The Development of SUMOylationInhibitors—A Novel Approach to

Combating Tumorigenesis

Mich Gehrig

Weds, May 14, 2014

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Drug Development Flowchart

Correlating an observation with an abnormality

Proof of concept (causation NOT

solely correlation) Alter the normal physiological

process (inhibitors)

Design/test inhibitors

Search for drug-like chemical scaffolds

Refine findings

Appropriate drug properties attained

(Rule of 5)

Clinical TestingDrug

Implementation

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What is SUMOylation?

Common Post – translational Modifications

• Phosphorylation/dephosph-orylation

• Acetlyation/deacetylation

• Ubiquitination/deubiquitin-ation

• Neddylation/Deneddylation

• SUMOylation/deSUMOyla-tion

SUMOylation

• Small ubiquitin-like modifier (SUMO)

• First discovered bound to RanGAP1

• Involved in:– Nuclear organization

– +/- transcriptional regulation

– +/- transcription factors

– Altered binding partners

– Protein Stability

– DNA repair

– Cell signaling 3

• Closely parallels SUMOylation• Can serve as an introduction to the SUMOylation pathway• More studied, better understood• Cross-talk between the two

NLP*Cl Laboratory (2007). http://e3miner.biopathway.org/help_intro.html

Ubiquitination Overview

Proteasome

Target Protein is degraded

Ubiquitin recycled

~ 25 E2Enzymes

~ 9 E1Enzymes

Hundreds of E3EnzymesProblems:

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SUMO-1

(A) Ribbon diagram of the 3D structure of SUMO-1(B) Surface representation

Song J. (2004) PNAS.

ubiquitin-like-protein-processing (Ulps) enzymes

Mature SUMO-1C-terminal

N-terminal

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SUMOylation Overview

Bettermann, K. et al. (2012) Cancer Lett.

Critical Points

Start

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Why are SUMOylation Enzymes Potential Therapeutic Targets?

Edited from: Alarcon-Vargas, et al. (2002) Cancer Biol Ther.

• Only one E1 enzyme (Aos1/Uba2) and one E2 enzyme (Ubc9) are known

• Found across prokaryotes and eukaryotes

• SUMO E1, Ubc9, SUMO E3, and SENPs highly expressed in cancers

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Drug Development Flowchart

Correlating an observation with an abnormality

Proof of concept (causation NOT

solely correlation) Alter the normal physiological

process (inhibitors)

Design/test inhibitors

Search for drug-like chemical scaffolds

Refine findings

Appropriate drug properties attained

(Rule of 5)

Clinical TestingDrug

Implementation

8

INTEGRAL PROBLEM

If we can inhibit SUMOylation, will it have the desired physiological

effects? (eg. Tumor supression)

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SUMOylation-Dependent Myc-driven Tumorigenesis

• c-Myc (Myc) is overexpressed in about ~25% of breast cancers

• Studies have shown that Myc is required for tumor maintenance and progression

Kessler, J.D. et al. (2011) Science

tamoxifen

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Selectively silence genes

Inducible Mycexpression

Turn Myc on (tamoxifen) or off

Death in “Myc ON” only

SUMOylation interference synthetically lethal with hyperactivated Myc

Kessler, J.D. et al. (2011) Science11

• Inhibiton of SUMO E1 resulted in increased population doubling time

• Reinsertion of wild-type cDNA restores cells to normal cancer state

SAE2 (E1) depletion with Mychyperactivation impairs proliferation

Kessler, J.D. et al. (2011) Science

• Increase in number of cells with aberrant DNA

• More spindle Defects

• Increased abnormal mitosis

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SAE2 (E1) depletion with Mychyperactivation impairs proliferation

Kessler, J.D. et al. (2011) Science13

• (Dox-inducible) SUMO E1 inhibition reduced tumor volume

• Low E1 levels correlated to higher survival probability in breast cancer patients

Destruction of p53 via Mdm2 SUMOylation

• p53 = tumor suppressor protein

• Mdm2= an E3 ubiquitin ligase that targets p53

14Buschmann, T. et al. (2000) Cell.

