The Alpha Project- Stepping Towards Predictive Biology

Michael B. GonzalesSenior Research FellowMolecular Sciences InstituteBerkeley, CA

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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

Magritte

Goal: To combine genomic and computational research in order to make predictive models of biological systems.

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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.

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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

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a

a factor

factor

zygotea/

Sex in the lab

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Response to Pheromone

Bright field image Fluorescent image

2 hour pheromone treatmentReporter: Prm1-YFP

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Ste5

Ste11

Fus3

Ste7 MAPK Cascade

Ste12 Transcriptional Activation

G1 Arrest Morphogenesis, fusion

/GpaI /Ste4 /Ste18

Ste20

PP

PPP

The pheromone response pathway

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CreditsXimena AresKirsten BenjaminRoger BrentIan BurbulisKirindi ChoiTina ChinAlejandro Colman-LernerJay DoaneMichael B. GonzalesAndrew GordonLarry LokAndrew MendelsohnOrna ResnekovEduard SerraDavid SoergelKumiko YamaguchiRichard Yu

UCSFMatt JacobsonBrian ShoichetKevan Shokat

UC BerkeleyJulie Leary (Chem)Stuart Russell (CS)

PNNLRichard Smith (Chem)Robert Maxwell

MITDrew Endy (ex MSI)Ty Thomson (BioEng)Gerry Sussman (CS)Tom Knight (CS)

CaltechShuki Bruck (CS)

Sandia NLSteve Plimpton (P, CS)Danny Rintoul (P, CS)

In search of a Gpa1-specific inhibitor

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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

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Gtpase sequence conservation in yeast

Gpa1 SpliceGpa1Gpa2

Sar1Arf3Cin4Arf2Arf1Arl1

*Arrows indicate GTP binding residues in Gpa1.

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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

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Gpa1 Contact Residues Conserved

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Gpa1 model based on 1CIP

Hinge

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Gpa1 models with/out cofactors

Gold = built with cofactorsAqua = built without cofactorsRMSD = 0.1079

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Gpa1 with bound GNP

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Initial screen• Screened ~500 molecules from Chembank library (thanks

Ilya)

• Used GTP, GDP, GNP, GTPS, ATP, ADP as “controls”

• Glide - standard speed/precision

• Docked into 2 Gpa1 (spliced) models based on 1CIP1) Built with Mg cofactor and GNP ligand2) Built without Mg cofactor and GNP ligand

• Docked into 1CIP

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Cofactors play critical role in ligand dock scores

Ligand +Cofactors -Cofactors

GTP 2 4

GNP 1 92

GDP 5 45

GTPS 417

ATP 3 8

ADP 9 54

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Gpa1 pocket built with/out cofactors

RMSD = 0.159

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GTP binding poses nearly identical

Mg

No cofactors

With cofactors

GTP binding poses in Gpa1 models built with/without cofactors

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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

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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