK8l CpFIKKd PfFIKK8l CpFIKKd PfFIKK8l CpFIKKd

11.0±0.8 48.0±0.9 16.0±0.3 78.0±0.5 1.6 1.6

40

vector (http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php). DNA constructs were

sub-cloned from the full-length constructs and appended with an N-terminal hexa-histidine tag

including an integrated TEV cleavage site (MHHHHHHSSGRENLYFQ*G) into the pET15-

MHL vector. Small-scale test expression was performed with all the cloned constructs according

to Savitsky et al. (2010).

All constructs were grown and purified as previously described (Vedadi et al. 2007) using the

Lex bioreactor system (Harbinger Biotechnology and Engineering Corp. (Epiphyte3), Toronto,

ON, Canada) and BL21(DE3)-V2R-pACYC-LamP as the expression host, which includes a

plasmid for co-expression of λ-phosphatase to suppress protein phosphorylation. To mitigate the

effect of the high number of cysteines, 2 mM TCEP was included in every purification steps to

minimize aggregation. Both PfFIKK8 and CpFIKK kinase domains eluted as monomers from a

Superdex S200 gel-filtration column (GE Life Sciences). The identities of the purified proteins

were verified by mass spectrometry analysis (ESI-TOF, Agilent Technologies, Toronto, ON,

Canada), which also confirmed the absence of phosphorylation. Proteins were concentrated to >8

mg/mL in a buffer containing 10 mM HEPES (pH 7.5), 500 mM NaCl and 2 mM 2-

mercaptoethanol or TCEP, and stored at –80oC.

2.4.2 Differential static light scattering (DSLS)

To study the stability of the purified FIKK samples and to identify potential ligands, we assayed

samples using differential static light scattering. This was carried out using the StarGazer

instrument (Harbinger Biotechnology and Engineering Corp. (Epiphyte3), Toronto, Canada).

The assay was performed using 2 µM of protein and 2 mM ligand in 384-well plates. Samples

were buffered in 100 mM HEPES (pH 7.5), and included 150 mM NaCl. The experiments were

conducted between 20°C to 85°C at a heating rate of 1°C per minute. The recorded scattered

light reads were fitted to the Boltzmann sigmoid function using Bioactive software and plotted

against the temperature (Figure 2.3), and the inflection point is termed Tm.

41

2.4.3 In vitro phosphorylation

For phosphorylation reactions, 20 µM of purified PfFIKK8 and CpFIKK were incubated for 12

h, 24 h and 48 h at room temperature in reaction buffer (10 mM HEPES (pH7.5), 500 mM NaCl

and 2 mM TCEP (tris(2-carboxyethyl)phosphine) containing 2 mM ATP and 2 mM MgCl2, and

the reactions were stopped by adding 10 mM EDTA. To measure MBP phosphorylation, 100 µM

of MBP (Sigma) was added to 20 µM FIKK proteins in reaction buffer containing 2 mM ATP

and 2 mM MgCl2 and incubated for 12 h at room temperature. Phosphorylation was detected by

mass spectrometry, by measuring an 80 Da mass increase using an Agilent electrospray-

ionisation time-of-flight (ESI-TOF) mass spectrometer. Prior to injection into the mass

spectrometer, the protein was resolved from small molecules by liquid chromatography on a C3

reverse-phase column with 0.1% formic acid and eluted with a methanol gradient. MS data were

analyzed with Agilent TOF Protein Confirmation Software.

Phosphorylated residues on FIKKs and MBP were mapped by LC-MS-MS in the Mass

Spectrometry facility of SickKids, Canada. For this experiment, proteins were digested with

trypsin (13 ng/µL trypsin in 50 mM ammonium bicarbonate and 10 mM DTT) for 3 hours at

37oC. The digested peptides were loaded onto a 150 μm ID pre-column (Magic C18, Michrom

Biosciences) at 4 μL/min and separated over a 75 μm ID analytical column packed into an

emitter tip containing the same packing material. The peptides were eluted over 60 min. at 300

nL/min. using a 0 to 40% acetonitrile gradient in 0.1% formic acid using an EASY n-LC nano-

chromatography pump (Proxeon Biosystems, Odense Denmark). The peptides were eluted into

an LTQ-Orbitrap hybrid mass spectrometer (Thermo-Fisher, Bremen, Germany) operated in a

data dependent mode. MS was acquired at 60,000 FWHM resolutions in the FTMS and MS-MS

was carried out in the linear ion trap. 6 MS-MS scans were obtained per MS cycle. The Raw data

was searched using Mascot (Matrix Sciences, London UK). The final data were analyzed with

SF3 software (Proteome Software, Scaffold, www.proteomesoftware.com/products/scaffold)

2.4.4 Kinase substrate peptide assay

This work was performed in collaboration with the laboratory of Dr. Benjamin Turk at Yale

University, USA. The substrate specificities of PfFIKK8 and CpFIKK were determined by

42

incubating 20 nM of each purified enzyme with an array of 200 peptide mixtures (50 µM in 50

mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1% Tween 20, 50 µM ATP

including 0.03 µCi/µl [γ-33

P]ATP) as described (Yeh et al. 2011). Most (198) peptide mixtures

had the general sequence Y-A-X-X-X-X-X-S/T-X-X-X-X-A-K-K(biotin), where X indicates an

equimolar mixture of the 17 amino acids excluding Cys, Ser and Thr, and S/T indicates an

equimolar mixture of Ser and Thr. In the context of this sequence, a single X position was fixed

as one of the 20 unmodified amino acids, pThr or pTyr. Two additional peptides having the

sequence Y-A-X-X-X-X-X-Z-X-X-X-X-A-K-K(biotin), in which Z was either Ser or Thr, were

included to assess phosphoacceptor residue preference. The assay was performed in 1536-well

plates (2 µL/well). After incubating 2 hr at 30 ºC, aliquots (200 nl) from each well were spotted

onto a streptavidin membrane (SAM2 biotin capture membrane, Promega), which was washed,

dried and exposed to a phosphor imager screen. Spot intensities were quantified using the

software accompanying the imaging system (QuantityOne, BioRad). The intensity data were

normalized by dividing each value by the average of all values corresponding to a single position

in the peptide. Heat maps were generated from log transformed quantified data (average of two

separate experiments for each kinase) using Microsoft Excel.

2.4.5 Enzymatic assay

Kinase activity was measured using an NADH-coupled LDH-PK ATPase assay according to the

method previously described by Kiianitsa et al. (2003). A 96-well format was used and the

method was adapted for use with high ATP concentrations. Peptides were obtained from Peptide

2.0 (Peptide 2.0 Inc., http://www.peptide2.com). The proteins at different concentrations (1 nM,

5 nM and 10 nM) for different peptides were pre-incubated in reaction solution (including 0.5

mM ATP, 150 µM NADH, 30 µM PEP, 10 mM MgCl2 and a LDH-PK mix from Sigma (with 3

units of LDH per ml)) for 30 mins at room temperature. A 2 fold serial dilution of peptides

starting from 100 µM were added to initiate the reaction. For measuring Km of ATP, 50 µM of

PT (RAPSFYR) was used as phospho-acceptor substrate; and ATP was diluted serially from 250

µM (2 fold serial dilution). In this case, the reaction was initiated by adding ATP to the reaction

solution. Initial rates were calculated and at least triplicate data were analyzed using SigmaPlot 9

(Systat Software Inc., www.sigmaplot.com).

43

Chapter 3 Part of this chapter has been submitted in the Journal of

Medicinal Chemistry and is currently under review.

Identification of potent inhibitors of CpFIKK 3

A potent, selective and cell permeable inhibitor, or a ‘chemical probe’, aids both in

understanding the biological function of a protein and in providing evidence for its suitability as

a drug target (Arrowsmith et al. 2015; Frye 2010). Protein kinase inhibitors, especially the most

common type I inhibitors, are usually very promiscuous due to the highly conserved binding

sites of the kinase, and there are numerous reports as evidence of misusing promiscuous

compounds as chemical probes (reviewed by Arrowsmith et al. 2015). Developing potent and

selective inhibitors against human protein kinases is a subject of extensive ongoing studies

(Knapp et al. 2013), and the highest quality chemical probes against protein kinases are being

recommended by a chemical probes portal (www.chemicalprobes.org/search/site/kinase). A

selective CpFIKK inhibitor would be very useful for understanding CpFIKK biology.

