High-Throughput Screens for Identifying Novel Anti-cancer Agents

1. High-Throughput Screens for Identifying Novel Anti-cancer Agents University Health Network F-F Liu MD, FRCPC Radiation Oncologist/Senior Scientist

2. Outline Introduction Forward Chemical Screen Reverse Screen Conclusions

3. HTS Test large number of molecules simultaneously via miniaturization and automation of assay protocols 1. Drug discoveries 2.Understand biological processes

4. Approaches to HTS 1. Reverse screens 2. Forward screens

5. Mutate Gene Screen for chemical binding Insert in vivo Add compound in vivo Look for phenotype Look for phenotype Reverse Genetics Reverse Classical Genetics Reverse Chemical Genetics

6. Reverse Chemical Genetics

7. Random mutagenesis Select mutant with phenotype Identified mutated gene Forward Genetics Forward Classical Genetics

8. Plate Cells Add 1 compound/well Select compound that produces phenotype Identify protein target Forward Genetics Forward Classical Genetics Forward Chemical Genetics Random mutagenesis Select mutant with phenotype Identified mutated gene

9. Forward Chemical Genetics Target Identification 1. Biotin labeling 2. Y3H 3. Micro-array

10. Forward Chemical Genetics

11. Outline Introduction Forward Chemical Screen Current Reverse Screen Conclusions

12. Forward HTS for Head & Neck Cancer Therapeutics

13. Day 2 (Add Compounds) (1/well) Day 4 (MTS - viability) Screening Procedure Day 1 (Seed Cells) • Choose cells that double every 21 h • reduce bias • Screen cancer vs. normal • choose “hit” criteria with caution • Lower dynamic range than other assays, but less manipulation

14. 64 hits screened by FaDu cell viability NIH/3T3 viability DECREASE 35 compounds 29 compounds 3 novel 1 studied Screening Procedure LOPAC Library (1280 compounds) Prestwick Library (1120 compounds) 23 compounds NO Anti-microbial YES 6 compounds

15. C666-1 viability 1 compound DECREASE 4 compounds 5757 viability DECREASE 1 compound 5 compounds 3 novel 1 studied Screening Procedure 6 compounds

16. HNC Therapeutics Forward small molecule HTS: 3 existing antimicrobials with novel anticancer properties: 1. Benzethonium Chloride Yip et al,Clin Cancer Res 15, 5557, 2006 2. Alexidine Dihydrochloride Yip et al,Mol Cancer Ther 5, 2234, 2006 3. Cetrimonium Bromide Ito et al, (manuscript under review)

17. Cetrimonium Bromide (CTAB) • Quaternary ammonium compound • Delocalized lipophilic cation • Lipophilic; delocalized positive charge • Penetrates hydrophobic cellular membranes • Accumulates in mitochondria due to negative M • Interacts with mitochondrial H+-ATP synthase

18. Anti-Cancer Specificity

19. Combination Therapy

20. CTAB Induces Apoptosis

21. CTAB Induces Apoptosis

22. CTAB Induces Apoptosis FaDu: Hypopharyngeal SCC C666-1: NPC GM05757: Primary normal fibroblast

23. CTAB Induces Apoptosis

24. CTAB Inhibits Mitochondrial ATP Synthase Activity

25. CTAB Reduced Intracellular ATP Levels

26. M of Cancer vs. Normal Cells

27. Anti-Cancer Specificity

28. CTAB Ablates In Vivo Tumour-Forming Capacity

29. CTAB Reduces Growth of Established Tumours

30. Proposed mode of action • CTAB is concentrated in tumor mitochondria due to M •  CTAB-ATP synthase interactions + - M - + P

31. Proposed mode of action Outer Mitochondrial Membrane • H+-gradient across IMM • dissipates •  M is sensed by PTP M Inner membrane • PTP opening induces MOMP • Mitochondrial dysfunction • Apoptogenic factors released - MOMP • Caspase activation • Apoptosis Matrix + MOMP Apoptosis Adapted from Wikipedia

32. Conclusions 1. Identified a novel mitochondria- mediated apoptogenic anti- cancer agent using a forward HTS. 2. Selective in vitro and in vivo efficacy against HNC models • Rooted at the mitochondria • M differences between cancer vs. normal cells

33. Outline Introduction Forward Chemical Screen Current Reverse Screen Conclusions

34. Objective Design HTS to identify novel genes that can selectively sensitize cancer cells to radiation when suppressed

35. Proposed Modifications RT+ Readout? 1.Target-driven siRNA-based approach 2.Incorporate RT into screen 3.Utilize a more appropriate readout for determining radiosensitization Adapted from Nature Rev Genetics 7:373, 2006

36. Kassner; Comb Chem & HTS; 11:175, 2008

37. The multidisciplinary approach of high-content screening (HCS) requires a combination of different expertise. Korn & Krause; 11:503, 2007

38. Specific Aims 1.Define experimental parameters for target-driven siRNA-based HTS 2.Conduct the screen using siRNA libraries to identify novel radiosensitizing targets 3.Validate and characterize hits

39. Specific Aims 1.Define experimental parameters for target-driven siRNA-based HTS 2.Conduct the screen using siRNA libraries to identify novel radiosensitizing targets 3.Validate and characterize hits

40. siRNA Transfection Parameters

41. Katz et al; Biotechniques; 44:9, 2008

42. Katz et al; Biotechniques; 44:9, 2008

43. Automated Clonogenic Assay Conclusions: • Effective in measuring effects of cytotoxic agents on cancer cells in vitro • Limitations • Low cell # reduces dynamic range • Unable to identify sensitization Katz et al; Biotechniques; 44:9, 2008

44. BrdU Cell Proliferation Assay • Bromodeoxyuridine (BrdU) • Thymidine analogue • Incorporated into newly synthesized DNA strands of actively proliferating cells (S phase) • Non-radioactive alternative to [3H]-thymidine incorporation • Examines cellular effects with long-term kinetics that are more reflective of therapeutic response

45. Positive siRNA Control Scrambled siRNA DNA Ligase IV siRNA

46. Specific Aims Define experimental parameters for target-driven siRNA-based HTS Conduct screen using siRNA libraries to identify novel radiosensitizing targets Validate and characterize hits

47. siRNA Libraries Dharmacon human siARRAY libraries: • 4 siRNAs pooled/gene • Protein Kinase siRNA library • 800 genes • Druggable siRNA library • 6080 genes

48. siRNA Screens

49. Specific Aims Define experimental parameters for target-driven siRNA-based HTS Conduct screen using siRNA libraries to identify novel radiosensitizing targets Validate and characterize hits

50. Validation of siRNA Hits siRNA library (6880 siRNAs) 0 Gy 2 Gy Decreased surviving fraction • 188 siRNAs (2 Gy) Increased surviving fraction No effect Eliminate Transfect ± RT (0 vs. 2 Gy) 137 siRNAs 51 confirmed hits Top 15 hits