This document summarizes the development of novel inhibitors of PAK1 for the treatment of breast cancer. It outlines assays to identify small molecules that promote PAK1 dimerization or stabilize its autoinhibited state. Hits from cytotoxicity screening will undergo PAK1 activity, dimerization, and phosphorylation assays. Promising leads will be characterized in vitro and tested in a mouse efficacy model. The goal is to develop PAK1 inhibitors with improved specificity and efficacy compared to existing drugs.

1. Breast Cancer pHortunepHarmaceuticals Biochem 4H03 November 19th, 2010

2. Breast cancer is the top cancer in women worldwide…

3. Worldwide Incidence 1.3 million diagnoses annually 519,000 deaths (2004)

4. and is increasing particularly in developing countries where the majority of cases are diagnosed in late stages Breast cancer is the top cancer in women worldwide - World Health Organization, 2010

5. Cancer Loss of cell cycle regulation Caused by environmental or genetic factors 4-stage tumor classification Benign or Malignant Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.

6. Classification of Breast Cancer Non-Invasive Lobular (LCIS) Ductal (DCIS) Invasive Lobular Ductal Inflammatory Paget’s

7. Anatomy www.breastcancer.org

8. Breast Cancer Kills Metastatic cells redirect resources from non-cancerous cells Metastasis to vital organs is lethal Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.

9. Treatment Options Ogden, Joy. Understanding Breast Cancer. UK: John Wiley & Sons Ltd, 2004.

10. What is our goal Target should be: Highly expressed Involved in cell proliferation Involved in cell motility Anti-apoptotic Identify novel inhibitory mechanism

11. identification of novel PAK1 inhibitors

12. p21-Activated Kinase 1 Key regulatory enzyme for cell signaling Cell Membrane Kichina, J.V., (2010) Expert Opinion Therapeutic Targets.14 (7): 703-725

13. PAK1 as a Target 57% of breast tumoursoverexpressPAK1 55% protein overexpression in breast cancer cells Constitutively expressed PAK1 stimulates carcinogenesis in mice. No current drugs on the market Can be targeted through different mechanisms Dummler, B, et al. (2009) Cancer Metastasis Rev.28: 51-63. Kumar, R ., et al. (2006) Nature Reviews Cancer6: 459-471

14. PAK1 PAK1 p21 P PAK1 PAK1 P P p21 P Activation of PAK1 Lei, M., et al. (2000) Cell. 102: 387-397

16. CEP-1347

17. OSU-03012

18. DW12

19. FL172PAK1 p21 P P P P Current PAK1 inhibitors Poor Specificity and Efficacy Yi, C., et al. (2010) Biochem. Pharmac. 80: 683-689

20. PAK1 Inhibitors of Monomerization p21 T423 Phosphorylation Inhibitors PAK1 p21 P Our Drug Target

21. Small Molecules Promote Dimerization Nitric Oxide Synthase (NOS) is an active homodimer Imidazole and 1-phenol imidazole promotes dimerization Given this finding, the promotion of dimerization by small molecules is feasible = imidazole or 1-phenol imidazole NOS NOS Sennequier, N. (1999) J.Biol. Chem. 274, 930-938.

22. Promoting Autoinhibition im Bcr-Abl Imatinib An inhibitor that binds to the autoregulatory loop of the kinase, Bcr-Abl Imatinibstabilizes the autoinhibitedstate of the protein by interacting with Asp381 Found to be highly selective and reasonably effective (Ki = 37 nM) Bcr-Abl active Bcr-Abl Imatinib stabilized autoinhibited inactive confirmation Cheetham, G.M. (2004).Curr. Opin. Struct. Biol. 14, 700–705.

23. Assay Design In vitro assay! Hits Leads Dimerization Activity Cytotoxicity Leads T423 ELISA NoHits Stage 1 Leads: PAMPA, IC50, MIC Cross-Reactivity Preliminary Lead In vivo mouse efficacy model Stage 2 Leads: CYP-450, Solubility, pKa Advance Lead

24. Cytotoxicity Assay Goal: determine cytotoxic molecules MCF-7 & MDA MB-134 cell lines Screen Canadian Compound Collection 30,720 small molecules 10uM bioactivity cut off for compounds Add cells Add CCC compounds (10uM) incubate Read OD590 HITS DMSO – negative controlTaxol (20nM) – positive controlTest wells

25. PAK1 Activity Assay Non-inhibited Reaction: Light Cdc42 Cdc42 PAK1 PAK1 P MBP MBP PAK1 PAK1 ATP Luciferin cmpd Luciferase + ADP Reduced Chemiluminesence Inhibited Reaction: ATP Light Cdc42 Cdc42 MBP Luciferin MBP Luciferase cmpd ATP ATP Luciferin + Luciferase Chemiluminesence ADP