Even though there is no generally accepted definition of a chemical probe, some common and

basic features define the characteristics of inhibitor molecules that can be used as a chemical

probes (Frye 2010): the enzymatic inhibition potency should strongly associate with cellular

and/or in vivo potency; molecular mechanisms should be clearly defined; the probe should be

freely available for research; and the probe should have enough data on its chemical and physical

properties; and the probe should have proven utility to address a biological question.

One of my aims in developing enzymatic and biophysical assay platforms for screening the

CpFIKK proteins was to identify and develop chemical inhibitors of CpFIKK. The first step to

obtain a chemical probe is to identify a chemical compound(s) that binds and inhibits the target

protein. The approach I used was to screen purified CpFIKK against a library of ~2,500 small

molecules enriched for type 1 kinase inhibitors, to select and validate potential hits and to

optimize the validated hits into more selective inhibitors in collaboration with medicinal

chemists. I employed biophysical and biochemical techniques to screen and develop potent and

selective inhibitors against C. parvum FIKK kinase. This chapter describes the results.

44

3.1 Inhibitor screening yielded multiple naphthyridine-based CpFIKK-inhibitors

In order to identify inhibitors of CpFIKK, I screened focused libraries of ~2500 compounds at

100 µM concentration with recombinant and purified CpFIKK kinase using two orthogonal

methods: binding using differential static light scattering (DSLS) and inhibition using enzyme

assays (Materials and Methods). From these studies, I identified two compounds 4a and 4j

(Figure 3.1) as potent inhibitors of CpFIKK kinase; these had IC50 of 3 nM and 224 nM,

respectively. These compounds, both 1,5-naphthyridine derivatives, were derived from the

Published Kinase Inhibitor Set (PKIS) assembled and distributed by GSK (Drewry, Willson, and

Zuercher 2014).

The PKIS set was particularly useful to me because the inhibition and selectivity profiles of each

of these small molecule inhibitors was already known against the human kinome (Elkins et al.

2015), and one of my aims was to develop a FIKK inhibitor with minimal activity against human

enzymes. The compounds have a common scaffold of a 1,5-naphthyridine, an amino-pyrazole

and a phenyl moiety (Figure 3.1)

4a

PKIS ID: GW780159X

IC50 = 3 nM

4j

PKIS ID: GW785804X

IC50 = 224 nM

Figure 3.1 Molecular structures of compounds 4a and 4j. PKIS IDs and IC50 inhibition

potency against CpFIKK are shown.

45

3.2 Selectivity of 4a within C. parvum kinome

Compound 4a was tested for selectivity against 14 Cryptosporidium kinases (Table 3.1) by

DSLS and also, if available, using enzyme assays. The C. parvum kinases were cloned and

characterized either by me or other members of our laboratory. I developed biochemical assays

for some of these enzymes (CpCDPK1 [cgd3_920], CpCDPK3 [cgd5_820], CpCDPK4

[cgd7_40], CpGSK [cgd4_240], CpCDK [cgd5_2510].). My approach to monitor selectivity was

to screen all the compounds for binding to the purified kinases first, and then run enzyme assays

with the binders against the kinases for which I had managed to develop enzyme assays.

Compound 4a stabilized and inhibited only CpCDPK1 significantly (IC50 84 nM); it showed

<2ºC shift against all the other kinases and >10 µM IC50 in the assays.

Interestingly, compound 4a had already showed some selectivity when tested against 300 human

kinases, inhibiting only HsALK5 at 10 nM (Gellibert et al. 2004), HsKDR at 100 nM (33%

inhibition) and two others with >50% inhibition at 1µM (KDR, 67%; MAP4K4, 60%; P38a

MAPK, 50%) (Elkins et al. 2015). In summary, compound 4a was a very promising hit, with

Table 3.1 Cryptosporidium kinases tested for selectivity with compound 4a. The

thermal shift and IC50 values are shown.

46

potent activity against the target kinase and minimal off-target activity against human kinases.

In order to optimize its selectivity and potency, I performed a hit expansion by systematically

exploring the structure-activity-relationship (SAR) for the CpFIKK inhibition and selectivity

against CpCDPK1. The results are described here.

3.3 Structure activity relationship of CpFIKK inhibitors

Compound 4a is a known inhibitor of human HsALK5 kinase (IC50 10 nM) (Gellibert et al.,

2004) and the active sites of CpFIKK kinase and HsALK5 are predicted to be very similar.

Therefore, in the absence of a crystal structure for CpFIKK, I used the inhibitor-bound crystal

structures of HsALK5 (e.g. PDB ID 1VJY) to guide the initial SAR. The idea was to design

compounds by exploring the regions whose modification changed HsALK5 activity and binding

in previous studies (Gellibert et al., 2004).

HsALK5 had been co-crystallized with naphthyridine derivatives that are type I kinase inhibitors

and bind at the ATP binding site of the kinase by interacting with the hinge region. In the crystal

structures, the N1 of the naphthyridine ring and the backbone NH of His-283 make a hydrogen

bond in the hinge region and this bond is critical for the activity of the majority of the known

kinase inhibitors as well as for the ATP binding (Figure 3.2). I did not vary the naphthyridine

ring during my initial exploration of the SAR. Two hydrogen bonds are formed between the

pyrazole moiety and aspartate (D351) from the DFG motif and catalytic lysine (K232). My goal

was to perform the SAR until I obtain a very potent inhibitor. Initially, substitution patterns at the

phenyl group, which extends towards the selectivity pocket the HsALK5 structure (Figrue 3.2),

were explored systematically. All the compounds synthesized were tested for activity against the

CpFIKK kinase and for selectivity against CpCDPK1. The chemical synthesis was performed by

Dr. Juntao Ye and colleagues in the laboratory of Dr. Mark Lautens at the University of

Toronto. The results are summarized in the table 3.2.

47

Interaction of a naphthyridine compound (golden) at the HsALK5 catalytic cleft is shown. A)

features the electrostatic bonds observed between the compound and active site residues. The

naphthyridine group of the compound interacts with the hinge residue H283. Two additional

bonds were observed between the imidazole moiety and the invariant Lys (K232) and Asp

(D351). B) shows the hydrophobic region (purple) where the phenyl moiety is poised. The phenyl

moiety is also very close to the so called gatekeeper residue (S280). (PDB ID 1VJY)

Figure 3.2 The naphthyridine derivative with HsALK5 active site (Gellibert et al. 2004).

48

Table 3.2 CpFIKK kinase activity of compounds 4a-i and selectivity against CpCDPK1.

Compounds Ar CpFIKK IC50 (nM)

CpCDPK1 IC50 (nM)

4a

3 ± 0.3 84 ± 7

4b

0.2 ± 0.1 190 ± 9

4c

42 ± 12 190 ± 20

4d

220 ± 40 210 ± 15

4e

180 ± 20 260 ± 20

4f

4100 ± 750 1500 ± 230

4g

1200 ± 620 350 ± 70

49

4h

>10000 74 ± 15

4i

>10000 >10000

4j

200 ± 40 220 ± 30

4k

3260 ± 820 375 ± 25

4l

>10000 2600 ± 370

Figure 3.3 The enzymatic inhibition of CpFIKK and CpCDPK1 by 4b.

50

By analyzing the activities of the various derivatives, I found that small hydrophobic groups in

the meta position are preferred for CpFIKK activity, and increase selectivity against CpCDPK1.

