Biochemical and bioinformatic investigations of potential drug targets in Plasmodium and other pathogens Gregory J. Crowther, Ph.D. Acting Instructor Division of Allergy & Infectious Diseases Department of Medicine University of Washington images: grand-illusions.com; Nucleus Medical Art,
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Biochemical and bioinformatic investigations of potential drug targets in Plasmodium and other pathogens
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Biochemical and bioinformatic investigations of potential drug targets
in Plasmodium and other pathogens
Gregory J. Crowther, Ph.D.Acting Instructor
Division of Allergy & Infectious DiseasesDepartment of MedicineUniversity of Washington
images: grand-illusions.com; Nucleus Medical Art, Inc.
Wes’ World: target-based drug development
• Distinct from “cell-based” (or “phenotypic”) drug development, which proceeds without knowledge of compounds’ specific subcellular targets.
• Targeting a specific protein (often with a known 3D structure) should enable rational, rapid development of selective inhibitors.
• Sequencing/analysis of genomes should bring many new targets into play.
• Few examples so far of drugs created via target-based approaches:- Relenza (zanamivir) – for influenza virus- difluoromethylornithine – for T. brucei- protease inhibitors – for HIV
Nelfinavir in the active site of HIV-1 protease. Image from C.M. Henry, C&EN 79: 69, 2001.
Wes’ World: target-based drug development
Identify good drug targets (proteins).
Express and purify targets.
Solve targets’ 3D structures with bound compounds.Confirm that compounds kill
Identifying and prioritizing good drug targets: TDRtargets.org
• database to facilitate target-based drug development for neglected diseases (African sleeping sickness, Chagas disease, filariasis, Leishmaniasis, leprosy, malaria, schistosomiasis, toxoplasmosis, tuberculosis)
• funded by TDR division of World Health Organization (WHO)
• other group leaders:- Fernán Agüero (U. of San Martín)- Matt Berriman (Sanger Institute)- Stuart Ralph (U. of Melbourne)- David Roos (U. of Pennsylvania)
• 2 major uses:- find info on a protein of interest- scan a genome for proteins with desirable traits
TDRtargets.org: find info on a protein of interest
(Search page)
TDRtargets.org: find info on a protein of interest
(Example of a gene page)
TDRtargets.org: scan a genome for proteins with desirable traits
Example of a weighted search of the P. falciparum genome:
Criterion Weight
is a protein 1
is an enzyme 100
has associated PubMed publications 35
has a molecular weight < 100 kilodaltons 20
has no transmembrane domains 20
has a solved crystal structure 50
has a ModBase 3D model 30
lacks an ortholog in humans 25
has been genetically validated as a target 50
has an ortholog that is essential in ≥1 model organism 50
has a druggability index > 0.6 35
has a compound desirability index > 0.3 35
has a precedent for assayability 35
is present in both P. falciparum and P. vivax 25
TDRtargets.org: scan a genome for proteins with desirable traits
TDRtargets.org: final points
• Orthology is used to make inferences about incompletely studied proteins (D. Roos).
• Druggability is predicted from protein binding pockets and similarities to known targets (A. Hopkins, B. Al-Lazikani, J. Overington).
• Weighting allows many criteria to be used in searches without discarding proteins that lack some desired criteria.
• Users supply search criteria according to their own interests and expertise.
• Further information: F. Agüero et al., Nature Rev. Drug Discov. 7: 900, 2008.
• Future work: linking targets to compounds through informatics.
• published info on Km’s, optimal buffers, etc. is available for many enzymes
Limitations:
• radioactivity, absorbance at UV wavelengths, HPLC, etc. are impractical for high-throughput screening
• useless for noncatalytic proteins – and for enzymes whose substrates aren’t available or whose activity can’t be easily measured
Screening for compound-target associationsvia enzyme activity assays
Examples of high-throughput screening assays:
dUTPaseReaction: dUTP => dUMP + PPi
Coupling reaction (Pyrophosphatase): PPi => 2Pi
Detect ↑Pi via malachite green kit (absorbance at 620 nm).
OMP DecarboxylaseReaction: OMP => UMP + CO2
Coupling reaction (CMP Kinase): UMP + ATP => UDP + ADPDetect ↓ATP via Kinase-Glo luminescence, or detect ↑ADP via fluorescence polarization.
