Application of High-Throughput Methodology to Human Drug Targets Duncan E. McRee, Ph.D. President, ActiveSight

ActiveSight Background Founded in 2003 by Duncan McRee, Ron Swanson and Les Tari Funded by Rigaku Americas Corp. 14 Personnel -10 Humans and 4 Robots Mission: Advance medicine by providing a portfolio of validated drug discovery target proteins for structure-based drug design and providing these as a service to biotech and pharma.

Structural Genomics Progress PDB Fruit Stand

Chemical Genomics

Fragment Based Screening

Fragment Screening Small fragments are screened by X-ray Small compounds ~200 MW at 10 mM or higher Several compounds are soaked at once to increase efficiency Hit rates are typically 3-4%

Small Fragments Have a higher hit rate Have higher ligand efficiency Leave more chemical space Need a smaller library

Chemical Space 50 100 150 200 500 Molecular Weight H C N O S Cl Fl Br

= Free energy per heavy atom Ligand Efficiency Δg / #Heavy Atoms = Free energy per heavy atom Hopkins, Groom and Alex, Drug Disc. Today (2004) 19, 430-1 IC(50) = 1 mM, 8 heavy atoms LE = 0.5 IC(50) = 10 nM, 38 heavy atoms LE = .29 kcal/mol

Ligand Efficiency From Wenlock et al (2003), J Med Chem 46, 1250-6

Potency vs. Efficiency HTS Hit Kd = 10 uM LE = 0.1 Fragment Hit

Connecting Fragments K(A) = 1 mM K(B) = 1 mM K(AB)=K(A)K(B)L ≤ 1 uM

ActiveSight Fragment Library 420 compounds expert selected, checked for solubility and validated at ActiveSight - typical molecular weights 100-200Da - rigid, low complexity, single core

Example: targeting hsp90 Target the “closed” conformation Data collection soaking 2-30 minutes, 2-10 mM concentrations in house data sets ~2Å, 30min/dataset Structure Solution Python script

Hsp90 Screening Results Compound RA1014

Inhibitor built from adenine core PDB entry: 1UY6 JRNL AUTH L.WRIGHT,X.BARRIL,B.DYMOCK,L.SHERIDAN,A.SURGENOR, JRNL AUTH 2 M.BESWICK,M.DRYSDALE,A.COLLIER,A.MASSEY,N.DAVIES, JRNL AUTH 3 A.FINK,C.FROMONT,W.AHERNE,K.BOXALL,S.SHARP, JRNL AUTH 4 P.WORKMAN,R.HUBBARD JRNL TITL STRUCTURE-ACTIVITY RELATIONSHIPS IN PURINE-BASED JRNL TITL 2 INHIBITOR BINDING TO HSP90 ISOFORMS JRNL REF CHEM.BIOL. V. 11 775 2004 JRNL REFN ASTM CBOLE2 UK ISSN 1074-5521

Scripps Fragment Screening Collaborations AIDS Protease with Holly Heaslet, Bruce Torbett, John Elder and Dave Stout AIDS is an enormous epidemic – a global health crisis AIDS protease inhibitors are highly successful at slowing disease but resistant strains of virus always arising so that new drugs must be continuously developed We are developing an affinity grid of the active sight to facilitate drug design efforts

Scripps Fragment Screening Collaborations SARS Structural Genomics with Peter Kuhn and Ray Stevens A number of SARS proteins have been solved that have unknown function With fragment screening we hope to identify binding sites and inhibitors for functional studies

Automated Structure Solution

MIAutoStructure Automated system Python script MIFit GUI CommandLine Automated system or or Keyworded parameter file Python script Launch, analyze intermediate results, log External applications (CCP4, SHELX)

MIFit Ligand Density

For more information go to Acknowledgements Robin Rosenfeld Les Tari Ron Swanson Isaac Hoffman Dan Bensen John Badger Paul Collins Bradley Smith For more information go to http://www.rigaku.com and http://www.active-sight.com http://www.molecularimages.com