Malaria Libre

2st project meeting12th August 2020

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3

Agenda

• Introductory session – if new members join

• Recap of last meeting

• Work flow and early lead criteria

• Project update

• How to contribute- new members?

• Discussion

MMV: Partnerships for health impact

Discover, develop and deliver safe,

effective and affordable

antimalarial medicines

https://www.mmv.org/research-development/mmv-supported-projects

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There are new ways of working

together….

1. Wells, T. N. C. et al. Nature Reviews Drug Discovery, 15, 661-662 (2016)2. Van Voorhis, W. C. et al. PLOS Pathogens, 12, e1005763 (2016)3. Williamson, A. E. et al. ACS Cent. Sci., 2, 687−701 (2016)

Open Innovation1

…..project partners sharing all information

Open Source1,3

…..shared data and open

participation

Open Science1

…..deposition of data in the public

domain

Open Access1,2

…..free access to data and materials

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Malaria Libre - New way of working

MalariaLibre

Open access

No patents

Suggestions are best form

of criticism

Opportunity to seek free

expertise

Not owned by any

lab

Data is public

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Malaria

Libre

People

resources

ProcessesInformation

sharing

• Open access to datahttps://www.mmv.org/mmv-open/malaria-libre/malaria-libre-

data-repository

• Suggestions:

post on LinkedIn

or email to

malaria.libre@mmv.org • Centrally coordinated and community developed

• Simple processes

• Periodic discussions

• Focussed efforts to targeted deliverables

• Core team to support the project(1st tier assays, synthesis)

• Engage scientists from industry and academia in

fortnightly/monthly discussions

• Expand network

Malaria Libre – Connect and Integrate

Objective: Identification of preclinical candidate(s)

Criteria for stage up : https://www.mmv.org/research-development/information-scientists

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Validated hit criteria checklist

Compound Property Criteria Aryl imidazoles Aryl piperazine

Potency Invitro potency Pf < 1uM yes yes

SAR Evidence with change in

potency

Yes Yes

Cytotoxicity assay (HepG2) >10 fold Yes Yes

Genotoxicity risk based on hidden

structures

No obvious risks Hidden aniline is replaceable No flag

Mechanism of action Novel based on structure

and phenotype

Probably novel Probably novel

Additional activity Pb/Pc/Pf/Pv liver, male&

female gamete formation

Some evidence of activity in

stageV gametocytes and Pb

liver schizonts- needs to be

confirmed further

MMV024406are active against

Pf and Pb liver stages but

transmission blocking activity to

be confirmed

Profile in invitro ADME assays to understand the issues Issues - metabolic stability Issues- CYP inhibition and

metabolic stability

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Workflow

Synthesis in individual labs &TCGLS

Registration of compounds in MMV database : Science

Cloud*

Purity >90%

Send the compounds to TCGLS for 3D7LDH and

HepG2 assay**

Data updated on webpages

Tier 1 ADME assays @TCGLSScreening in panel of

strains (Shailja)

*

** CDRI to screen compounds synthesised in house in 3D7SYBR and K1

Blood stage specificity assay(Shailja)

1-2 compounds/scaffoldto understand the potential

1-2 compounds/scaffoldto understand the potential

In vivo PK(FNDR, TCGLS)mouse and rat

Tier1 assays: solubility, logD, h mic, rhep

CYP inhibition, PPB, blood/plasma partitioning

hERG(manual patch clamp)Ames(2 strain)in case of aniline

In vivo Pharmacology models

Invitro PRR

Early lead compound(s)

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MMV Compound NumberTarget Product Profile (Treatment or Chemoprevention)Target Candidate Profile List TCP claimsCompound PropertiesCompounds resynthesised (Y/N)? Y

Full analytical data (1H, MS, RT, absolute stereochemistry known).pdf reports showing data and written out

electronicallyMolecular Weight (MWt) <500Salt/ adductSample MWt (including salt or adduct)cLogP/ ALogP (Science Cloud)LogD pH 7.4 <5No. of H-bond donors <5No. of H-bond acceptors <10tPSANo. of stereogenic centres <3If chiral, racemic or single isomer? Single isomerClear SAR (Y/N)? YNo. of synthetic steps from commercial starting points Simple, short routeMaximum scale synthesised to dateSolubility PBS/ SGF/ FASSIF/ FESSIF/ pH 6 (mM) ≥10mM (PBS)Melting point MeasuredChemical stability (pH 2 and pH 8) stable

Genotoxicity risks based on structure e.g. hidden anilines present?No. If "Yes" then explain and give data in safety

sectionLigand efficency (pIC50/ per heavy atom count) For info

Scifinder search performed on exact structure and simplified coreState whether novel based on Scifinder search

of exact molecule or simplified core

Early lead criteria – structural aspects

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Potency at in vitro biochemical target (Ki) (Pf, Pv, Pb, Hs) - if relevant Ki<100nM against PfSelectivity vs relevant related host target (fold) >100 foldPotency Erythrocyte assay (EC50) - (minimum 7 strains including lab derived and clinical mutants)

