University of Michigan Center for Chemical Genomics CCG

1. University of MichiganCenter for Chemical Genomics (CCG) A collaboration between LSI and Dept of Pharmacology to make High Throughput Screening (HTS) available to all UM Faculty LSI Website http://www.lsi.umich.edu/ccg

2. CCG Personnel David Sherman (Director), Rick Neubig (Assoc. Dir.), Susan Johnson (Admin) HTS � Stuart Decker & Martha Larsen HTP � Janet Smith, Jim DelProposto Chemical Diversity � David Sherman Informatics � Rick Neubig, Renju Jacob (LSI IT staff)

4. Inhibiting Bak/Bax Binding to Bcl-2

6. Chemical Genomics vs. Genetic Models With robotic instrumentation and compound libraries, it is often possible to quickly identify relatively specific inhibitors. The use of a small molecule allows the study of the kinetics of a response in a more subtle, graduated way that is not possible with gene disruption techniques and avoids problems with embryonic lethality often observed with genetic knockouts. Small molecules have the advantage of being easily distributed among different laboratories. The small molecule may be further developed as a therapeutic agent. Represents an opportunity to integrate areas of expertise across the university such as technology development (engineering), structure based design, nanobiology, chemistry, etc.

8. Assays The assay is the most critical aspect of success High Throughput Screening (10,000 � >1,000,000 compounds) depends on simple, robust assays Many different types are used effectively Fluorescence, luminescence, colorimetric Biochemical or cell-based (mammalian or yeast) Must be done in small volumes (10-50 ul) and with simple automated readouts

9. Compound Libraries CCG now has 35,000 diverse compounds from Chembridge, Maybridge, and ChemDiv Natural product library Additional 20-30,000 Maybridge compounds through Fisher agreement Virtual library >2 million compounds in database

10. Screening Formats In vitro/biochemical (for enzymes, protein-protein interactions, ligand binding, etc) Assay readouts such as Fluorescence intensity, FP,TRF, ABS, LUM, BRET, FRET Cell Based (mammalian cells, yeast, zebra fish, etc): Enzymatic/ELISA readouts (luciferase reporter, alkaline phosphatase, b-gal complementation, FRET, etc) 2. �High content� microscopy-based screens measuring protein translocation, neurite outgrowth, GPCR signaling, cell motility, cell proliferation, apoptosis chromosome imbalance, vesicular trafficking, others)

11. HTS Instrumentation Biomek FX liquid handling robot Multidrop dispensers ELx plate washer PHERAstar multimode plate reader

12. Informatics

13. Some Approaches to Small Molecule High Throughput Screening

15. New Opportunities for CCG Fisher funding initiative for novel technologies (CCG website): Collaborative Pilot Project Proposals Expand screening technologies and libraries, i.e. NMR Fragment-based screening Robotic integration for UHTS

16. Ras/MEK/ERK Signaling

17. PD98059 MEK Inhibitor

18. Effects of PD98059 on the Morphology of Ras Transformed Cells

20. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo

21. Three-dimensional representations of the ternary complex of MEK1 bound to PD318088 and MgATP.

24. SPCs Subtilisin-Related Proprotein Convertases Yeast protease (Kex2) Human homolog (furin) Enzyme cleave C-terminally Arg (KR, RR, RXKR) Drug Targets Kex2: fungi Furin: toxins, viral, cancer Known inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin Yeast Kex2 protease and human furin are Subtilisin-Related Proprotein Convertases (SPCs) Enzyme- process proproteins by cleavage C-terminally to Arg residues (e.g., KR, RR, RXKR) in eukaryotic secretory pathway Drug Target- Kex2: virulence,cell wall integrity of pathogenic fungi Furin: activation of bacterial toxins, viral glycoproteins and implicated cancer metastasis Known protein and peptide inhibitors for Kex2 and furin

