One Ring to Rule Them All. Linda H. Malkas, PhD October 23, 2018

Transcription

1 ne Ring to Rule Them All Linda H. Malkas, PhD ctober 23, 2018

2 Disclosures I do not have any disclosures.

3 Current Cancer Molecular Therapeutics It is a disease exhibiting heterogeneity making it difficult to treat successfully even with the most intensive multi-modal therapies available Identify tumor-associated characteristics for cancer selectivity defects in protein structure and interactions errors in cell signaling identify genome, transcriptome, and protein biomarkers that are relevant to cancer therapy and precision medicine search for- or improvement of - clinical agents that can selectively target tumors

4 Cancer: unmet need and a novel therapeutic strategy ewer inhibitory therapeutic strategies largely rely on the targeting of a single enzymatic activity in cancer cells, resulting in general good initial tumor response followed by the development of drug resistance A better approach to anticancer therapeutics would be targeting of key hub factors in molecular pathways critical for cancer cell growth and evolution such as DA repair Targeting of molecular hubs would result in the shutting down of a myriad of functions in cancer cells and making it more difficult for these cells to develop resistant clones Targeting of molecular hubs in cancer cells could effectively lead to cancer cures

5 Why DA Repair is Important

6 Eukaryotic DA replication fork

7 Click to edit Master title style

8 Cancer Cell s DA Replication Apparatus Is Altered in Function and Structure Human Cell DA Synthesome MCF 10A MCF 7 Hs578Bst Hs578T MDA- MB468 PCA Profile in Human Breast Tumor

9 PCA IDCL Interactions Facilitate DA Replication and Repair Protein-Protein Interaction Site Disordered Structure

10 Mapping the cancer associated domain

11 The L126-Y133 region is structurally altered and more exposed to protein-protein interaction in cancer-associated PCA

12 Antibody Detects Cancer Cells in Tissue ormal DCIS Invasive Metastatic

13 capcaab Staining of Individual Slides

14 Therapeutic Drug Discovery o-o Rules Drugs can only be made against proteins with enzymatic activities Drugs shouldn t be made against disordered regions in proteins Drugs can t be made to block protein-protein interactions

15 capca Target Therapeutics Two Approaches Being Pursued - Peptide - Small molecules

16 ur Strategy + DA damaging agent Damaged DA PCA + Repair Proteins Repaired DA DA damaging + Damaged DA agent capca Ca PEP + Repair Proteins Damaged DA PCA has many protein binding partners

17 How I See PCA

18 What happened when you take a hub out?

19 Selective cytotoxicity of R9-caPep in BC cells

20 Verifying capep sequence itself is cytotoxic to cancer cells

21 R9-cc-caPeptide treatment effectively reduces tumor volume in a mouse model R9-caPep

22 capep inhibits DSB repair in cancer cells

23 Inhibition of DA replication and induction of S-phase arrest and apoptosis by R9-caPep

24 R9-caPep interferes with replication fork extension

25 BRCA1 deficient BC cells are especially sensitive to capep

26 capep enhances the activity of cisplatin

27 R9-caPep works synergistically with Chk1 inhibitors

28 Dysregulation of replication initiation and induction of DA damage and apoptosis by R9-caPep and Chk1 inhibitors

29 capep action 1. Functional and structural basis: The L126-Y133 region of PCA is functionally important and structurally altered in cancer cells. 2. Target validation: A cell permeable peptide harboring the L126-Y133 sequence selectively kill cancer cells, without causing significant toxicity to normal cells. ( 4 publications on 3 cancer types) 3. Druggability: The L126-Y133 region delineates a known small molecule binding pocket.

30 capeptide forms a cavity within capca The Caldera

31 capep and its binding partners

32 Drug Discovery

33 AH2 AH18 AH20 AH34 AH37 AH39 AH45 AH52 AH95 AH2 AH18 AH20 AH34 AH37 AH39 AH45 AH52 AH95 AH2 AH18 AH20 AH34 AH37 AH39 AH45 AH52 AH95 AH2 AH18 AH20 AH34 AH37 AH39 AH45 AH52 AH95 AH2 AH18 AH20 AH34 AH37 AH39 AH45 AH52 AH95 Cytotoxic Effect of AH Compounds HCC1937 MCF7 SK--AS um um um um um um um um um SK--BE2c SK--DZ um um um 100 um um 100 um 0 0

34 Discovery and characterization of AH39

35 Exploratory Chemistry H H H H H H H #1174 IC50 > 30 um #1164 IC50 = 183 um A B C #1160 IC50 = 0.15 um m #1162 IC50 > 50 um H H H #1175 IC50 > 30 um #1165 IC50 = 35 um H H H H #1177 IC50 > 30 um AH39 IC50 = 1.10 um m H H H H #1176 IC50 > 30 um #1161 IC50 = 21.6 um H H #1166 IC50 = 34 um H H H H H #1178 IC50 = 1.10 um m #1180 IC50 > 30 um #1179 IC50 > 30 um