Compared with the unsubstituted compound (4c), addition of a chloro group (4a) improves

CpFIKK activity by about 15 fold and addition of a methyl group (4b) significantly improves

FIKK activity (IC50 0.2 nM) and selectivity (940 fold) against CpCDPK1 (Figure 3.3). Larger

alkyl groups, ethyl (4h) and isopropyl groups (4i), result in complete loss of CpFIKK activity

and interestingly the addition of an ethyl group (4h) maintains activity for CpCDPK1. Fluoro

(4d) and methoxy (4e) additions decrease CpFIKK activity by 4-5 fold and the more polar cyano

(4f) and trifluoromethy (4g) groups decrease CpFIKK activity significantly. We observed no

significant impact of polar groups at this position on selectivity against CpCDPK1.

At the para position, the fluoro group is tolerated with a moderate drop in CpFIKK activity

(compare 4c and 4j) but did not improve selectivity against CpCDPK1. The chloro (4k) and

methoxy (4l) groups lead to a >100 fold drop (compare 4c and 4k, 4l) in CpFIKK activity. The

effect of di-substitution at the phenyl ring was also explored and the results are summarized in

Table 3.3. Addition of a para fluoro group (4m and 4n) to the most potent CpFIKK compounds,

4a and 4b, lead to a moderate drop in CpFIKK activity and no improvement in selectivity against

CpCDPK1. Both dimethyl (4o) and dichloro (4p and 4q) groups lead to significant drop in FIKK

activity, but as observed with meta ethyl group (4h), dimethyl group (4o) maintains potent

CpCDPK1 activity.

Table 3.3 CpFIKK kinase activity of compounds 4m-q and selectivity against CpCDPK1.

51

Compounds Ar CpFIKK IC50 (nM)

CpCDPK1 IC50 (nM)

4m

25 ± 6 450 ± 74

4n

140 ± 50 73 ± 9

4o

2900 ± 850 19 ± 2

4p

750 ± 60 >10000

4q

>10000 3500 ± 340

52

Table 3.4 CpFIKK kinase activity of compounds 4r, 5a, and 6 and selectivity against

CpCDPK1.

Compound CpFIKK IC50 (nM)

CpCDPK1 IC50 (nM)

4r >10000 400 ± 180

5a >10000 330 ± 60

6 3600 ± 287 1510 ± 195

Within the limited variations explored at the napthyridine and aminothiazole rings, none of the

substitutions are tolerated, indicating that there is only a small pocket in this region of FIKK.

Methyl substitution at the naphthyridine ring (4r) and acetyl (5a) and methyl (6) substitutions at

the aminogroup at the thiazole ring led to almost complete drop in CpFIKK activity (Table 3.4).

However, compounds 4p and 5a retain moderate CpCDPK1 activity. These results suggest that

there are very limited options of exploring at the aminothiazole and naphthyridine groups to

obtain active compounds, whereas the phenyl ring variations is the principal determinant of the

potency.

3.4 Orthogonal confirmation of CpFIKK inhibition

I used differential static light scattering (DSLS) to confirm the binding of representative CpFIKK

inhibitors identified using biochemical assays (Kiianitsa, Solinger, and Heyer 2003). All

CpFIKK inhibitors, including the most potent inhibitor 4b (Figure 3.4), showed a significant

53

increase in thermal stabilization, and the inactive compound 4o showed no significant thermal

stabilization of the CpFIKK protein upon binding (Table 3.5).

Table 3.5 Thermal stabilization of CpFIKK by inhibitors.

Compound 4a 4b 4l 4m 4o

Thermal shift (ºC) 5.1 5.2 4.7 3.6 0.82

0

200

400

600

800

1000

1200

34

35

36

37

38

39

40

41

42

43

44

45

46

Temperature (oC)

Lig

ht

scat

teri

ng

inte

nsi

ty (

AU

)

5.2oC

+ Compound 4b

No Ligand

Figure 3.4 CpFIKK stabilization by 4b in differential static light scattering (DSLS or

thermal melt) assay.

54

3.5 Selectivity profile of CpFIKK inhibitor 4b against human kinases

To begin to understand the selectivity of the newly synthesized inhibitors against the human

kinome, 4b was screened against a panel of 140 human kinases (~25% of the human kinome) at

the International Centre for Kinase Profiling, MRC, UK (Bain et al. 2007).

Only 6 protein kinases showed >50% inhibition of activity when screened with the compound at

500 nM, >100 times the potency for CpFIKK (Figure 3.3, Table 3.6). The complete list of the

kinases and their inhibition activities are shown in Table 3.6. Not surprisingly, ALK5

(TGFBR1), KDR (VEG-FR2) and P38A-MAPK showed 86%, 59% and 91% inhibition of

activity, respectively, by 4b. These three kinases were also inhibited by 4a in a previous study

(Elkins et al. 2015). In addition, four other kinases, TAK1, RIPK2, PKD1 and Yes1, were also

inhibited by 4b significantly (60%, 70% and 91% and 47%, respectively at 500 nM). These

results suggest that 4b is a moderately selective inhibitor.

55

The figure was reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

The kinases that have been used in this screening assay are marked with red (<50% inhibition),

blue (50 – 90% inhibition) and green (>90% inhibition) circles. 6 enzymes that were inhibited

>50% of their activity by compound 4b are shown in the table. 500 nM 4b was used.

Figure 3.5 Selectivity of 4b against 140 human kinases shown on the human protein kinase

tree.

56

Table 3.6 Percentage of inhibition of the activities of human kinases in the presence of 500

nM 4b.

Human Protein Kinases % of inhibition MAP4K5 0

S6K1 0

MARK2 0

JNK1 0

GCK 0

DYRK1A 0

HIPK2 0

RSK1 0

PKBb 0

JAK2 0

TTBK2 0

CAMKKb 0

SGK1 0

PKCz 0

ERK8 0

MARK3 0

TLK1 0

PRK2 0

Aurora A 0

AMPK (hum) 0

OSR1 0

CK2 0

MEKK1 0

PINK 0

PAK6 0

ULK2 0

PDK1 0

MAP4K3 0

TTK 0

MARK4 0

TSSK1 0

p38g MAPK 0

DYRK2 0

EF2K 0

PKA 0

MSK1 0

IR 0

PKBa 0

MKK1 0

IKKb 0

CK1γ2 0

p38d MAPK 0

MST4 0

GSK3b 0

SRPK1 0

57

ROCK 2 0

PKCa 0

MKK2 0

EIF2AK3 0

CAMK1 0

TTBK1 0

BRSK1 0

EPH-B1 0

IRAK4 0

MST2 0

IRAK1 0

DYRK3 0

CHK1 0

NEK6 0

MAPKAP-K2 0

PLK1 0

WNK1 0

PHK 0

PAK5 0

MLK1 0

CDK9-Cyclin T1 0

ASK1 0

ERK1 0

TIE2 0

PAK4 0

HIPK1 0

ULK1 0

PRAK 0

BTK 0

SIK2 0

Src 0

HIPK3 0

SmMLCK 0

EPH-A4 0

CDK2-Cyclin A 0

IGF-1R 0

PKCγ 0

PAK2 0

NEK2a 0

LKB1 0

IKKe 0

CHK2 0

MLK3 0

PIM2 0

ERK5 0

RSK2 0

MAPKAP-K3 0

MNK1 1

CSK 1

MPSK1 1

EPH-B4 1

EPH-A2 1

58

TAO1 1

PDGFRA 1

MNK2 2

CK1δ 3

PIM1 3

ZAP70 3

TrkA 3

STK33 3

MKK6 4

DAPK1 4

SYK 5

EPH-B2 5

IRR 6

MELK 9

MARK1 9

PIM3 9

MST3 9

DDR2 9

JNK3 10

Aurora B 11

BRSK2 11

EPH-B3 11

TESK1 13

SIK3 14

NUAK1 14

Lck 15

FGF-R1 15

ABL 19

p38b MAPK 20

ERK2 20

JNK2 21

TBK1 23

CLK2 24

MINK1 26

BRK 26

HER4 40

YES1 47

VEG-FR 59

TAK1 60

RIPK2 70

TGFBR1 86

p38a MAPK 91

PKD1 91

59

3.6 Structural understanding of inhibitor binding

Protein structures in complex with small molecule inhibitors provide insights into the interaction

and inhibition mechanism and opportunities to design more selective and potent inhibitors.

