Building New Research Models in

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1 Building New Research Models in Drug Discovery Arena: A Tough Integration of Clinically Relevant Signaling Pathways with Translational Small Molecules Prabhat Arya Science Club, School of Life Sciences University of Hyderabad November 30, 2016

2 ur current understanding of molecular pathways is insufficient as a platform for effective pharmaceutical discovery. Novartis Boston (2005)

3 Several biotechnology companies have focused on the known elements of a few key pathways to target them with new medicines. But for the genome to be translated into medicines with any reliability l and regularity, far more work needs to be done. Defining the role of pathways in complex diseases will undoubtedly take many years.

4 Traditionally, how we have been doing in the drug discovery arena - we all started like this Defining the target (could be enzymes or isolated protein(s) Structural information on the target finding the pocket Well defined Compact Deep pocket small molecule binder Structural guided chemistry /med chem Lipinski s s Ruleof5 February 22,

5 From Genomic Knowledge to Signaling Pathways tough journey in understanding the complexity and its impact! Complex, multiple protein-protein interactions Dynamic and temporal processes! Regulation (normal) and de-regulation (disease) 5

6 The sequencing of complete genomes provides a list that includes the proteins responsible for cellular l regulation... However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. Science 2003, 300,

7 Science 2009, 326,

8 Looking at the domains only The human genome encodes 120 SH2 domains embedded in a variety of Proteins! 523 kinases and 138 phosphatases encoded by human genome! Science 2009, 326,

9 76 amino acid At least 20 structurally distinct dstnct ubiquitin-binding domains (UBDs) are embedded in a variety of proteins. (mm bindings) There are ~600 ubiquitin E3 ligases and more than 90 deubiquitinating proteinases encoded d by the human genome. Science 2009, 326,

10 Challenges in Neuro Drug Discovery: Literature Perspective 10

11 Underestimated the complexity of human biology! February 22,

12 In the Context of Neurological Disorders Why Alzheimer's Drugs Keep Failing DRUG CANDIDATES HAVE A 99.6 PERCENT FAILURE RATE, AND PR EARLY DETECTIN METHDS MAKE CLINICAL TRIALS DIFFICULT AND CSTLY dementia has become a graveyard for a large number of promising drugs. A recent study looked at how 244 compounds in 413 clinical trials fared for Alzheimer's disease between 2002 and f those 244 compounds, only one was approved. The researchers report that this gives Alzheimer's disease drug candidates one of the highest failures rates of any disease area 99.6%, compared with 81% for cancer. Dementia is a ticking bomb costing the global economy 350 billion and yet progress with research is achingly slow Sci. Am February 22,

13 February 22,

14 February 22,

15 2016 Steve Haggarty February 22,

16 2015 February 22,

17 ur Translational Chemical Biology Journey Related to Cancer Drug Discovery 17

18 Chemical Biology Model: Step B Step A Modern rganic Synthesis Novel Chemical Toolbox Biologically Relevant Chemical Space Cell Signaling Biology (Biological l Questions/ Novel Assays) Cellular/Zebrafish Studies In Vivo Studies Natural Product-like Natural Product-inspired Hybrid Natural Products Understanding! Biophysics, and Structural Biology SPR Protein NMR Computational Tools Step C Translational Path! 18

19 Chemical Biology Model Revised Version: Patient-derived Questions Modern rganic Synthesis Novel Chemical Toolbox Biologically Relevant Chemical Space Natural Product-like Natural Product-inspired Hybrid Natural Products Understanding! Cell Signaling Biology (Biological l Questions/ Novel Assays) Cellular/Zebrafish Studies In Vivo Studies Biophysics, and Structural Biology SPR Protein NMR Computational Tools Translational Path! 19

20 Map large surface area Shallow surface Combination of several weak interactions Extended hydrophobic interactions Possible hot spots May 14, 2014 Issue Theme: Chemical Biology of Protein-Protein Interactions 20

21 Molecules (natural products) for pathways present complex 3D architectures H 2 N dense display of stereo-defined groups challenging task in placing them on the drug discovery path! excellent track record as small molecule modulators of protein-protein interactions can serve as a good source of inspiration for developing novel scaffolds in the drug discovery arena! H Eib Eribulin H H H 21

22 H H H H H H Me Me H H H H Halichondrin B H H Me H Me H H Nature, 2010 February 22,

23 Step A: Building A Natural Product-inspired i d Chemical Toolbox 23

24 ur chemical toolbox-based working model Bioactive Natural Products Key features in our design: Natural Product Inspired Sub-structures t Natural Product Fragments as Sub-structures 3D architectures sufficient complexity stereochemical and skeletal diversity synthesis in a reasonable time-scale easy to follow-up medicinal chemistry studies Exploring Macrocyclic Chemical Space! Cyclic compounds Large surface area Pre-organization Less freely rotating bonds Enhanced cell permeation From Arya Research Team: Chem. & Biol. 163 (2005) Curr. pin. Chem. Biol. 247 (2005) Chem. Rev (2009)

25 Nat Chem Biol 2010,, 6,, 861 Too complex Too simple

26 Three Inspirational Natural Products for Building a Chemical Toolbox H 2 N H Eib Eribulin H H H Geldanamycin Epothilone 26

27 Building a Macrocyclic Toolbox Based on Eribulin Sub-structures Mahendar Khatravath th Ravi Jimmidi H 2 N H H H H Saidulu Konda Naveen Kumar PhD awarded

