Greg Crowther

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Drug discovery for neglected tropical diseases

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

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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
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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.).
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TDRtargets.org allows users to zoom in on
protein targets of particular interest
Prioritization of drug targets
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and create genome-wide rankings of targets
based on the users own criteria.
Prioritization of drug targets
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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)
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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).
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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,
Alzheimers, diabetes inhibitors kill T. brucei
(PubMed ID 18644955) current work testing in
animal models
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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
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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).
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Melting temperature (Tm) reflects protein
stability
Identifying targets of cell-active compounds
A compound that targets a particular protein
should bind to it and stabilize it, shifting the
melting curve and Tm to the right.
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Preliminary validation of thermal melt approach
Identifying targets of cell-active compounds
DHODH inhibitors cause dose-dependent increases
in DHODHs Tm Negative controls HSP90
inhibitors dont change DHODHs Tm
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Thermal melt assays for target identification
Identifying targets of cell-active compounds
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
Limitations false positives ligands bind to
protein and raise its Tm but dont inhibit it
false negatives not all substrates increase Tm
not all inhibitors do either?
Crowther et al. (2009), J. Biomol. Screen 14 in
press.
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Enzyme activity assays for target identification
Identifying targets of cell-active compounds
Advantages direct readout of target
inhibition published info on Kms, 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
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Identifying targets of cell-active compounds
Examples of high-throughput enzyme activity assays
dUTPase (PF11_0282) Reaction dUTP gt dUMP
PPi Coupling reaction (Pyrophosphatase) PPi gt
2Pi Detect ?Pi via malachite green kit
(absorbance at 620 nm). Glycerol-3-Phosphate
Dehydrogenase (PFL0780w) Reaction
glycerol-3-phosphate NAD gt dihydroxyacetone
phosphate NADH Coupling reaction (Diaphorase)
resazurin NADH gt resorufin NAD Detect
?resorufin via fluorescence (excite at 560 nm,
emit at 590 nm). OMP Decarboxylase
(PF10_0225) Reaction OMP gt UMP CO2 Coupling
reaction (CMP Kinase) UMP ATP gt UDP
ADP Detect ?ATP via Kinase-Glo luminescence, or
detect ?ADP via fluorescence polarization. S-Aden
osylhomocysteine Hydrolase (PFE1050w) Reaction
S-adenosylhomocysteine gt homocysteine
adenosine Coupling reaction (Adenosine
Deaminase) adenosine gt inosine Detect
?homocysteine SH via ThioGlo fluorescence
(excite at 379 nm, emit at 513 nm).
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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
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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
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Questions? Comments? If were out of time, feel
free to send email to crowther_at_u.washington.edu
or talk to me tonight at dinner.