Greg Crowther & Wes Van Voorhis Department of Medicine University of Washington

1. Drug discovery for neglected tropical diseases Greg Crowther & Wes Van Voorhis Department of Medicine University of Washington

2. Protein-based (target-based) projects 1. Prioritization of drug targets • TDRtargets.org 2. Structural genomics of pathogen proteins • MSGPP.org • SSGCID.org 3. “Piggy-back” approach to target-based drug design • protein farnesyltransferase • glycogen synthase kinase 4. Identifying targets of cell-active compounds • thermal melt assays • enzyme activity assays PDB: 1tqx Fluorescence Temperature

3. Prioritization of drug targets TDRtargets.org is a database of protein data relevant to drug discovery research. Other team leaders: Fernán Agüero (U. of San Martin), Matt Berriman (Sanger Institute), Stuart Ralph (U. of Melbourne), David Roos (U. of Pennsylvania), Sam Sia (Columbia U.).

4. TDRtargets.org allows users to “zoom in” on protein targets of particular interest … Prioritization of drug targets

5. … and create genome-wide rankings of targets based on the users’ own criteria. Prioritization of drug targets

6. Structural genomics of pathogen proteins Medical Structural Genomics of Pathogenic Protozoa (MSGPP.org) • selection of potential drug targets • expression, crystallization, 3D structure determination, ligand binding • organisms: Plasmodium, T. brucei, T. cruzi, Leishmania, T. gondii, E. histolytica, Giardia, Cryptosporidium • collaborators: Fred Buckner, Erkang Fan, Wim Hol, Ethan Merritt, Christophe Verlinde (all UW) Seattle Structural Genomics Center for Infectious Disease (SSGCID.org) • like MSGPP, but focused on biodefense-related pathogens and (re-)emerging diseases • other team leaders: Peter Myler (SBRI), Lance Stewart (deCODE), Gabriele Varani (UW), Garry Buchko (Battelle)

7. us Big Pharma Image: NASA / Tom Tschida “Piggy-back” approach to target-based drug design Drug development is difficult and expensive when starting from scratch. Look for promising drug targets where a lot of development has already been performed. See if the existing drugs show promise against “our” diseases, then piggy-back onto existing efforts. Collaborators: Fred Buckner, Mike Gelb, K.K. Ojo, Christophe Verlinde (all UW).

8. “Piggy-back” approach to target-based drug design • Protein Farnesyltransferase • • adds farnesyl (C15H25) groups to proteins (important for localization, etc.) • • target for oncology -- in Phase III trials • • inhibitors cure rodent malaria (PubMed ID: 17606674) • • current work: optimizing pharmacokinetics • Glycogen Synthase Kinase • • phosphorylates glycogen synthase and signaling-related proteins • • target for mania, Alzheimer’s, diabetes • • inhibitors kill T. brucei (PubMed ID: 18644955) • • current work: testing in animal models

9. Identifying targets of cell-active compounds • Thousands of antimalaria compounds have been identified in screens of chemical libraries. • Their subcellular targets are unknown, making optimization difficult. How do you improve activity against the parasite without hurting the host? • Try to identify targets of some of compounds in order to facilitate optimization. Our (high-throughput) approaches: thermal melts and enzyme activity assays. • Collaborators: Roger Wiegand et al. (Broad Institute); Kip Guy (St. Jude); Kelli Kuhen, Richard Glynne, Achim Brinker et al. (Genomics Institute of Novartis Research Foundation). Images: trampledunderfoot.co.uk; tulane.edu/~wiser; clipartof.com

10. Temperature Identifying targets of cell-active compounds Thermal melt: Heat protein, watch it unfold. Solvent-accessible hydrophobic surface area (measured with fluorescent dye) Adaptation of a figure by Martin C. Stumpe and Helmut Grubmuller (www.mpibpc.mpg.de).

11. less stable (no ligand) more stable (with ligand) Fluorescence Temperature DTm Identifying targets of cell-active compounds Melting temperature (Tm) reflects protein stability A compound that targets a particular protein should bind to it and stabilize it, shifting the melting curve and Tm to the right.

12. Identifying targets of cell-active compounds Preliminary validation of thermal melt approach • DHODH inhibitors cause dose-dependent increases in DHODH’s Tm • Negative controls: HSP90 inhibitors don’t change DHODH’s Tm

13. Identifying targets of cell-active compounds Thermal melt assays for target identification Limitations: • false positives ligands bind to protein and raise its Tm but don’t inhibit it • false negatives not all substrates increase Tm; not all inhibitors do either? Advantages: • can be applied to most Plasmodium proteins* • a standard buffer (100 mM HEPES, 150 mM KCl, pH 7.5) works well for many proteins* *Crowther et al. (2009), J. Biomol. Screen14: in press.

14. Identifying targets of cell-active compounds Enzyme activity assays for target identification Advantages: • direct readout of target inhibition • published info on Km’s,optimal buffers, etc. is available for many enzymes Limitations: • useless for noncatalytic proteins • radioactivity, absorbance at UV wavelengths, HPLC, etc. are inappropriate for high-throughput screening • substrates may not be available • each enzyme is different

15. Identifying targets of cell-active compounds Examples of high-throughput enzyme activity assays dUTPase (PF11_0282) Reaction: dUTP => dUMP + PPi Coupling reaction (Pyrophosphatase): PPi => 2Pi Detect ↑Pi via malachite green kit (absorbance at 620 nm). Glycerol-3-Phosphate Dehydrogenase (PFL0780w) Reaction: glycerol-3-phosphate + NAD+ => dihydroxyacetone phosphate + NADH Coupling reaction (Diaphorase): resazurin + NADH => resorufin + NAD+ Detect ↑resorufin via fluorescence (excite at 560 nm, emit at 590 nm). OMP Decarboxylase (PF10_0225) Reaction: OMP => UMP + CO2 Coupling reaction (CMP Kinase): UMP + ATP => UDP + ADP Detect ↓ATP via Kinase-Glo luminescence, or detect ↑ADP via fluorescence polarization. S-Adenosylhomocysteine Hydrolase (PFE1050w) Reaction: S-adenosylhomocysteine => homocysteine + adenosine Coupling reaction (Adenosine Deaminase): adenosine => inosine Detect ↑homocysteine –SH via ThioGlo fluorescence (excite at 379 nm, emit at 513 nm).

16. Identifying targets of cell-active compounds Thermal melt and enzyme activity assays in the context of drug discovery Screen for protein-compound associations: thermal melts, enzyme activity assays Anti-parasitic compounds: inhibit P. falciparum + promising chemical properties High-Throughput Screen: validated proteins that need new scaffolds Test whether the protein is a target of the compound in parasites: a) substrate buildup? b) select resistance = mutations in target? c) overexpress in Plasmodium = increase in ED50? Hit optimization: directed by target

17. Summary • Target-based drug discovery has not yet led to many new drugs for neglected tropical diseases. • Nevertheless there are reasons for optimism. - New genomic/bioinformatic data (e.g., via TDRtargets.org): more possible protein targets better prioritization of targets - New biochemical methods (e.g., thermal melts): more “screenable” proteins - New 3D protein structures (e.g., via MSGPP and SSGCID): more structure-based drug design - New private-sector involvement (e.g., Novartis): better compound libraries more screening horsepower more piggy-backing opportunities

18. Questions? Comments? If we’re out of time, feel free to send email to crowther@u.washington.edu or talk to me tonight at dinner.