28 Inspiration from Epothilone Jasplakinolide Chondramide H S N Epothilone H Bhanudas Dasari Cytoskeleton

29 Nat Rev Cancer 2007 February 22,

30 Ansamycins as HSP90 Inhibitors Nat Chem Biol

31 Inspiration from Ansamycins Ansamycin-inspired Geldanamycin Srinivas Jogula 31

32 Where are we going now? Patient-derived Cancer Stem Cells Pathways Leading to Selective Cell Death and Cell Migration 32

33 Apoptosis (Programmed Cell Death) pro anti Bcl-family: Set of closely related Anti and proapoptotic proteins 33

34 Nature 2005

35 Nature Medicine 2013

36 Identification of the cell type capable of initiating and sustaining growth of the tumor the cancer stem cell (CSC) While the existence of CSCs was first proposed over 40 years ago, only in the past decade have these cells been identified and characterized in hematological og malignancies.

37 Nat. Rev. Drug Discovery July

38 Nat. Rev. Drug Discovery July

39 Cell 2009 Salinomycin HC H H H H H H H 39

40 Cell 2014 Kitambi Karolinska 40

41 ur Collaborative Roadmap Isolation of Cancer Stem Cells (CSCs) from human brain tumor. Validation of Cancer Stem Cells. Identification of small molecules as highly selective killers of CSCs. Understanding of specific pathways affected by small molecule hits! Cell survival assays using CSCs vs cancer cells. Cell survival, proliferation and migration assays with CSCs in zebrafish. 41

42 Scientific Components Cellular Assays Chemistry Focused natural product-inspired macrocyclic compounds Cell viability and cell death assays using patient-derived primary cancer cells, tumorspheres and cancer stem cells Cellular Assays Cell migration assays using Patient-derived primary cancer cells, tumorspheres and cancer stem cells Tumor Xenograft Patient-derived primary cancer cells, tumorspheres and cancer stem cellsbased tumor xenograft studies

43 Scientific Team Gopal Kundu NCCS Pune Subhadra Dravida Transcell Biologics Satish Kitambi Hyderabad Karolinska Inst Sweden Patient-derived primary cancer cells, Various tumorospheres and cell death cancer stem cells assays (Brain, Breast, Head and Neck) February 22, 2017 Assays for cell viability and cell death through Caspase 3 activation Kiranam Chatti DRILS Cell migration assays Harinarayana Rao Reliance Life Sciences Patient-derived xenograft 43

44 Cell Death Studies with Patient-derived Brain Tumor Cells German Patient H N H N NH Me Me PMB 7 PMB 7 H N Me Me H N Me Me PMB H N NH H H TBDPS H PMB TBDPS Me Me PMB H N H H TBDPS And several other intermediate compounds from this series (in total, 35 compounds are tested to date) In collaboration with the Kitambi Lab, Karolinska Inst 44

45 ur Early Data with Patient-derived Brain Tumor Cells ATP measurement assay black spots: DMS as control red spots: staurosporin as a control blue spots: Compounds from this 10.0 um nly 30 compounds are tested to date! In collaboration with Satish Kitambi Karolinska 45

46 ur Early Data with Patient-derived Brain Tumor Cells ATP measurement assay Data with A7 In collaboration with Satish Kitambi Karolinska 46

47 Multiplex Immunostaining Assays with DMS with compound A7 Red: Phalloidin Green: Apoptotic Cells (through Caspase 3 activation) Blue: Nucleus % apoptotic cells DMS A7 In collaboration with Satish Kitambi Karolinksa 47

48 2016

49 ur Next Level Study! Indian Patient Before Xenograft 12 Samples After Xenograft 12 Samples Patient t 1 Breast PCC TS Tissues-X PCC-X Patient 2 Breast PCC TS Tissues-X PCC-X Patient 3 Patient 4 Brain Brain PCC PCC TS TS Tissues-X Tissues-X PCC-X PCC-X Patient 5 Patient 6 H&N H&N PCC PCC TS TS Tissues-X Tissues-X PCC-X PCC-X PCC: Primary Cancer Cells // TS: Tumorospheres // H&N: Head and Neck Cancer 49

50 Cancer Stem Cells: Proliferation and Cell Migration in Zebrafish Model! Lateral view of 2 days old zebrafish larvae with DAPI (stains all the nucleus as blue) and the newly transplanted CSC in red Dorsal view of approximately 1 month old zebrafish transplanted with CSCs labelled red brain brain eye Cancer Stem Cells Kitambi Karolinska 50

51 Attempts to Addressing the Mode of Action? Cell Chemical Biology February 22,

52 Sincere Thanks to Arya Academic Research Team in Canada ( ) NRC Canada Genomics and Health Initiative Biochemistry Dept, McGill University Chemical Biology Program Broad Institute t of Harvard and MIT

53 MoES/CSIR-CDRI Graduate Students (2009-) Madhu Aeluri ( ) Srinivas Chamakuri ( ) Ravikumar Jimmidi ( ) Shiva Krishna Reddy ( ) Bhanudas Dasari ( ) Srinivas Jogula ( ) Saidulu Konda ( ) Mahender Khatravath ( ) Naveen Kumar ( ) Jagan Gaddam (2010-) Science and Technology Park University of Pune

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