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The Alpha Project - Stepping Towards Predictive Biology Michael B. Gonzales Senior Research Fellow Molecular Sciences Institute Berkeley, CA 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 Goal: To combine genomic and computational research in order to make predictive models of biological systems. Magritte 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. 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 Sex in the lab a factor a a factor zygote a/a a Response to Pheromone 2 hour pheromone treatment Reporter: Prm1-YFP Bright field image Fluorescent image The pheromone response pathway P P P P P Ste2 a/GpaI b/Ste4 g/Ste18 Ste20 Ste11 S t Ste7 MAPK Cascade e Fus3 5 Ste12 Transcriptional Activation G1 Arrest Morphogenesis, fusion 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 MIT Drew Endy (ex MSI) Ty Thomson (BioEng) Gerry Sussman (CS) Tom Knight (CS) UC Berkeley Julie Leary (Chem) Stuart Russell (CS) Caltech Shuki Bruck (CS) Sandia NL PNNL Richard Smith (Chem) Steve Plimpton (P, CS) Danny Rintoul (P, CS) Robert Maxwell In search of a Gpa1-specific inhibitor 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 Gtpase sequence conservation in yeast Gpa1 Splice Gpa1 Gpa2 Sar1 Arf3 Cin4 Arf2 Arf1 Arl1 *Arrows indicate GTP binding residues in Gpa1. 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 Gpa1 Contact Residues Conserved Gpa1 model based on 1CIP Hinge Gpa1 models with/out cofactors Gold = built with cofactors Aqua = built without cofactors RMSD = 0.1079 Gpa1 with bound GNP Initial screen • Screened ~500 molecules from Chembank library (thanks Ilya) • Used GTP, GDP, GNP, GTPgS, 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 Cofactors play critical role in ligand dock scores Ligand +Cofactors -Cofactors GTP 2 4 GNP 1 92 GDP 5 45 GTPgS 4 17 ATP 3 8 ADP 9 54 Gpa1 pocket built with/out cofactors RMSD = 0.159 GTP binding poses nearly identical With cofactors No cofactors Mg GTP binding poses in Gpa1 models built with/without cofactors 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 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