• Self-ubiquitinated: p53 not destroyed

• SUMOylated: autoubiquitinationblocked; p53 degradated

Drug Development Flowchart

Correlating an observation with an abnormality

Proof of concept (causation NOT

solely correlation) Alter the normal physiological

process (inhibitors)

Design/test inhibitors

Search for drug-like chemical scaffolds

Refine findings

Appropriate drug properties attained

(Rule of 5)

Clinical TestingDrug

Implementation

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SUMOylation Inhibitors:4 previously known classes (SUMO E1 Enzyme)

• Class I (2004)– Adenoviral Protein GAM1

• First ever reported E1 inhibitor

• Class II (2010)– Semi-synthetic mechanism-based proteins

• Mimic adenylate or tetrahedral bond formation during thioester bond formation

• Class III (2009)– AMP mimics

• Class IV (2009)– Natural products (ginkgolic acid, anacardic acid

kerriamycin B) 16

Problems We Currently Face• Need to identify small molecule inhibitors

• Need to have better drug-like properties:

– Cellular entry, Solubility, Activity/Half maximal inhibitory concentration (IC50)

Kumar A. (2013) J Chem Inf Model

Chemical Name

IC50 (µM)

Class IV

Ginkgolicacid

3.0

Anacardicacid

2.2

KerriamycinB

11.7

Class III MLN4924 8.2

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Lower IC50 = greater drug potency

The Search #1Kumar, A. et al. (2013) J Chem Inf Model

• Maybridge screening collection downloaded (77,931 compounds) and compared against downloaded crystal structure of SUMO E1– Each compound docked in

ATP binding site of SUMO E1• 5000 docked positions

generated for each ligand!

– Separated by geometrics, energetics

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SUMO activating enzyme (SUMO E1) crystal structure

Yishuzhang (2014) http://commons.wikimedia.org/wiki/File:SUMO_activating_enzyme_wi

th_Mg_and_ATP.png

ATP Binding domain

The Search #2Kumar, A. et al. (2013) J Chem Inf Model

• Top 5% kept

• Evaluted:

– Ligand-binding free energy calculated via surface molecular mechanics analysis

• 24 compounds selected to be purchased from vendors

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The Search #3 (overview)Kumar, A. et al. (2013) J Chem Inf Model

In silico screening scheme used to identify inhibitors of SUMO E120

SUMOylation Assay, 100µMKumar, A. et al. (2013) J Chem Inf Model

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SUMOylation Assay, 20µMKumar, A. et al. (2013) J Chem Inf Model

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IC50 of Compound 21Kumar, A. et al. (2013) J Chem Inf Model

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N,N’’-[methylene-bis(4,1-phenylene)]bis(N’’-phenylurea)scaffolding

Binding of Compound 21 to SUMO E1Kumar, A. et al. (2013) J Chem Inf Model

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Reducing agent

Compound 21

• Disappearance of biotinylated SUMO appearance of E1-SUMO

• Reinforces that compound 21’s mode of action is achieved by targeting SUMO E1

Results: Not So GoodKumar, A. et al. (2013) J Chem Inf Model

Chemical Name IC50 (µM)

Class IV Ginkgolic acid 3.0

Anacardic acid 2.2

Kerriamycin B 11.7

Class III MLN4924 8.2

SmallMolecule

Compound 21 14.4 ± 1.3

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The Search #4 (second search)Kumar, A. et al. (2013) J Chem Inf Model

• Compounds with similar structure to compound 21

– ZINC database (~18 million commercially available molecules)

• Same approach as before

– 37 compounds purchased

Compound 21

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ResultsKumar, A. et al. (2013) J Chem Inf Model

21Parent Molecule

Chemical Name IC50 (µM)