26. PAK1 Activity Assay Positive Control = Luciferase + Luciferin + ATP Negative Control = Luciferase + Luciferin Activity Control = Luciferase + Luciferin + ATP + PAK1 + MBP + sphingosine + GTP + test compound Test Well = Luciferase + Luciferin + ATP + PAK1 + MBP + cdc42 +GTP + test compound

27. Dimerization Assay cdc42 cdc42 cdc42 cdc42 P P P PAK1 PAK1 PAK1 PAK1 PAK1 P cmpd P P P P Venus Cerulean Cerulean Venus PAK1 475nm cmpd Fluorescence 528 nm Cerulean Venus

29. Negative Control: Same conditions except test compounds

30. Activity Control #1: Compound with PAK1 (Cerulean)

31. Activity Control #2:Compound with PAK1 (Venus)

32. Test Well: PAK1(Cerulean)+ PAK1 (Venus) + cdc42 + ATP + GTP + test compoundsPositive control Negative control Test wells Activity control #1 Activity control #2 Background wells Rizzo, M.A., et al. (2004) Nature Biotech.22 (4): 445-449

33. Activation Loop Phosphorylation Assay

35. Negative Control: No compoundsPositive control Negative control Test wells

36. Assay Design In vitro assay! Hits Leads Dimerization Activity Cytotoxicity Leads T423 ELISA NoHits Stage 1 Leads: PAMPA, IC50, MIC Cross-Reactivity Preliminary Lead In vivo mouse efficacy model Stage 2 Leads: CYP-450, Solubility, pKa Advance Lead

37. In vivo ?

38. Current Methods of Administration Intravenous Herceptin Oral Tamoxifen Imatinib Kinase inhibitor specific for cancer cells

39. Conclusion PAK1 is overexpressed more than receptor required for Herceptin Proposed robust strategy and novel mechanism of inhibition of PAK1

40. $8.7 billion in sales worldwide (2009) Projected $16.5 billion in 2016 Herceptin revenue: $4.8 billion (2009) GlobalData. Breast Cancer – Drug Pipeline Analysis & Market Forecasts to 2016 Maggon, K. et al. (2008). Global Cancer Market Review: World Best Selling Top 10 anti-Cancer Drugs. Knol publishing.

41. Worldwide Incidence

43. Stages

44. PAK1 biology - briefly Controls adhesion-induced Rac1 activation and cell spreading by regulating Rac1-b-Pix interaction. Modulates cytoskeleton dynamics and cell mobility at the leading edge through phosphorylation of multiple substrates. During mitosis, PAK1 is recruited to the centrosomes where it becomes activated and phosphorylates Aurora-A and Plk1, both important regulators of mitotic events. Promotes cell proliferation through phosphorylation of c-Raf and MEK PAK protects cells from apoptosis via multiple mechanisms.

45. PAKs as therapeutic targets Expression of a constitutively active PAK1 mutant increases cell motility, anchorage-independent growth, and invasiveness in MCF-7 breast cancer cells and leads to development of metastatic mammary tumors and other types of breast lesions in a transgenic mouse model. NF1 and NF2 are both pathologically and molecularly distinct diseases, but they both involve mitogenic signaling pathways that are downstream of Ras and impinge upon the regulation of PAK activities. In NF1, PAKs are upstream effectors that result in the full activation of the Ras-MAPK pathway, which is one of many oncogenic signaling cascades. In NF2 patients, loss of Merlin is associated with elevated levels of Rac-GTP accompanied by abnormal PAK1 activation