Hence, I attempted to co-crystallize active compounds with CpFIKK, CpCDPK1, and its

homologue TgCDPK1 (CDPK1 from T. gondii). I was able to obtain co-crystals structures of

TgCDPK1 with multiple hit compounds (PDB ID 5DVT, 5DVU and 5DVR), including the most

potent CpFIKK inhibitor 4b, at 2.05Å resolution (5DVT). Compound 4b inhibits recombinant

TgCDPK1 at 19 nM IC50 and shows >5oC thermal shift upon binding.

As described above, naphthyridine compounds are ATP-competing type I class of kinase

inhibitors and bind to the active site of the kinases by anchoring with the hinge region (Gellibert

et al. 2004). The co-crystal structure of TgCDPK1-4b shows that the naphthyridine moiety

interacts with the backbone of tyrosine (Tyr131) at the hinge region via a hydrogen bond that

anchors the compound at the active site (Figure 3.4B). Two residues, Asp195 from the DFG-

motif and Ser61 from the Gly-rich loop, are also at close vicinity (3-4 Å) and form weak

electrostatic bonds with the amino group of the aminothiazole moiety; these probably act as

secondary anchors in the interaction. The phenyl ring is located at the hydrophobic selectivity

pocket where the so called ‘gatekeeper’ residue is a smaller residue (Gly128). The gatekeeper

residue often plays role in inhibitor selectivity and the smaller gatekeeper of TgCDPK1 and

CpCDPK1 (also Gly) has been exploited in multiple studies to design selective inhibitors of

these kinases (S. M. Johnson et al. 2012; Kuhlenschmidt et al. 2016; Castellanos-Gonzalez et al.

2013). CpFIKK also contain a smaller residue (Ser516) at the gatekeeper position suggesting a

common mechanism of inhibition.

I superposed the co-crystal structures of NM-PP1-bound CpCDPK1 (PDB ID 3NCG) with the

4b-bound TgCDPK1 (5DVT) to understand the interaction patterns of the compounds in

CpCDPK1 (Figure 3.4 C). The kinase domains of the two kinases show great sequence similarity

(~70%) and not surprisingly, the two ligand interaction pockets in 3D space are almost identical.

One exception is the position of Gly-rich loop of the empty CpCDPK1 active site, which is

slightly drifted away from the binding site, probably because no charged groups in the compound

60

(NM-PP1) anchor the Ser in the Gly-rich loop of CpCDPK1. Sequence comparison suggests that

the ligand binding pocket is also similar between CpCDPK1 and CpFIKK, except for the

residues contributing from the first two subdomains (Table 3.7). The first two subdomains of

CpFIKK kinase domain align poorly with CpCDPK1 and any other classic eukaryotic kinases,

and the alignment we used (Clustal Omega run and then manual curating) is probably not very

reliable at this region. Other exceptions include the presence of a cysteine (Cys) in CpFIKK at

the -1 position of the canonical DFG-motif (DFA motif in CpFIKK), which is an Ile in

CpCDPK1; Met112 of CpCDPK1 is replaced by a Gln in CpFIKK too. These differences

probably confer the differences in affinity between the CpFIKK and CpCDPK1 proteins with the

inhibitor compounds.

Table 3.7 Probable inhibitor interacting residues in TgCDPK1, CpCDPK1 and CpFIKK.

Some residues have been omitted in CpFIKK due to lack of sequence similarity with

canonical kinases.

A) Surface representation of the ligand binding pocket of TgCDPK1 (PDB ID 5DVT). B)

Interacting residues of TgCDPK1 (grey) with 4b and superposed CpCDPK1 (golden). Hinge

binding by the naphthyridine group and an electrostatic interaction between the aminothiazole

moiety and the invariant Asp (D195) was observed. C) Superposed structures of 4b-TgCDPK1

(grey) and apo-CpCDPK1(golden) are shown.

TgCDPK1 L57 S61 V65 A78 M112 L114 L126 G128 Y131 L181 I194 D195

CpCDPK1 L S V A M L I G Y I I D

CpFIKK L - - - N A L S Y I C D

Figure 3.6 Structure analysis of parasite kinases with compound 4b.

61

A.

B. C.

Hinge

DFG motif

Gly-rich loop

62

The residues and residue-numbers in TgCDPK1 sequence are indicated by red fonts on top of

the alignment.

To understand if smaller gatekeeper at the active site plays role in 4b binding in CpFIKK, I

generated and purified a gatekeeper mutant version of the protein, CpFIKKΔS516M, in which

the serine was replaced by methionine. The mutant protein was completely active. When I tested

the mutant using a binding assay (Figure 3.8), I found that the mutant enzyme bound AMPPNP,

a non-hydrolyzable ATP analog, but not 4b. 4b also did not inhibit the enzymatic test of the

gatekeeper mutant. This suggests that the Ser gatekeeper in CpFIKK creates a pocket that is

important for the inhibition by naphthyridine compounds.

Figure 3.7 Sequence alignment of the kinase domains of TgCDPK1, CpCDPK1 and

CpFIKK.

63

Thermal shift of the protein is observed with AMPPNP, but not with compound 4b.

3.7 Materials and methods

3.14.1 Enzymatic inhibition assay

An NADH coupled LDH-PK ATPase assay was used to measure the kinase activity according to

procedures described in the Chapter 2. The concentrations of proteins in the reaction were 1.2

nM (CpCDPK1) and 10 nM (CpFIKK). Inhibitor compounds were solubilized at the stock

concentration of 10 mM in 100% DMSO and were applied in the reaction from 25 µM

downward with 2 fold dilutions. Syntide 2.0 (PLARTLSVAGLPGKK) for CpCDPK1 and P5

(RRRAPSFYRK) for CpFIKK were the substrates used as the phosphoacceptor peptides. The

reaction solution contained 0.25 mM of ATP and 0.1 mM of peptide substrates. The CpCDPK1

Figure 3.8 Thermal melt assay with CpFIKK-gatekeeper-residue-mutant CpFIKKΔS516M.

0

500

1000

1500

2000

2500

3000

3500

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

No ligand

4b

AMPPNP+MgCl2

Inte

nsi

ty (

AU

)

Temperature (oC)

64

reaction contained 2 mM CaCl2 in addition to the original reaction mixture. Inhibition parameters

were plotted and calculated by using Sigmaplot.

3.7.2 Screening with human kinases

This work was performed in collaboration with International Centre for Kinase Profiling (ICKP),

MRC, UK (www.kinase-screen.mrc.ac.uk/kinase-screen-express). For this assay, I provided 25.5

µM of compounds in 100% DMSO. The compounds were diluted 51 fold to make the final

reaction concentration of 500 nM. The screening assays were performed following the protocol

described by Bain et al. (2007).

3.7.3 X-ray crystallography

A purified sample of the tag-intact TgCDPK1 kinase domain was crystallized using the sitting

drop vapour diffusion method in the presence of inhibitors at room temperature. All crystals

were obtained by mixing one part of protein solution (10 mM HEPES pH 7.5, 500 mM NaCl, 2

mM TCEP) at 20 mg/mL containing 1 mM inhibitor with one part of reservoir solution. The

reservoir solution contained 20% PEG3350 and 0.2 M di-sodium-tartarate (pH 7.5). Large plate-

shaped crystals appeared within four weeks of incubation and were cryo-protected in glycerol

supplemented mother liquor before being flash cooled in liquid nitrogen. The diffraction data

were collected at 1.9 Å resolution using an X-ray source (APS BEAMLINE 24-ID-E) equipped

with CCD ADSC QUANTUM 315 detector, processed with XDS (Kabsch 2010) and extracted

with PDB_EXTRACT 3.15 (Yang et al. 2004).