25. Crystal structures: structural conservation Kex2: Homologues in Candida albicans and glabrata Furin: processes growth factors, receptors, serum, matrix proteins & metalloproteinases. eg. furin inhibitors and anthrax Crystal structures: catalytic domains of Kex2 and furin complexed with covalent inhibitory peptides has revealed extensive structural conservation Kex2: Homologues in pathogenic fungi such as Candida albicans and glabrata required for virulence Furin: processes growth factors, receptors, serum, matrix proteins & metalloproteinases. eg. furin inhibitors block processing of Anthrax Protective Antigen and hence inhibit toxins effect on macrophagesCrystal structures: catalytic domains of Kex2 and furin complexed with covalent inhibitory peptides has revealed extensive structural conservation Kex2: Homologues in pathogenic fungi such as Candida albicans and glabrata required for virulence Furin: processes growth factors, receptors, serum, matrix proteins & metalloproteinases. eg. furin inhibitors block processing of Anthrax Protective Antigen and hence inhibit toxins effect on macrophages

26. Objective Identify small, organic inhibitors of Kex2 and furin drug models lead compounds conjugates for organic metal-chelate inhibitors

27. Method Use secreted, soluble forms of Kex2 and furin with fluorogenic peptide substrates 384-well low volume plates (25ul) HDR �pin tool� (0.2�l)

28. Assay Kex2, 1.55nM Add compounds Add substrate Boc-VPR-MCA,18.75�M Add stop reagent Read fluorescence (380nm EX / 470nm EM) Controls Positive: substrate only & known inhibitor (spiked) DMSO >1% barcoded plates: Chembridge & ChemDiv [4.2-15�M] Dose-response (50�l), IC50 (PRISM) Statistics: mean, SD and CV for control wells (n=16-32) Z�>0.5 for all plates and for assay Kex2 (15�l, 1.55nM final) in 200mM BisTris, pH 7.0 containing 1mM CaCl2 and 0.01% Triton X-100 was added using the Multidrop dispenser (Thermo Labsystems) to 384-well low volume plates (Corning, #3677). Compounds (or controls) were added using the HDR �pin tool� (Biomek FX, Beckman) in a volume of 0.2�l. After a 15 min incubation (RT), the substrate, Boc-Val-Pro-Arg-MCA (tert-butoxycarbonyl-Val-Pro-Arg-methylcoumarinamide, Bachem) was added (5�l, 18.75�M final). Controls were DMSO (1% final) and the known inhibitor, Dec-Arg-Val-Lys-Arg-cmk (decanoyl-Arg-Val-Lys-Arg-chloromethylketone, Bachem). Buffer 1 without Triton X-100. After 15 in incubation (RT), acetic acid (5�l, 0.5mM final) was added and fluorescence was measured (380nm, ex/ 470nm, em) within 45 min using the PHERAstar high-throughput fluorescence microplate reader (BMGLabtech). Gain was set on the DMSO negative control at 50% with substrate alone as positive control (full inhibition). Kex2 inhibitor was added to select well as a control. All plates were barcoded for identification and linked to compounds from stock plates (Chembridge and ChemDiv libraries). Final compound concentrations were 4.2-15�M. Dose-response data was obtained with 384-well plates (Corning, #3710) using same concentration of enzyme and substrate but final reaction volumes of 50�l. See poster Center for Chemical Genomics for further details on equipment and compounds tested. Statistics: Assay quality was determined by calculating mean, standard deviation and coefficent of variation (CV, SD/mean) for control wells on each plate (n=16-32). Z�>0.5 for all plates and for assay(12) (Fig. 4). Any plates Z�<0.5 were repeated. Dose response values were calculated using Prism (GraphPad). Kex2 (15�l, 1.55nM final) in 200mM BisTris, pH 7.0 containing 1mM CaCl2 and 0.01% Triton X-100 was added using the Multidrop dispenser (Thermo Labsystems) to 384-well low volume plates (Corning, #3677). Compounds (or controls) were added using the HDR �pin tool� (Biomek FX, Beckman) in a volume of 0.2�l. After a 15 min incubation (RT), the substrate, Boc-Val-Pro-Arg-MCA (tert-butoxycarbonyl-Val-Pro-Arg-methylcoumarinamide, Bachem) was added (5�l, 18.75�M final). Controls were DMSO (1% final) and the known inhibitor, Dec-Arg-Val-Lys-Arg-cmk (decanoyl-Arg-Val-Lys-Arg-chloromethylketone, Bachem). Buffer 1 without Triton X-100. After 15 in incubation (RT), acetic acid (5�l, 0.5mM final) was added and fluorescence was measured (380nm, ex/ 470nm, em) within 45 min using the PHERAstar high-throughput fluorescence microplate reader (BMGLabtech). Gain was set on the DMSO negative control at 50% with substrate alone as positive control (full inhibition). Kex2 inhibitor was added to select well as a control. All plates were barcoded for identification and linked to compounds from stock plates (Chembridge and ChemDiv libraries). Final compound concentrations were 4.2-15�M. Dose-response data was obtained with 384-well plates (Corning, #3710) using same concentration of enzyme and substrate but final reaction volumes of 50�l. See poster Center for Chemical Genomics for further details on equipment and compounds tested. Statistics: Assay quality was determined by calculating mean, standard deviation and coefficent of variation (CV, SD/mean) for control wells on each plate (n=16-32). Z�>0.5 for all plates and for assay(12) (Fig. 4). Any plates Z�<0.5 were repeated. Dose response values were calculated using Prism (GraphPad).