EC50<100nM (48h, 72h)

Free EC50 in 72h 3H NF54 erythrocyte assay NF54 72h 3H EC50 hill slopePotency Cytotoxicity assay (EC50) - (minimum two cell lines) (µM) >100 foldAsexual intraerythrocytic blood stage specificity (ring vs. Trophozoites vs. schizont) Based on microscopyIn vitro PRR (Log10), onset of action (h) and Hill Slope

Mechanism of actionNovel based on resistance panel or strategic

imperative if knownPb/ Pf liver schizont EC50 EC50<100nM for causal TCP4Pc/ Pv hypnozoite EC50 EC50<1000nM for TCP3Pf NF54 Gametocyte stage I/ III EC50 MeasuredPf NF54 Gametocyte stage V EC50 EC50<100nM for TCP5Pf Male gamete formation assay (exflagellation): incubation/ incubation and wash out/ no pre-incubation EC50s For TCP5: EC50<100nM on male and/or femalePf Female gamete formation assay: incubation/ incubation and wash out/ no pre-incubation EC50s

For TCP5: EC50<100nM on male and/or female

TCP 3 Evidence of in vitro block of relapseTCP 1 Calcualed Human MPC from in vitro data

Good to have: MIR; Pf and Pv clinical field isolate values

Early lead criteria – Pharmacology

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Stability in plasma (mouse, rat, dog, human) stable Permeability: Caco-2 (A-->B; B-->A, ER plus units Measuredin vitro met stab Clint - mouse/ rat/ dog/ human microsomes Measuredin vitro met stab Clint - mouse/ rat/ dog/ human hepatocytes MeasuredPlasma protein binding (mouse, rat, dog, human) MeasuredBlood/ plasma partitioning (mouse, rat, dog, human non-infected blood) MeasuredAlbumax Bindidng MeasuredCYP450 inhibition (CYP1A2, 2D6, 2C9, 2C19, 3A4) + those that overlap with current therapies IC50uM 3/5 >10uM and none <1uM

Mouse i.v. (dose), Clearance (ml/min/kg), t1/2, Volume (L/Kg) Measured

Mouse p.o. (dose), Tmax, Cmax, AUC, F%, t1/2F%>20% using stable suspension / solution in

acceptable vehicle

Rat i.v. (dose), Clearance (ml/min/kg), t1/2, Volume (L/Kg) Measured

Rat p.o. (dose), Tmax, Cmax, AUC, F%, t1/2F%>20% using stable suspension / solution in

acceptable vehicle

TCP1 Dose to clear 9 / 12 Logs parasites from single/ three qd dose(s) For TCP1

TCP4 Dose to give t>prophylactic concentration for 7 days from single doseFor TCP4

Predicted adult human t1/2, Cl and V hours, mL/min/Kg, L/Kg

hERG IC50 uM (manual patch clamp) >1uM; some potent examples >10uM

Early lead criteria – DMPK and Toxicity

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SAR around aryl piperazine (MMV024406) - recap

Challenges:High h microsomal clearance

High CYP inhibition

MMVID 024406Pf NF54/3D7(µM): 0.26, 0.42eLogD: 3.7Clint(hmic,uL/min/mg): 20.6Caco2: 2.1Sol (PBSbuffer, µM) <2.5CYP 1A2, 2C9, 2D6, 3A4 % Inhibition@10 µM: 45,69,72,84

1803900-, 8.2NDNDNDND

ND

024408-, 0.43

4.318.1ND7.2

IC50>30µM

1803901-/23

High h microsomal clearance

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MMV024406

yDHODH assay

neither DHODH nor bc1 inhibitor

Rate of killing

2-time points FACS rate of killing 2-time points PRR

MMV690906

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MMV024408(series 1a) – Challenges and way forward

Pf3D7,IC50: 0.43uMelogD: 4.3clogP: 3.2H mic (clint, uL/min/mg; t1/2, min):18.1, 77R hep (clint, uL/min/mg; t1/2, min): 232.7, 3

[O]

[O][O]

[O]

Putative sites of metabolism

CYP mediated putative metabolites can be assessed using GLORY

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SAR around aryl piperazine - MMV024408(series 1a)

Objective: Improve potency(3D7LDH IC50<100nM)

Address poor metabolic stability and solubility

Lower log D; modifications of metabolic hot spots

1804685

3.18

1804634

3.50

MMVIDPf3D7 IC50 uM

1804684

2.56

1804459

1.13

1804508

0.19

1804345

1.17

MMVID 1804508Pf 3D7(µM): 0.19eLogD: 3.8Clint(hmic,uL/min/mg;t1/2,min): 33;42Clint(rhep,uL/min/106;t1/2,min): 138;5(PBSbuffer, µM) 7.45CYP 1A2, 2C9, 2D6, 3A4 % Inhibition@10 µM: <20

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SAR around aryl piperazine - MMV024406(series 1c)

Objective: Improve potency(3D7LDH IC50<100nM)