The structure was determined by molecular replacement. Inactivated TgCDPK1 crystal structure

(PDB ID 3KU2) served as a search model for solving the inhibitor-TgCDPK1 co-crystal

structures. Phaser (McCoy 2006; McCoy et al. 2007) was used for the molecular replacement

calculations, while model building was performed with COOT (P. Emsley et al. 2010; Paul

Emsley and Cowtan 2004) and the structures were refined with Phenix (Adams et al. 2010) and

data scaling was performed with Aimless 0.5.15 (Evans and Murshudov 2013) from the CCP4

suite of programs (Winn et al. 2011). Relevant data collection and refinements statistics are

65

shown in Table 3.8. The coordinates for the structure and their structure factors have been

deposited with the Protein Data Bank (http://www.pdb.org) (Berman et al. 2000).

Table 3.8 Data collection, phasing, and refinement statistics for the TgCDPK1 co-

crystal structures.

66

Chapter 4 Part of this chapter has been submitted in the Journal of

Medicinal Chemistry and is currently under review.

Phenotypic effects of CpFIKK inhibitors on parasites 4

My aim in generating high affinity inhibitors was to use them to understand the biology of the

FIKK enzymes and to explore their suitability as drug targets. In the last Chapter, I described the

discovery of a potent selective in vitro CpFIKK inhibitor. However, many drugs or inhibitors

that show efficacy in vitro are not bioavailable or show off target phenotypic effects, and thus it

is critical to link phenotypic observation with inhibition of the specific protein target. At this

point, with the potency of the compounds confirmed enzymatically, with the scaffold derived

from a drug-like compound, and with promising results about selectivity, I set out to find a

biological system to analyze cellular effects of CpFIKK inhibition. For this I explored the effects

of the CpFIKK/CpCDPK1 inhibitors on the growth of two parasites C. parvum and T. gondii by

taking advantage of the assays and genetic advances made by our collaborators. This chapter

describes the results.

4.1 Evaluation of CpFIKK inhibitors in C. parvum growth inhibition

Inhibitors against CpFIKK proteins, if bioavailable, would likely disrupt the FIKK-mediated

functions in the parasites. However, we showed previously that CpFIKK inhibitors also inhibit

recombinant CpCDPK1 potently. Therefore, if administered on parasites we would expect to see

dual protein specific phenotypic changes in the parasites at higher concentrations, e.g. at >1 µM.

However, because compound 4b has a huge selectivity window due to enzymatic potency

between CpFIKK and CpCDPK1 (>1000 fold potent towards CpFIKK) I expected CpFIKK-

specific phenotypic effect on the parasites at lower concentrations.

Understanding the biological roles of proteins in C. parvum is especially difficult because most

of the different stages of life cycle have not been recapitulated in vitro (Striepen 2013). At this

moment, with our collaborator’s laboratory settings, the only phenotypic effect that can be

scored is inhibition of parasite growth at the asexual life cycle stage.

67

Selective chemical inhibition of CpCDPK1 shows growth inhibition of C. parvum

(Kuhlenschmidt et al. 2016; Castellanos-Gonzalez et al. 2013). However, since we do not know

the biology of CpFIKK, we do not know if we will see any growth related phenotypic effect by

chemically knocking down FIKK gene function, although this is the only assay currently

available. To understand if CpFIKK has any growth related phenotypic effects in C. parvum, I

collaborated with the laboratory of Dr. Christopher Huston, Vermont University, to test the

impact of the compounds on C. parvum growth in vitro.

In order to understand the role of FIKK in C. parvum growth, I evaluated (in collaboration) the

in vitro parasitic activity of 5 compounds with varying CpFIKK activity (4a, 4b, 4d, 4o with

CpFIKK IC50 3 nM, 0.2 nM, 221 nM, 2930 nM respectively) including a FIKK inactive

compound 4q (CpFIKK IC50 >10,000 nM).

For this inhibition experiment, the compounds were added to the host cell (HCT) prior to

infection with the parasites. All the CpFIKK active compounds inhibited parasite growth with

EC50 ≤5.5 µM but the CpFIKK inactive compound 4q had no effect, suggesting that the

inhibition was target based. However, the observed EC50 did not correlate with the CpFIKK

inhibition IC50. The most CpFIKK active compounds 4a and 4b required concentrations to kill

the parasites that were 1840 and 12800 fold higher than the IC50 for CpFIKK inhibition and only

66 and 14 fold higher concentrations than the IC50 to inhibit CpCDPK1, respectively (Table 4.1).

Moreover, even though 4d and 4o showed significant growth inhibition at 6 and 1.3 fold higher

concentrations than enzymatic potency, these concentrations were also 6-204 fold higher than

their CpCDPK1 inhibition IC50 (Table 4.1). Therefore, it is highly likely that inhibition of

CDPK1, or perhaps another cellular target, is responsible for the cell killing. Inhibition of

CpFIKK likely has no effect in C. parvum growth.

Inhibitors that target CpCDPK1 kill the C. parvum parasite (Castellanos-Gonzalez et al. 2013;

Kuhlenschmidt et al. 2016), and CpCDPK1 was posited as a potential drug target for

Cryptosporidiosis. However, I found no strong correlation between CpCDPK1 inhibition and

growth inhibition, suggesting that the CDPK1 inhibitors are also killing through a different

target(s). This corroborates a previous study with another set of small molecule inhibitors

(Kuhlenschmidt et al. 2016). CpFIKK selective inhibitors from different structural classes will be

needed to establish the involvement of CpFIKK in parasite growth and can be a subject of future

68

studies. This said, CpFIKK-inhibitors from this study are already suitable to study the role of the

protein in other stages of life cycle, when other stage-specific biological assays become

available. In conclusion, our potent and CpFIKK inhibitor 4b could serve as a useful tool for

understanding the biological role of FIKK in C. parvum. Even though compound 4b and the

other inhibitors showed parasite growth inhibition, the disconnection between the enzymatic and

cellular data suggests strongly that FIKK is not essential for parasite growth.

Table 4.1 Parasite invasion inhibition profiles of CpFIKK inhibitors.

Compounds EC50 (µM) EC50 potency compared

to CpFIKK IC50 (Fold)

EC50 potency compared

to CpCDPK1 IC50 (Fold)

4a 5.52 1840 66

4b 2.56 12800 14

4d 1.33 6 6

4o 3.88 1.3 204

4q No inhibition Not applicable Not applicable

69

Activity data of most potent CpFIKK- inhibitor 4b, equally potent CpFIKK/CpCDPK1- inhibitor

4d and inactive compound 4q are shown here.

4.2 Chemical genetics experiments with FIKK knock out (KO) strains of T. gondii

Genetic manipulation of Cryptosporidium parasites has been notoriously difficult and only

recently has there been some progress (Vinayak et al. 2015). In the absence of suitable

Cryptosporidium FIKK knock-out strains, I wanted to test the CpFIKK-compounds in another

apicomplexan that has a single member of FIKK and where gene manipulation was better

developed. One such well-studied apicomplexan parasite is T. gondii. Since the CpFIKK and

Figure 4.1 Inhibition of C. parvum growth in vitro.

70

TgFIKK are thought to be orthologous proteins and the single members of FIKKs are retained

evolutionarily in these genomes, similar biological roles might be expected. My collaborators

from the laboratory of Dr. Dana Mordue (NYMC, USA), recently generated a FIKK knock out

(KO) strain of T. gondii (Pru strains) and discovered that FIKK is essential for cyst formation

and survival under stress, but apparently is not essential for completing the asexual life cycle

(Skariah et al. 2016). We found previously that the CpFIKK-inhibitors also inhibit CDPK1 from

T. gondii; compound 4b inhibits the recombinant TgCDPK1 at 19 nM IC50. TgCDPK1 plays an

essential role in exocytosis and chemical inhibition of TgCDPK1 shows growth inhibition (S. M.

Johnson et al. 2012; Hui, El Bakkouri, and Sibley 2015). Therefore, because compound 4b is

predicted to have dual FIKK/CDPK1 activity, the addition of 4b to T. gondii would be predicted

to inhibited cell growth due to TgCDPK1 inhibition and obstruct cyst formation due to TgFIKK

inhibition. The Mordue lab system allowed me to test the inhibitors against Toxoplasma species

to test if chemical knock down phenocopied the genetic KO and the growth inhibition occurs.