29. Z� score Z� from representative assay. + control: substrate, - control: Kex2 +substrateZ� from representative assay. + control: substrate, - control: Kex2 +substrate

30. Screen results The primary screening with Kex2 identified 29 compounds as �hits� (=30% inhibition). Dose response assays confirmed 14 hits, excluding 15 false positives (Table 1, Fig. 5). Preliminary screening for furin showed that furin inhibitors may represent a distinct set of compounds(data not shown). Given that the specificities of the enzymes are quite similar, inhibitors may be interacting with unique features of the target enzymes exclusive of substrate-recognition sites. If true, high throughput screening may yield inhibitors that detect subtle differences between members of the SPC family. The primary screening with Kex2 identified 29 compounds as �hits� (=30% inhibition). Dose response assays confirmed 14 hits, excluding 15 false positives (Table 1, Fig. 5). Preliminary screening for furin showed that furin inhibitors may represent a distinct set of compounds(data not shown). Given that the specificities of the enzymes are quite similar, inhibitors may be interacting with unique features of the target enzymes exclusive of substrate-recognition sites. If true, high throughput screening may yield inhibitors that detect subtle differences between members of the SPC family.

32. Dose response of CCG-22129

33. Kex 2 Screen Results HTS on Kex2 Z�>0.7 14 �hits� (0.047%) IC50: 27nM-1.5�M 8 �hits� structural similarity MW<400 Furin Bioassays The University of Michigan Center for Chemical Genomics (CCG) of the Life Sciences Institute has made possible the identification of small molecule inhibitors of the eukaryotic proprotein convertases by high throughput screening (HTS) of large compound libraries. HTS using Kex2 as a target identified 14 hit compounds after primary screens and secondary (dose response) assays with ideal statistics (Z� scores > 0.70 ). These hits (0.047% of 30,000 compounds screened) were not identified in other screens with other assays at the CCG. The best value of IC50 for Kex2 was 27nM with CCG-29212. Eight (b) out of 14 hits have a common structural core making initial structure-activity comparisons possible (Table 1). Another strucural core, (a), was found twice (Table 1). Molecular weights of these compounds are less than 400, and structural features suggest they should be efficiently internalized into cells. Future goals will be to confirm inhibition using alternative (non-fluorescence) assays, to characterize modes of inhibition, to identify higher-affinity derivatives, to determine selectivity among the SPC family members and to use compounds in bio-assays in yeast and mammalian cells.The University of Michigan Center for Chemical Genomics (CCG) of the Life Sciences Institute has made possible the identification of small molecule inhibitors of the eukaryotic proprotein convertases by high throughput screening (HTS) of large compound libraries. HTS using Kex2 as a target identified 14 hit compounds after primary screens and secondary (dose response) assays with ideal statistics (Z� scores > 0.70 ). These hits (0.047% of 30,000 compounds screened) were not identified in other screens with other assays at the CCG. The best value of IC50 for Kex2 was 27nM with CCG-29212. Eight (b) out of 14 hits have a common structural core making initial structure-activity comparisons possible (Table 1). Another strucural core, (a), was found twice (Table 1). Molecular weights of these compounds are less than 400, and structural features suggest they should be efficiently internalized into cells. Future goals will be to confirm inhibition using alternative (non-fluorescence) assays, to characterize modes of inhibition, to identify higher-affinity derivatives, to determine selectivity among the SPC family members and to use compounds in bio-assays in yeast and mammalian cells.

34. Acknowledgements CCG and HTS staff Bob Fuller and Tomoko Komiyama LSI support