1. The Alpha Project - Stepping Towards Predictive Biology Michael B. Gonzales Senior Research Fellow Molecular Sciences Institute Berkeley, CA

2. molecular sciences institute The Molecular Sciences Institute Founded in 1996 by Nobel Laureate Dr. Sydney Brenner Independent, non-profit research laboratory that combines genomic experimentation and computer modeling Core research activity - The Alpha Project Currently ~20 senior research fellows - molecular biology, physics, chemistry, mathematics

3. Magritte Goal: To combine genomic and computational research in order to make predictive models of biological systems. molecular sciences institute

4. The Alpha Project Five-year, multidisciplinary research effort The focus of the Alpha Project is to examine extra/intra-cellular information flow and processing Collaborators include California Institute of Technology, the Massachusetts Institute of Technology, the University of California, Berkeley, University of California, San Francisco and Pacific Northwest National Laboratory.

5. molecular sciences institute Why baker’s yeast? S. cerevisiae mating provides a level of system description greater than that for almost any other eukaryotic process Alpha pheromone signal pathway is GPCR mediated and analogous to higher eukaryotes Yeast are highly tractable experimentally; facilitating the development of new experimental methods Well-suited to rapid iterative experimental cycles linking new experimentation to new computation

6. molecular sciences institute a  a factor  factor zygote a/  Sex in the lab

7. molecular sciences institute Response to Pheromone

8. molecular sciences institute Ste2 S t e 5 Ste11 Fus3 Ste7MAPK Cascade Ste12 Transcriptional Activation G1 ArrestMorphogenesis, fusion  /GpaI  /Ste4  /Ste18 Ste20 P P P P P The pheromone response pathway

9. molecular sciences institute Credits Ximena Ares Kirsten Benjamin Roger Brent Ian Burbulis Kirindi Choi Tina Chin Alejandro Colman-Lerner Jay Doane Michael B. Gonzales Andrew Gordon Larry Lok Andrew Mendelsohn Orna Resnekov Eduard Serra David Soergel Kumiko Yamaguchi Richard Yu UCSF Matt Jacobson Brian Shoichet Kevan Shokat UC Berkeley Julie Leary (Chem) Stuart Russell (CS) PNNL Richard Smith (Chem) Robert Maxwell MIT Drew Endy (ex MSI) Ty Thomson (BioEng) Gerry Sussman (CS) Tom Knight (CS) Caltech Shuki Bruck (CS) Sandia NL Steve Plimpton (P, CS) Danny Rintoul (P, CS)

10. In search of a Gpa1-specific inhibitor

11. molecular sciences institute Gpa1 Background Key regulatory protein in pheromone signalling pathway Tethered to the plasma membrane via interaction with heptahelical receptor (GPCR) No crystal structure Several good crystallized homologs Rat ~66% ID, 45% Sim, 1.5 Angstroms Divergent insert aa 130-234 does not include binding site - removal has no effect on activity

12. molecular sciences institute Gtpase sequence conservation in yeast Gpa1 Splice Gpa1 Gpa2 Sar1 Arf3 Cin4 Arf2 Arf1 Arl1 *Arrows indicate GTP binding residues in Gpa1.

13. molecular sciences institute Identifying Selective Inhibitor for Gpa1 Evaluate sequence conservation within S. cerevisiae Evaluate crystal structures for homology model building Build/Evaluate homology model Dock small molecules Perform small molecule screen

14. molecular sciences institute Gpa1 Contact Residues Conserved

15. molecular sciences institute Gpa1 model based on 1CIP Hinge

16. molecular sciences institute Gpa1 models with/out cofactors Gold = built with cofactors Aqua = built without cofactors RMSD = 0.1079

17. molecular sciences institute Gpa1 with bound GNP

18. molecular sciences institute Initial screen Screened ~500 molecules from Chembank library (thanks Ilya) Used GTP, GDP, GNP, GTP  S, ATP, ADP as “controls” Glide - standard speed/precision Docked into 2 Gpa1 (spliced) models based on 1CIP 1)Built with Mg cofactor and GNP ligand 2)Built without Mg cofactor and GNP ligand Docked into 1CIP

19. molecular sciences institute Cofactors play critical role in ligand dock scores Ligand+Cofactors-Cofactors GTP24 GNP192 GDP545 GTP  S417 ATP38 ADP954

20. molecular sciences institute Gpa1 pocket built with/out cofactors RMSD = 0.159

21. molecular sciences institute GTP binding poses nearly identical Mg No cofactors With cofactors GTP binding poses in Gpa1 models built with/without cofactors

22. molecular sciences institute Moving forward Evaluate the use of multiple (homology) models to enhance the rank scores *Dock into multiple representative structures *Perform simple scoring function across all ranked molecules - I.e. average score, energy, etc. Evaluate the impact of cofactors/ligands on homology model docking scores *Build homolgy models of protein with many known ligands (Cdk2) - build with and without cofactors/ligands *dock into several resolved crystal structures as well as homology models

23. molecular sciences institute Small molecule identification - the old fashioned way Perform small molecule screens on S. cerevisiae in the lab Powerful genetic tools make assay for inhibitor molecules very straightforward 1000 - 5000 molecules can be screened in ~1month