100% inhibition reached at 1 µM concentrations. No detectable difference observed between the

two samples.

Perc

enta

ge o

f gr

ow

th

[4b] (μM)

0

20

40

60

80

100

120

10 1 0.1 0.01 0.001 0.0001 0

WT

FIKK-KO

Figure 4.2 In vitro growth inhibition results of wild type (WT) and FIKK knocked out

(KO) strains of T. gondii with compound 4b.

71

My goal was to compare the phenotype of the CpFIKK-inhibitor treated wild type parasites with

the phenotype of TgFIKK-KO strains. The active compounds were tested against wild type (WT)

and FIKK-KO T. gondii. For growth inhibition assays, the compounds were administered at

different concentrations on the host cells (HFF) before the parasite infection. For the cyst

inhibition assay, the compounds at 1 µM were administered just before the induction of the cyst

production in the infected host cells. Compounds 4a, 4b, 4o and 4q were used for these assays.

At concentrations predicted to inhibit both FIKK and CDPK1, I found that compound 4b

inhibited the growth of the parasites but did not affect T. gondii cyst production (Figure 4.2). The

results, therefore, are not completely conclusive at this point, mainly because we do not know for

certain that these compounds inhibit TgFIKK. That said, the most likely scenario is that the

growth inhibition is due to TgCDPK1 inhibition.

4.3 Materials and methods

4.3.1 C. parvum growth inhibition assay

This experiment was performed in collaboration with the Huston lab at Vermont University.

Compound inhibition of C. parvum growth was tested using a previously developed microscopy-

based assay. Human ileocecal adenocarcinoma (HCT-8) cells (ATCC) were cultured in RPMI-

1640 medium (Invitrogen) supplemented with 10% heat inactivated FBS (Sigma), 120 U/mL

penicillin and 120μg/mL streptomycin (ATCC). Three-fold increasing concentrations of

compounds from 0.004 to 25 µM diluted in culture media were added to 100% confluent HCT-8

cells in clear well, back-walled 384-well plates (BD Falcon), followed by addition of

approximately 5500 excysted C. parvum Iowa strain oocysts (Bunchgrass Farms, USA) per well.

Oocysts were excysted with 10 mM HCl for 10 min at 37° C, followed by 2 mM solution of

sodium taurocholate (Sigma) for 10 min at 16°C and then diluted in culture media and added to

each well. After 48 h of infection, the wells were washed three times with 111 mM D-galactose,

fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.25%

Triton X100 in PBS for 10 min at 37°C, washed three times with PBS containing 0.1% Tween

20 and then blocked with 4% bovine serum albumin (BSA) (Sigma) in PBS for 2 h at 37°C.

Parasitophorous vacuoles of C. parvum were stained with 1.33 µg/mL fluorescein labeled Vicia

villosa lectin (Vector Laboratories) diluted in 1% BSA in PBS with 0.1% Tween 20. After 1 h

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incubation at 37°C, the nuclear stain Hoechst 33258 (Anaspec) was added at 0.09 mM diluted in

water for another 15 min, followed by five washes with PBS containing 0.1% Tween 20 before

imaging. A Nikon Eclipse Ti2000 epifluorescence microscope with an EXi blue fluorescent

microscopy camera (QImaging, Canada) and NIS-Elements Advanced Research software

(Nikon, USA) was programmed to focus on the center of each well and acquire a three-by-three

composite of 20X field images. Nuclei and parasite numbers were quantified on exported .tif

files using previously developed macros and the batch function in ImageJ (National Institutes of

Health).

4.3.2 T. gondii growth inhibition and cystogenesis inhibition assay

This experiment was performed in Dana Mordue lab at New York Medical College (NYMC)

according to the protocol described by Maubon et al. (2010). Type II T. gondii wild type (WT)

and FIKK-KO Pru strain was used for both 50% effective concentration (EC50) determination

and in vitro cyst experiments. Knocked out strains were prepared according the protocol

describes by Skariah et al. (2016). Strains were maintained by serial passage in an HFF

monolayer under tachyzoite growth conditions at 37oC with 5% CO2 in Dulbecco’s modified

eagle medium (DMEM) supplemented with 10 fetal bovine serum (FBS, Invitrogen), 4 mM

glutamine, 500 U/mL penicillin and 250 U/mL streptomycin. On day 3, potential plaques were

counted under microscope to determine the inhibitory concentration of the compounds. Serial

dilution of compounds in concentrations ranging from 10 µM to 100 pM were used and results

were plotted from triplicated experiments.

Cyst induction or trachyzoite to bradyzoite conversion assay was performed according to

protocol previously described (Maubon et al. 2010). Cystogenic Type II strains were used.

Extracellular tachyzoites were allowed to invade HFF cells and natural cystogenesis was

achieved after 7 days. For inhibition assay the medium was replaced with 1 µM inhibitor

containing medium before the induction of cystogenesis. The cysts were assessed by

immunofluorescence assay (IFA).

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Chapter 5

Discussion 5

Most biomedical studies, especially those on human signaling networks, have focused on

relatively few protein targets, and this is best exemplified in bibliometric studies of research on

the human kinome (Edwards et al. 2010). Parasite research is not very different. In order to

expand the parasite research scope, I chose to work on a relatively unknown kinase family, the

FIKK kinases from apicomplexan parasites, for my PhD project. My goal was to contribute to

the understanding the biochemical processes of signaling networks in parasites and thus shed

light on their biology. My ultimate goal was to explore the suitability of FIKK proteins as a

potential drug target in C. parvum, one of the relatively less studied human and animal parasites

of significant global health burden.

In this study, I developed methods to express and purify FIKK kinase domains from C. parvum

and P. falciparum. In so doing I identified an adjacent region to the kinase domain that is

essential for bacterial expression of CpFIKK and PfFIKK8 Using biochemical and screening

approaches, I characterized the enzymatic properties of the enzyme, as well as its substrate

preferences and developed a screening assay using which I identified small molecule inhibitors

of the CpFIKK enzyme, and confirmed their inhibition using orthogonal assays. Next, I

collaborated with a chemistry group (Lautens Lab) at the University of Toronto to generate new

compounds and analyzed their structure-activity relationship. Potent compounds were screened

against panels of Cp_kinases and human kinases to identify those that were most selective.

Finally, I investigated the effects of the most potent and selective CpFIKK inhibitors on the

cellular growth of the parasites. I discovered that CpFIKK is an active kinase and prefers Ser as a

phosphor-acceptor residue flanked by Arg at -3 and +3 positions on the substrate. A series of

naphthyridine-based compounds can inhibit the CpFIKK activity and the most potent inhibitor,

called the compound 4b, is also a CpCDPK1 inhibitor. Growth inhibition of the parasites in vitro

was also observed by these compounds, but there was little correlation between FIKK inhibition

and inhibition of cell growth. I conclude FIKK is not likely to be a drug target, at least for the

growth stages tested.

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5.1 The role of N-terminal extension (NTE)

Protein kinases are regulated in a variety of ways. For example, some possess regulatory

subunits. These can modulate enzymatic activity in different ways, for example by binding to

specific molecules or ligands to activate the catalytic domain. For example, human PKA kinase

has two catalytic subunits and two regulatory subunits in a dimer; cAMP binding at the

regulatory subunits promotes a conformational rearrangement and releases the catalytic subunits

to phosphorylate their substrates (Huse and Kuriyan 2002; Nolen, Taylor, and Ghosh 2004). In

some cases, the regulatory subunits or domains are controlled through phosphorylation. For

example, in Src tyrosine kinase the linker between the non-catalytic SH2 and SH3 domains and

the C-terminal tail are phosphorylated to bring conformational changes, unblock the activation

site and thus, regulate their activity (L. N. Johnson, Noble, and Owen 1996; Huse and Kuriyan

2002). Another example of allosteric regulation by ligand binding and phosphorylation at the

non-catalytic domain/sub-unit is the EGF receptor kinase, where substrate binding at the non-

catalytic domain (EGF binding domain) promotes dimerization and subsequent phosphorylation

of the tail segment to drive the kinase towards active conformation (Nolen, Taylor, and Ghosh

2004; Endicott, Noble, and Johnson 2012).