Address poor metabolic stability, poor solubility and high CYP inhibition

Lower log D; modifications of metabolic hot spots

18038990.373.645.2;372.9;242ND49

10,15,20,37

MMVID 024406Pf 3D7,LDH(µM): 0.42eLogD: 3.7Clint(h mic,uL/min/mg;t1/2,min): 20.6; -Clint(r hep,uL/min/106;t1/2,min): NDCaco2: 2.1Sol (PBSbuffer, µM) <2.5CYP 1A2, 2C9, 2D6, 3A4 % Inhibition@10 µM: 45,69,72,84

MMV 024408

Series 1a

Explore substitution pattern – impact on potency and metabolic stability

Introduction of linker – impact on potency(synthesised few compounds, results awaited)

Reverse amide- potency improvement?heterocycles

MMV 1803899Series 1c Modulation of pKa to modulate activity

NH2, Me

Plans around aryl piperazine scaffold

isosteres of amide

Improving metabolic stability

Reverse amide- moving away from anilines

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Snapshot of SAR around aryl imidazole (MMV023227)

Challenge:• Improve metabolic stability of compounds while improving activity

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Rate of kill – PRR studies

MMV693239

MMV1794035 : 2 pt. FACS assay ongoing

MMV1794035

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Addressing poor metabolic stability of MMV023227

[O]

[O]

[O] MMV023227

MMV1794034

MMV893302

MMV1804344

MMV892881

MMV ID 023227 1794034 892881 893302 893303 1804344

Pf3D7(SYBR/LDH,IC50 uM)

0.46/1.1 0.86/- 0.72/- 0.92/- 0.7/2.4 -/1.4

HLM, Clint 205 246 54 98 70.3 ND

r hep,Clint 206 163 ND 22.4 13.4 ND

MMV893303

Putative metabolites

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MMV 892881 Synthesise and characterise 5-7

diverse compounds to confirm SAR

and DMPK properties wrt to

MMV023227

MMV 884798

2 analogs in amides indicate a

reverse pattern as compared to amines and

ethers - synthesise 4-5 diverse compounds with

low log D

Modifications to improve potency

Explore role of linker lengthAza indoles and substituted

phenyl

Substitution with CH2OH

Oxazole/thiazole

Plans around aryl imidazole scaffold

Few representative compounds

X= O,NH; R= H,F

Proposed molecules by Gloria

Update on MMV023227 & MMV024406 by CSIR-CDRI

Approach to MMV023227 analogues

Approach to MMV024406 analogues

Amination reactions

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Update on MMV024406 by CSIR-CDRI

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Action Items and responsible teams

Action items Responsible team/group Status

Design and Synthesis of analogues CDRI(Sanjay, PP, Kishore); Gloria; TCGLS

Ongoing at CDRI and TCGLS; Gloria to initiate

Primary screening(3D7 screening) LDH assay: TCGLSSYBR assay: CDRI (in house compounds)

TCGLS: ongoingCDRI : to initiate after 15th Aug – Kiran to send 2 compounds from each scaffold

Cytotoxicity(HepG2) TCGLS ongoing

Screening of representative compounds in ring stage assay

Shailja(JNU) Kiran to send 2 compounds from each scaffold- aryl piperazine analogs are prioritized

Screening of representative compounds in clinical isolates

Shailja(JNU) Kiran to send 2 compounds from each template for screening

In vitro ADME profiling(Tier 1) TCGLS Select new set of compounds

Life cycle assays Volunteers? Select compounds

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ESAC review

Early Discovery Review is on 26th and 27th October 2020

Each project is assigned an ESAC mentor and discovery review is done once a year

14 September: draft version of report to be sent to MMV project director28 September: deadline for final version of report to be sent to Angelique12 October : deadline for ESAC reviews to send questions (via MMV) project team19 October : deadline for project team written response to ESAC reviewer questions

https://www.mmv.org/about-us/people-governance/expert-scientific-advisory-committee-esac

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Ways of contribution

• Design and synthesis of compounds to achieve the objectives

• Synthesis of compounds

• Identification of putative metabolites for both scaffolds

• Carry out experimental in vitro Met ID

• Screen front runners in lab derived strains other than 3D7; asexual intraerythrocytic blood stage

assays, mechanism of action

Details of objectives and plans: https://www.mmv.org/mmv-open/malaria-libre/malaria-

libre-data-repository

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Together WE can – come join the community

Preclinical Candidate MedicinalChemistry

ParasitologyBiochemistry

DMPK

Invitrotoxicology

Structural Biology

Invivotoxicology Predictive

modelling

• Status update – synthesis, screening, SAR, ESAC review

• Where we are with respect to early lead

• ESAC presentation

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Tentative agenda for next meeting

Tentative date of meeting : 2nd September 2020

Time : 1530-1630 (Indian standard time)

Committed funders of MMV

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SAR around aryl piperazine (MMV024406) - recap

Challenges:• High h microsomal clearance

• High CYP inhibition

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SAR around aryl piperazine - MMV024408(series 1a)

Challenge:• High h microsomal clearance