Class IV

Ginkgolic acid 3.0

Anacardic acid 2.2

Kerriamycin B 11.7

Class III MLN4924 8.2

Small Molecule

Compound 21 14.4 ± 1.3

Compound 25 11.1 ± 3.1

Compound 26 13.0 ± 0.4

Compound 30 40.3 ± 7.4

Compound 37 11.7 ± 5.327

Take Note of the Phenylurea groups

Results: Predicted Docking of Compound 25Kumar, A. et al. (2013) J Chem Inf Model One phenylurea

protrudes into the hydrophobic

patch near Leu49 and Ile96

One phenylureais placed near

Ile384 and Thr141, the ATP binding pockets

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Results: DrugabilityKumar, A. et al. (2013) J Chem Inf Model

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The new

compounds

Discovered compounds have weaker activity, but higher drugability

Drug Development Flowchart

Correlating an observation with an abnormality

Proof of concept (causation NOT

solely correlation) Alter the normal physiological

process (inhibitors)

Design/test inhibitors

Search for drug-like chemical scaffolds

Refine findings

Appropriate drug properties attained

(Rule of 5)

Clinical TestingDrug

Implementation

30

Refining the Search

• Problem:

– discovered compounds barely fit within limits

– poor solubility (symmetrical)

– Active site of SUMO E1 not actually symmetric

• Methods

– Returned to Maybridge Chemical library

– Selected non-symmetrical compounds

31Kumar, A. et al. (2013) Bioorg. Med. Lett.

A New Class of SUMO E1 InhibitorsQuinazolinyloxyl biaryl urea

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Quinazolinyloxyl biaryl urea identified as new class of SUMO E1 Inhibitors

Compound 4

Kumar, A. et al. (2013) Bioorg. Med. Lett.

A New Class of SUMO E1 InhibitorsQuinazolinyloxyl biaryl urea

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Compound 9 the most potent version of compound 4

Compound 4

Cl binds in hydrophobic

pocket;quinazoline

N’s to Ile-96, Asp-48

This is where

adenosine in ATP binds

Kumar, A. et al. (2013) Bioorg. Med. Lett.

Overall Results

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Chemical Name IC50 (µM)

Class IV Ginkgolic acid 3.0

Small Molecule Compound 25 11.1 ± 3.1

Quinazolinyloxyl biarylurea Compound 9 13.4

Kumar, A. et al. (2013) Bioorg. Med. Lett.

Correlating an observation with an abnormality

Proof of concept (causation NOT

solely correlation) Alter the normal physiological

process (inhibitors)

Design/test inhibitors

Search for drug-like chemical scaffolds

Refine findings

Appropriate drug properties attained

(Rule of 5)

Clinical TestingDrug

Implementation

Drug Development Flowchart: Conclusion

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Summary

• SUMO E1, Ubc9, SUMO E3, and SENPs highly expressed in cancers

• Only one E1 enzyme (Aos1/Uba2) and one E2 (Ubc9) enzyme that are known

– Easy drug target

• SUMOylation inhibition (via E1 inhibition) prevents tumorigenesis

– Myc-dependent cancers

– Prevents ubiquitination of tumor-supressor p53

• Rapid advancement in the development of SUMO E1 inhibitors

– Establishment of Quinazolinyloxyl biaryl urea as a viable drug scaffold

• Need to:– Further refine inhibitors

– Clinical testing

– Drug implementation36

Kumar, A. et al. (2013) Bioorg. Med. Lett.

Thank you!

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Bettermann, K.; Benesch, M.; Weis, S.; Haybaeck, J. SUMOylation in carcinogenesis. Cancer Lett. 2012, 316, 113-125.

Buschmann, T.; Fuchs, S. Y.; Lee, C.; Pan, Z.; Ronai, Z. SUMO-1 Modification of Mdm2 Prevents Its Self-Ubiquitination and Increases Mdm2 Ability to

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