S-Adenosylhomocysteine HydrolaseReaction: S-adenosylhomocysteine => homocysteine + adenosineCoupling reaction (Adenosine Deaminase): adenosine => inosineDetect ↑homocysteine –SH via ThioGlo fluorescence (excite at 379 nm, emit at 513 nm).
Screening for compound-target associations via enzyme activity assays
MMV-funded collaboration between UW and Novartis (Kelli Kuhen, Richard Glynne et al.):
• 9 Plasmodium enzymes thought to be good drug targets were screened against ~5,500 anti-Plasmodium compounds. Goal: link some compounds to specific targets.
• Inhibitors were identified for 6 enzymes: adenylosuccinate synthase, choline kinase, guanylate kinase, N-myristoyltransferase, OMP decarboxylase, and S-adenosylhomocysteine hydrolase.
• Most inhibitors were less potent against enzymes (IC50) than against cells (EC50). Follow-up studies can show whether any of these compounds kill cells through their action on the enzymes studied. If not, they may still be useful as “tool compounds.”
• Manuscript in preparation….
An alternative screening method:thermal melting (heat protein, watch it unfold)
X axis: Temperature
Y axis:Solvent-accessible
hydrophobic surface area
(measured with fluorescent dye)
Adaptation of a figure by M.C. Stumpe and H. Grubmuller (www.mpibpc.mpg.de).
Thermal melt assays: melting temperature (Tm) reflects protein stability
Flu
ores
cenc
e
Temperature
less stable(no ligand)
more stable(with ligand)
Tm
A compound that binds to a particular protein will stabilize it, shifting the melting curve and Tm to the right.
Advantage:• “generic” – same readout for every protein
Limitation:• measures binding rather than inhibition per se
Thermal melt assays:application to screens of pathogen proteins
G.J. Crowther et al., J. Biomol. Screen. 14: 700, 2009:
• Tm could be measured for 58 of 61 Plasmodium proteins tested.
• A standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) works well for most proteins.
• Buffer optimization can reduce Tm variability in others, like phosphoethanolamine N-methyltransferase (top) and adenosine deaminase (bottom).
Thermal melt assays:application to screens of pathogen proteins
G.J. Crowther et al., Anal. Biochem. 399: 268, 2010:Melting curves predict the quality of enzymes like bovine xanthine oxidase (A) and E. histolytica cysteine protease 1 (B).
Thermal melt assays:application to screens of pathogen proteins
Collaborative project led by Kip Guy at St. Jude (Memphis):
• A screen of ~300,000 compounds revealed 172 new potent inhibitors of blood-stage P. falciparum (EC50 < 2 µM).
• UW contribution: these 172 compounds were screened against 61 Plasmodium proteins via thermal melt assays.
• Ligands (possible inhibitors) were identified for 7 proteins: 6-phosphogluconolactonase, 6-pyruvoyltetrahydropterin synthase, choline kinase, D-ribulose-5-phosphate 3-epimerase, glycogen synthase kinase, and thioredoxin.
• Paper will be published in Nature on May 20….
Thermal melt assays:application to screens of pathogen proteins
Conclusion
• Target-based drug discovery has not yet led to many new drugs for neglected 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 screening methods (e.g., thermal melt assays): ○ 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
Thank you . . . “Parasites! Excellent!”
. . . to my assay team:- Diana Chung- Panqing He- Kuzma Kovzun- Phil Rodenbough- Andrew Thomas
. . . to other inhabitants of Wes’ World:- Lynn Barrett (lab manager)- Fred Buckner’s lab- Mike Gelb (Dept. of Chemistry)- Wim Hol and MSGPP- Alberto Napuli’s expression/purification group
. . . to collaborating groups around the world:- Kip Guy (St. Jude)- Ray Hui (SGC, U. of Toronto)- Kelli Kuhen and Richard Glynne (Novartis)- TDR network (Fernán Agüero, Matt Berriman, Stuart Ralph, David Roos)- Roger Weigand and Dyann Wirth (Broad Institute)
. . . to funding agencies:- Medicines for Malaria Venture (MMV)- WHO/TDR