Greg crowther wes van voorhis department of medicine university of washington
Download
1 / 18

Greg Crowther & Wes Van Voorhis Department of Medicine University of Washington - PowerPoint PPT Presentation


  • 139 Views
  • Uploaded on

Drug discovery for neglected tropical diseases. Greg Crowther & Wes Van Voorhis Department of Medicine University of Washington. Protein-based (target-based) projects. 1. Prioritization of drug targets • TDRtargets.org 2. Structural genomics of pathogen proteins • MSGPP.org

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Greg Crowther & Wes Van Voorhis Department of Medicine University of Washington' - Albert_Lan


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Greg crowther wes van voorhis department of medicine university of washington

Drug discovery for neglected tropical diseases

Greg Crowther & Wes Van Voorhis

Department of Medicine

University of Washington


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


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.).


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

Prioritization of drug targets


… and create genome-wide rankings of targets targets of particular interest …

based on the users’ own criteria.

Prioritization of drug targets


Structural genomics of targets of particular interest …

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)


us targets of particular interest …

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).


“Piggy-back” approach to targets of particular interest …

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


Identifying targets of targets of particular interest …

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


Temperature targets of particular interest …

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).


Melting temperature t m reflects protein stability

less stable targets of particular interest …

(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.


Preliminary validation of thermal melt approach

Identifying targets of targets of particular interest …

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


Thermal melt assays for target identification

Identifying targets of targets of particular interest …

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.


Enzyme activity assays for target identification

Identifying targets of targets of particular interest …

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


Identifying targets of targets of particular interest …

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).


Identifying targets of targets of particular interest …

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


Summary targets of particular interest …

• 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


Questions? Comments? targets of particular interest …

If we’re out of time, feel free to send email to [email protected] or talk to me tonight at dinner.


ad