36 AH1160 inhibits growth of breast cancer cells and tumors

37 AH1160 in CI-60 panel screen Median IC50 = ~330 nm CCRF-CEM HL-60(TB) K-562 MLT-4 RPMI-8226 SR LX IMVI M14 MALME-3M MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 IGRV1 CI/ADR-RES VCAR-3 VCAR-4 VCAR-5 VCAR-8 SK-V-3 DU-145 PC A498 ACH CAKI-1 RXF 393 S12C TK-10 U CS Cancer Colon Cancer on-small cell Lung Cancer Leukemia Melanoma varian Cancer PC Renal Cancer

38 The effect of AH1160 on cell cycle progression and apoptosis

39 Relative length of DA segment AH1160 inhibits DA replication fork movement o Rx AH1160

40 AH1160 inhibits homologous recombination mediated DA repair EJ5-GFP DR-GFP

41 AH1160 sensitizes cancer cells to cisplatin treatment Relative number of colonies

42 AH small molecules have no thyroid hormone activity

43 Body Weight (g) AH1160 inhibits the growth of breast cancer xenograft tumors Vehicle AH1160 (40 mg/kg) Vehicle Days after tumor injection

44 Tinkering with molecules

45 AH1996 in CI-60 panel screen Median IC50=~320 nm

46 ormal cells R e la tiv e C e ll G ro w th Breast Cancer cells AH1996 inhibits the growth of human breast cancer cells M D A -M B M D A -M B H s t M C F H C C P B M C 7 S M o rm a l S A E C L o g [A H ] AH1996 IC50 (M) MDA-MB x 10-7 MDA-MB x 10-7 Hs578t 3.78 x 10-7 MCF x 10-7 HCC x 10-7 PMBC > 5 x SM0032 > 5 x 10-6 ormal SAEC > 5 x 10-6

47 Effect of AH1996 on xenograft tumor growth in mice Vehicle AH1996

48 P e r c e n t o f A p o p to tic C e lls AH1996 induces apoptosis in cancer cells DMS AH S M SM S K - -D Z 0.2 SK--DZ D M S A H

49 AH1996 causes cell cycle arrest in cancer cells

50 Relative length of DA strands Mechanism of action: Inhibition DMS AH MDA-MB [AH1996] (mm)

51 Mechanism of action: Inhibition

52 Potential of AH1996 in combination therapy of existing chemotherapeutic drugs

53 Relative AH1160 Concentrations Improved stability of AH1996 in liver microsomes 1.2 Fig 4. Stability of AH1160 in human Liver microsomes ADPH - ADPH Incubation time (min)

54 AH1996 is stable to amide hydrolysis

55 AH1996 is orally available to mice

56 14-Day ral Toxicity Study of AH1160 and AH1996 in Mice Clinical and Anatomic Pathology Report CCLUSIS: The objective of this study was to determine the toxicity of AH1996 in male and female Es1e/SCID mice following 14 days of oral, once daily administration. one of the observations in clinical pathology (hematology and clinical chemistry) parameters, macroscopic observations, or histopathology findings were considered a direct effect of the test article. The no-observed-adverse-effect level (AEL) for AH1996 in this study was 100 mg/kg/day, the highest dose level tested.

57 T u m o r V o lu m e (c m 3 ) Targeting capca for Cancer Treatment IMPACT Identified and validated a cancer-specific region (L126-Y133) of capca as an anti-cancer drug target Developed an orally active small molecule compound (AH1996), which targets this PCA region and selectively kills cancer cells at submicromolar concentrations Penetrates BBB and active against glioblastoma Virtual screen targeting the L126-Y133 region Vehicle only AH D a y s a fte r tu m o r im p la n ta tio n Medicinal chemistry to improve potency and metabolic stability Malkas Funding W81XWH-14-LCRP-CA Malkas W81XWH Hickey Reckamp Alex s Lemonade Stand Shared Resources APCF, DDSB, MMSBD Publications Gu et al. Plos ne 2014 Gu et al. EBioMedicine 2014 Smith et al. Mol Pharmacology 2015 Start-Up Company RLL, LLC Horne Synold Chung FDA pre-id package submitted and answered

58 Summary DA repair is a critical pathway for cell survival and a good target for anticancer drug development (ex. PARP inhibitors) Identified and validated a cancer-specific region of PCA as a therapeutic target Developed a potent small molecule compound (a snowstorm) that targets this PCA region and selectively kills a broad-spectrum of cancer cells Protein-protein interaction sites and disordered protein domains can be used for drug targets Targeting important molecular hubs in cancer cells can be a viable drug development strategy

59 Shutting down hub to hub (or huba huba)

60

61 Future Plans Complete ID-enabling preclinical studies and submit an ID application in Spring 2019

62 Acknowledgments City of Hope Dr. Robert Hickey Dr. Long Gu Dr. Shanna Smith Dr. Caroline Li Mr. Robert Lingeman Dr. David Horne Dr. Yate-Ching Yuan Dr. Yun Yen Dr. Jeremy Stark Dr. Tim Synold TGen Dr. Daniel Von Hoff Greater Baltimore Center Dr. Lauren Schnaper University of Maryland Dr. Pamela Bechtel Dr. Jennifer Sekowski Indiana University Dr. Sunil Badve Dr. George Sandusky Dr. Beamon Argawal Dr. Jianying Liu Dr. George Sledge Dr. Fei Shen Ms. Lacey Dobrolechi Torrey Pines Institute Dr. Gregg Fields Albany Molecular Research, Inc. (AMR)