The Alpha Project - Stepping Towards Predictive Biology

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

2. 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 molecular sciences institute

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

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. molecular sciences institute

5. 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 molecular sciences institute

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

7. Response to Pheromone molecular sciences institute

8. The pheromone response pathway P P P P P Ste2 / GpaI a / Ste4 / Ste18 b g Ste20 S Ste11 t Ste7 MAPK Cascade e molecular sciences institute Fus3 5 Ste12 Transcriptional Activation G1 Arrest Morphogenesis, fusion

9. 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) UCSF Matt Jacobson Brian Shoichet Kevan Shokat UC Berkeley Julie Leary (Chem) Stuart Russell (CS) PNNL Richard Smith (Chem) Robert Maxwell Credits Ximena Ares Kirsten Benjamin Roger Brent Ian Burbulis Kirindi Choi Tina ChinAlejandro Colman-Lerner Jay Doane Michael B. Gonzales Andrew Gordon Larry Lok Andrew Mendelsohn Orna Resnekov Eduard Serra David Soergel Kumiko Yamaguchi Richard Yu molecular sciences institute

10. In search of a Gpa1-specific inhibitor

11. 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 molecular sciences institute

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

13. 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 molecular sciences institute

14. Gpa1 Contact Residues Conserved molecular sciences institute

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

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

17. Gpa1 with bound GNP molecular sciences institute

18. 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 • Built with Mg cofactor and GNP ligand • Built without Mg cofactor and GNP ligand • Docked into 1CIP molecular sciences institute

19. Cofactors play critical role in ligand dock scores Ligand+Cofactors-Cofactors GTP 2 4 GNP 1 92 GDP 5 45 GTPS 4 17 ATP 3 8 ADP 9 54 molecular sciences institute

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

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

22. 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 molecular sciences institute

23. 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 molecular sciences institute