46. PAK1 and the Cell Cycle

47. P PAK1 PAK1 p21 P P P PAK1 in Action MBP MBP P P

48. A Good Schematic Representation of PAK1

49. PAK1 PAK1 p21 p21 P PAK1 PAK1 PAK1 PAK1 P P p21 P Activation of PAK1 Complete version

50. MCF-7 Culturing Protocol Use Eagle's MEM, supplemented with 10% FBS, 1% penicillin/streptomycin. Can also add non essential amino acids (0.1 mM), Insulin (10ug/mL) and Sodium pyruvate (1mM). Add 10nM estrogen to media for a 3-4x increase in cell numbers. Maintain temperature at 37°C in humidified, concentrated CO2 (5%) atmosphere. Once MCF-7 cells reach approximately 90% confluence on plates, remove media and passage cells by rinsing with 1xPBS twice. Add 2-3 mL of warm (37°C) 0.25% Trypsin- 0.53 mM EDTA solution to cells to disperse cell layer. Observe under an inverted microscope. Dispersal should happen between 5 and 15 minutes. If cells are not detaching properly, place flask back in 37°C incubation chamber. Do not incubate for more than 3 minutes or so. Note: Do not agitate the chills during dispersal, either by hitting or shaking the flask. This may cause clumping as the cells detach. Once MCF-7 cell layer is dispersed (3min at 37°C) deactivate Trypsin by adding 5 ml cells/Trypsin-EDTA to 10mL of complete growth medium (see step 1) in sterile tube. Aspirate cells by gently pipetting Centrifuge cells in growth medium for 5 minutes at 125x g-force. Remove trypsin/growth medium suspension from tube. Resuspend pellet (MCF-7 cells) in 10 mL fresh growth medium (see step 1) Plate 1 mL of suspension to each new plate containing 9 mL original growth medium (see step 1), and incubate at 37°C in humidified 5% CO2 atmosphere.

51. Cytotoxicity Assay Culture cell lines approximately 4-7 days according to standard protocols in Eagle Minimum Essential Media. Partition the cell culture into several 96-well plates with OD of 10,000 cells per well. ~ 40 plates a day 80 compounds per plate 10 days in total to partition all necessary wells for the 32000 compounds. 16 controls per plate. Positive Controls: 20 nM of Taxol need 0.06 mg  1mg = $40 something Negative Controls: DMSO Total volume in each well of 180 µL. Add compounds (10 µM) and compare ODs with control to decide which ones go through. Run this experiment in triplicates for the two cells lines.

52. Luciferase Assay Add ATP, PAK1, MBP, and cdc42-GTPys + previous hits to a 384-well plates + luciferase + luciferin. Positive Control: 1) ATP + Luciferase + Luciferin. Activity Control: 1) ATP + Luciferase + Luciferin + PAK1 + sphingosine + MBP + drug. Negative Control: 1) Luciferase + Luciferin + no ATP. 2 “active controls” per compound. 24 compounds per plate on a 96 well plate. (Assuming 1% hit…14 plates*3 (for triplicates) = 42 plates)

53. FRET Assay PCR and clone human PAK1 with cerulean and venus into expression vectors (2 vectors each with a tag) and transform E. coli cells with the vectors. Express and purify using Ni column and then cutting the HIS-tag out. With cerulean, add Cdc42-GTP + phosphatase to get monomers. In parallel, do the same with venus-tagged PAK1. Add venus-tagged PAK1 to Cerulean-tagged PAK1 + drug. Excite at 423nm (cerulean will absorb and emit at 475nm) and venus (YFP) absorb at 515nm and will emit at 528nm. Cerulean and Venus work in 5 nm radius; our protein is approximately 4.5 nm radius from N termini from one monomer to C termini of another. Controls 1) Two proteins that dimerize together with the cerulean and the venus tag. 2) No drug. 3) Cerulean fusion protein with drug. Excite at 423 and observe at 475 to make sure that the drug does not affect the abs/ems spectra. 4) venus fusion protein with drug. Excite at 515 and observe at 528 to make sure that the drug does not affect the abs/ems spectra.

54. FRET Assay

55. T423 Phosphorylation Assay Attach Ab specific for PAK1 to a high binding plate, and incubate it with PAK1. Incubate plate with drugs + cdc42-GTPys + ATP. Incubate primary Ab specific to T423, then add secondary Ab for detection. Negative Control: Everything except for ATP. Positive Control: Everything except for drugs.

57. Minimum Enzyme Activity: 4 x MIC [drug]

58. Maximum Enzyme Activity: No compound addedEnzyme Activity log[drug]

59. PAMPA Assay Donor Well Receiving Well Hexadecane Membrane

60. CYP450 Assay Concentrated liver microsomes + lead molecule + O2(g) + NADPH + H+ Incubate tube Analyze LC/MSMS Check for Potential metabolites

61. Solubility solute Measure concentration of solute in each UV/VIS Spectroscopy shake octanol water pKa

63. Drug Localization Low-resolution whole-body autoradiography Routine in preclinical ADME studies, but need high levels of drug accumulation Informs about high-capacity low-affinity binding sites High-resolution autoradiography Using this method, the focus of study can emphasize routes of delivery, adsorption, distribution, metabolism, and excretion Can create a drug homunculus Autoradiography combined with immunocytochemistry (co-localization) can further contribute to the characterization of target cells through simultaneous demonstration of radiolabelled drug and a cancer marker

64. Assay Design In vitro assay! Hits Leads Dimerization Activity Cytotoxicity Leads T423 ELISA NoHits Stage 1 Leads: PAMPA, IC50, MIC Cross-Reactivity Preliminary Lead In vivo mouse efficacy model Stage 2 Leads: CYP-450, Solubility, pKa Advance Lead