The kinase domain of FIKKs contains a ~40 residue long region at the N-terminal region that

was needed for the expression of the catalytic domains (Figure 5.1). This region is only

conserved in Plasmodium species, but not so conserved in FIKKs from other parasites. I found

that this extension is needed for the kinase domains of PfFIKK8 and CpFIKK to be expressed in

bacterial and insect cell expression system and to be soluble (Figure 2.1, Table 2.1). However,

the findings do not necessarily confirm that the N-terminal extension has any role in catalytic

domain activation or regulation. It is possible that this extension region is an integral part of the

kinase domain, or simply interacts somehow with the expression machinery, because I was

unable to express a protein construct lacking the exo-kinase domain or extension part. Therefore,

the possibility that this extension has regulatory activity is yet to be explored in FIKKs and might

be subject to future studies.

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* indicates the boundary between the N-terminal extension and the kinase domain.

5.2 FIKK kinases are unique in substrate preference

Protein kinases are essential for cell communication and they phosphorylate a wide variety of

substrates. The process of substrate recognition and phosphorylation by kinases is not random;

kinases are rather specific for certain substrate motifs. The phosphorylation motif refers to the

sequence around the phosphorylated residues S/T/Y on the protein/peptide substrates. Ser-Thr

kinases recognize either Ser or Thr or both on the suitable substrate. The specificity of substrate

preference is determined by the residues around the phospho-residue on the substrate. The

substrate recognition or phosphorylation motifs of different eukaryotic protein kinases (ePKs)

are provided in Table 5.1. FIKKs phosphorylate substrates that are rich in basic amino acids.

PfFIKK4.1 phosphorylates human dematin (Brandt and Bailey 2013), PfFIKK4.2 and PfFIKK12

phosphorylate MBP (Nunes et al. 2010)– both are rich in Arg content.

My initial study suggested that FIKKs might prefer Arg-rich substrates since they phosphorylate

Arg-rich MBP better than other generic substrates (Figure 2.4). The positional scanning peptide

array results suggest that both PfFIKK8 and CpFIKK favour Ser as a phosphoacceptor residue,

flanked by Arg at both -3 and +3 positions (Figure 2.5). Biochemical studies later confirmed

these results (Figure 2.6). This was the first time any of the FIKK members had been

Figure 5.1 Sequence alignment of the N-terminal extension of different apicomplexan

FIKKs.

76

biochemically characterized and the biochemical nature of FIKK’s substrate recognition

understood.

(Pinna and Ruzzene 1996; Songyang et al. 1994; Gui et al. 1994; Tessmer et al. 1977; Osman et

al. 2015).

X indicates any residue and ȹ indicates hydrophobic residues

5.3 CpFIKK/CpCDPK1-inhibitors: what makes them selective?

The most commonly used kinase inhibitors interact with the active site. The shape and

composition of kinase active sites allow for many possible interactions of kinase inhibitors, and

this has been exploited to generate selective inhibitors. Figure 1.3 shows the different interaction

regions in an active site of a typical protein kinase that can be exploited to design kinase

inhibitors. Here, I am attempting to explain the selectivity pattern of the naphthyridine

compounds.

Table 5.1 Substrate preference of different protein kinases.

77

5.3.1 The Hydrophobic region I selectivity: role of gatekeeper

One of the regions that is exploited during kinase inhibitor discovery attempts to gain selective

binding is the Hydrophobic region I, in which a bulky residue prevents binding by large

inhibitors. This bulky residue also has been termed the ‘gatekeeper’ residue. The size of the so-

called gatekeeper residue affects the accessibility of the hydrophobic region (Blencke, Ullrich,

and Daub 2003; Liu et al. 1999; Shah et al. 1997; Huang et al. 2009; Gorre et al. 2001). A

smaller residue at the gatekeeper position creates a relatively larger hydrophobic pocket and can

play role in selectivity of kinase inhibitors, since most of the eukaryotic protein kinases contain a

larger residue at this position. Approximately 20% of human kinases have a smaller amino acid

at this position and these kinases can be targeted by compounds having bulky moiety that fits

into the larger hydrophobic region. This is why the hydrophobic region close to the gatekeeper

is sometimes called ‘the selectivity pocket’. Many inhibitor discovery attempts targeting

TgCDPK1 are take advantage of the ability to use bulky compounds targeting the space left

unoccupied as a result of the small Gly residue as gatekeeper. Sequence alignment suggests that

CpFIKK contains a Ser (S516) at the gatekeeper position (Figure 3.5, Table 3.7), which might

also create a hydrophobic pocket for the inhibitors.

My compound screening and SAR studies uncovered a series of naphthyridine based compounds

with great potency against CpFIKK (Chapter 3). These were the first inhibitors reported for any

FIKK kinase family member. The most potent compounds were constructed with a naphthyridine

group, an amino-pyrazole moiety and a phenyl ring (Figure 3.1). In a previous study, Gellibert et

al. (2004) showed that the phenyl ring of similar naphthyridine compounds occupy the

Hydrophobic region I in HsALK5, which also contain a Ser as gatekeeper. Our TgCDPK1

structure also shows similar interactions with the naphthyridine inhibitors (PDB ID 5DVT,

5DVR, 5DVU). As previously pointed out, TgCDPK1 has a Gly (Gly128) at the gatekeeper

position allowing bulky kinase inhibitors (BKIs) to bind to this protein. If the residue is mutated

(modeled in Figure 5.3) to Met the chemical space at the hydrophobic region gets occupied by

the bulkier side chain and compound 4b will not be able to bind the active site. Indeed, I found

that a G128M mutated version of TgCDPK1 and a S516M mutated version of CpFIKK are

immune to compound 4b inhibition (Figure 3.8).

78

Golden shaded residues indicate Gly (left) and Met (right). The modeled G128M mutation shows

that there would be steric collision if Gly is mutated to larger residue, like Met.

However, a large hydrophobic pocket appears not obligatory for the activity of the compounds.

For example, among the mostly inhibited human kinases by compound 4b, at least two of them

have large gatekeeper residues (Table 5.2). Compound 4b shows 91% inhibition of the human

PKD1 kinase, which has a Met as the gatekeeper residue. These counter-intuitive results make it

is very difficult to predict what actually confers the potency and selectivity, and establishing the

reasons will require structural information about these proteins, especially HsPKD1. My

structure-superposition attempt suggests that gatekeeper Met of HsTAK1 (PDB ID 2EVA)

should have conflicted with the naphthyridine compounds. This needs to be further investigated

and a co-crystal structure of the compound 4b and HsPKD1 or HsTAK1 would answer some

questions about how the bulkier phenyl moitery would accommodate in the active sites having

larger residues and can be a subject of future studies.

Figure 5.2 Representation of the active site of TgCDPK1 occupied by compound 4b.

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5.3.2 Selectivity between CpFIKK and PfFIKK8

Although the smaller gatekeeper in CpFIKK can accommodate the naphthyridine compounds,

this is not the only determinant of selectivity and potency. PfFIKK8 also has a Ser at the

gatekeeper position, but is not inhibited potently by the naphthyridine compounds. This

selectivity probably achieved due to the differences in sequences between the two proteins. Even

though PfFIKK8 and CpFIKK are evolutionarily close, the sequence identity between the kinase

domains is only 38%, and sequence alignment shows that these two proteins differ in multiple

regions in the kinase domains (Figure 1.6). For example, sequences vary at the subdomain II, IV,

VIA and IX. CpFIKK has inserts in the activation segment and at subdomain IX, and these are

absent from PfFIKK8. Several residues from the subdomains I to VII contribute to the active site

and inhibitor interactions. Variations in these regions may play roles in the naphthyridine

compound selectivity. These results indicate that there are some additional determinants, other

than the gatekeeper residue, that contribute to the potency and selectivity of the naphthyridine

compounds.

Table 5.2 Highest inhibited protein kinases from human kinome by compound 4b at

500 nM and the gatekeeper residues.

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5.3.3 A common inhibitor for both CpFIKK and PfFIKK8 provides insights to design CpFIKK-selective inhibitors.

Although, I discovered very few hits from my screen of a ~2500 focused library, I obtained one

compound, called CGP060476, from a Novartis kinase inhibitor series that inhibits both CpFIKK

and PfFIKK8 at 5 µM and 3.3 µM IC50, respectively. This is a pan-kinase inhibitor targeting a

large numbers of human protein kinases, as well as CpGSK (cgd2_1060), CpCDPK2

(cdg7_1840), CpCDK (cgd_2510), CpCDPK1 and TgCDPK1 from my parasite recombinant

kinase panel.

Chemical structure of CGP060476 and its activity profile are shown in the right panel of Figure

A. ‘R’ moieties in Figure B are proposed to be added on the compound 4b.

A

Figure 5.3 A. Active site of the co-crystal structure of CGP060476-TgCDPK1 (top left)

and superposed structures of CGP060476 and 4b-bound TgCDPK1 (bottom left). B.

Proposed compound structures to occupy the Hydrophobic region II of CpFIKK.

81

B

The co-crystal structure of TgCDPK1-CGP060476 shows that this compound interacts with the

hinge and spans from the Hydrophobic region I to Hydrophobic region II (Figure 1.3, Figure 5.5,

golden). In this structure, I am especially interested about the chloro-phenyl moiety which

occupies the Hydrophobic region II of the active site, because this part is missing from the

naphthyridine compounds we created (Figure 5.5A, bottom). CpFIKK is targeted by

CGP060476; this means that the Hydrophobic region II of this protein can also be occupied by

hydrophobic moieties like alkyl or alkyl-aryls. Our SAR strategies did not explore inhibitors that

occupy the Hydrophobic region II and therefore, expanding the compounds at the naphthyridine

group with another hydrophobic hand might provide extra potency to the naphthyridine

compounds as well as achieve greater selectivity towards CpFIKK. Figure 5.5B shows some

examples of the chemical strategies that can be taken to design more compounds targeting the

Hydrophobic region II.

5.4 Parasite killing by compound 4b is probably due to CDPK1 inhibition

I tested the selectivity of CpFIKK inhibitor compound 4b with multiple C. parvum kinases

(Table 3.1), and found that compound 4b also showed significant potency against the CDPK1

orthologues from C. parvum and T. gondii (Chapter 3). When this compound was applied to C.

parvum and T. gondii parasites in vitro, I observed that growth inhibition was inhibited at

concentrations more consistent with inhibition of CDPK1 (or another target) than FIKK (S. M.

Johnson et al. 2012; Kuhlenschmidt et al. 2016; Castellanos-Gonzalez et al. 2013). Confidence

that the mechanism of killing was through CDPK1 was increased by my observation that

Compound 4q, which is inactive against both CpFIKK and CDPK1, showed no effect on growth.

Further supporting the hypothesis that FIKK is not a drug target was my observation that growth

82

inhibition did not correlate with biochemical inhibition of CpFIKK, when I tested a series of

FIKK inhibitors (Table 4.1). For example, the less potent CpFIKK inhibitor 4a kills the C.

parvum parasite with more potency than the more potent CpFIKK inhibitor 4b. The most likely

explanation for this is that CpFIKK is not essential in the growth at the lytic stage of the life

cycle.

Formally, I cannot be certain that the inhibitors made to CpFIKK also inhibited TgFIKK because

I was unable to express recombinant TgFIKK protein and test the activity of the inhibitors

directly. However it can be likely because the two proteins are evolutionarily related and share

considerable sequence similarity (Figure 1.6). The rationale to test my FIKK inhibitors in T.

gondii was to exploit its molecular genetics capabilities; T. gondii is the only parasite where the

FIKK protein has been knocked out. In KO strains, the absence of FIKK had no effect on the

lytic cycle, but was found to be important for tissue cyst production (Skariah et al. 2016). Hence,

the potent compounds were tested on T. gondii in vitro to find out any FIKK-specific target

engagement and phenotypic effect. We found no change in the tissue cyst production when

potent inhibitors (e.g. compound 4b) were applied; rather the only phenotypic effects we

observed was growth inhibition. Hence, the cellular assay suggests either that the naphthyridine

series of the inhibitors may not target TgFIKK potently enough to see a phenotype, that the

activity of FIKK is important for the phenotype or that the phenotype observed in the knock-out

is an off-target consequence of the gene disruption. We might also conclude that growth

inhibition is due to TgCDPK1 inhibition.

5.5 Utility of a chemical tool to understand the biological role of CpFIKK in parasites

Potent and specific small molecule inhibitors can be extremely useful tools to understand the

biological roles of the proteins, especially for the genes that are difficult to knock out. These

chemical tools can also be useful for other biological studies. A potent small molecule inhibitor,

like compound 4b, will be useful to find out the biological function of C. parvum, a parasite

where genetic manipulation is extremely difficult.

83

Unlike T. gondii, C. parvum does not produce tissue cysts during any stages of its life cycle. This

indicates that FIKKs might have been evolved to perform different functions in different

parasites. An alternate role for FIKKs might be especially possible for C. parvum because its

biology differs significantly from that in other apicomplexans (discussed in Chapter 1). Hence,

the role of FIKK in C. parvum might not be in growth but possibly in another stage of the

complex life cycle. Unfortunately, experimental set ups to investigate different life cycle stages

in C. parvum are not available currently due to difficulties in working with the parasites outside

living organisms. This restricts my ability to investigate FIKK’s role in different life cycle

stages. As newer assays of C. parvum biology become available, compound 4b, could be

extremely helpful in elucidating the function of FIKK.

A chemical probe (described in Chapter 3) would be an ideal tool for chemical biology

experiments on FIKK proteins. However, designing highly selective chemical probes for protein

kinases based on active site inhibitors is especially difficult because of the relatively conserved

binding sites of the kinases. Most “off the shelf” kinase inhibitors will target dozens of kinases,

and significant effort and characterization is required before a potent inhibitor will be useful as a

probe of a kinase function. Although the definition of a useful probe is evolving, we consider a

compound to be a kinase chemical probe if the compound –

has a potency less than 50 nM IC50 and potency confirmed by orthogonal assays

is >50 fold selective over other kinases and tested against proteins from other

families (GPCR, bromodomains etc.)

has cellular activity at < 1 µM in a target engagement assay and a cell –based activity

assay

is accompanied by negative control compounds

has no phenotype in cells harbouring the target kinase with an inactivating mutation

My CpFIKK inhibitor compound 4b meets some of the criteria, e.g. potency and negative control

tests, its cellular activity could not be tested, and thus we cannot be certain it engaged CpFIKK

in cells. The phenotypic changes that were observed at >1 µM were more likely due to off-target

activity. Moreover, we were only able to measure its off-target inhibition against 15% of the

Cpkinome. Among these kinases, two (CpFIKK and CpCDPK1) were inhibited by the

84

compound 4b, albeit the compound was far more selective for CpFIKK. Without fully exploring

the remainder of the kinome, it is not yet possible to approve consider 4b as a chemical probe.

Moreover, the same compound inhibits at least 5 human kinases with equivalent potency (Figure

3.3, Table 3.6).

It might be possible to improve the properties of 4b and meet chemical probe criteria. If I were to

do this, my strategies would be to –

1. determine the selectivity against other Cryptosporidium kinases by expanding the

kinase panel

2. create a mutant parasite harbouring an inactive FIKK enzyme as a control for cellular

activity

3. obtain permeability data on all the compounds tested on parasites

4. obtain target engagement data on all the compounds tested in parasites

5. identify a new chemical series by screening a wider variety of libraries. Compounds

from more than one chemical series will help to further validate or de-validate the

enzyme as a drug target.

85

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