is brugia malayi’s cofactor-independent phosphoglycerate mutase (ipgm) druggable?
DESCRIPTION
An oral presentation of research published in PLoS Neglected Tropical Diseases (http://www.ncbi.nlm.nih.gov/pubmed/24416464).TRANSCRIPT
Is Brugia malayi’s cofactor-independent phosphoglycerate mutase (iPGM) druggable?
Gregory J. Crowther1, Michael L. Booker2, Min He3, Ting Li3, Sylvine Raverdy4, Jacopo Novelli4, Panqing He1,
Natalie R. Grattan1, Amy M. Fife2, Robert H. Barker Jr2, Martin L. Kramer2, Wesley C.
Van Voorhis1, Clotilde K. S. Carlow4, Ming-Wei Wang3
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1Division of Allergy & Infectious Diseases, University of Washington, Seattle, WA, USA
2Genzyme Corporation, Waltham, MA, USA3The National Center for Drug Screening, Shanghai, China
4Division of Parasitology, New England Biolabs, Ipswich, MA, USA
Acknowledgments• Research effort
– Genzyme• Alexei Belenky• James Lillie
– UW• Steve Nakazawa Hewitt• David Leibly• Jack Mo• Christophe Verlinde
• Compounds– Novo Nordisk
• Funding– WHO/TDR– NIH (AI080625 and AI089441 to W.C.V.V.)
science.smith.edu/departments/Biology/SWILLIAM/
Lymphatic filariasis
• Caused by parasitic nematodes – Wuchereria bancrofti– Brugia malayi– Brugia timori
• 120 million infections
Lymphatic filariasis
• Caused by parasitic nematodes – Wuchereria bancrofti– Brugia malayi– Brugia timori
• 120 million infections• Adult worms cause most disfigurement and are
less susceptible to existing drugs (diethylcarbamazine, ivermectin, albendazole)
biocadmin.otago.ac.nz
Cofactor-independent phosphoglycerate mutase (iPGM)
• Part of glycolysis and gluconeogenesis
Cofactor-independent phosphoglycerate mutase (iPGM)
• Part of glycolysis and gluconeogenesis• Doesn’t require 2,3-bisphosphoglycerate• drug target for lymphatic filariasis?
– RNAi in C. elegans: severe phenotype
Cofactor-independent phosphoglycerate mutase (iPGM)
• Part of glycolysis and gluconeogenesis• Doesn’t require 2,3-bisphosphoglycerate• drug target for lymphatic filariasis?
– RNAi in C. elegans: severe phenotype – Distinct from host (mammals have dPGM)– B. malayi and C. elegans enzymes available for HTS– Druggability???
• Active site• Allosteric sites
HTS of iPGM at 2 sitesGenzyme (Boston) NCDS (Shanghai)
Compounds tested 220,000 160,000
Compound source(s) Preferred commercial vendors Novo Nordisk
Emphasis of compound library
druglikeness/leadlikeness (Rule of 5, Rule of 3, similarity to existing drugs), heterocycles, natural product analogs
heterocycles, lactams, sulfonates, sulfonamides, amines, 2° amides, natural product-derived compounds
1° screen enzyme C. elegans iPGM B. malayi iPGM
HTS assay strategy
Read absorbance at 340 nm in 384-well plates
Genzyme HTS/follow-up
Selective inhibitors of B. malayi iPGM
Compound ID StructureB. malayi iPGM, IC50
H. sapiens (or *P. falciparum) dPGM, IC50
C. elegans iPGM, IC50
C. elegans larvae, LC50
Genzyme-1 23.8 µM >30 µM 6.6 µM >25 µM
Genzyme-2 10.4 µM >30 µM 6.7 µM >25 µM
Genzyme-3 22.5 µM >30 µM 6.1 µM >25 µM
NCDS-1 38.3 µM >200 µM* N.D. N.D.
3
O O
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F F
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F FO
Why were there so few hits?
• problems with assay performance?– Z’-factors > 0.5
Why were there so few hits?
• problems with assay performance?– Z’-factors > 0.5
• problems with compound libraries?– 2 sites; both found good hits in other screens
Why were there so few hits?
• problems with assay performance?– Z’-factors > 0.5
• problems with compound libraries?– 2 sites; both found good hits in other screens
• problems with enzyme stocks?– 2 distinct stocks (C. elegans, B. malayi)– Reasonable MW, specific activity, Km
• low druggability of iPGM?
Non-druggable active site?• Bacillus stearothermophilus iPGM structure:
– 2-PG, 3-PG interact only with (9) hydrophilic residues– small, buried site + peptide “gate” = limited access
Jedrzejas et al., EMBO J 2000
What about allosteric sites?
• Advantage: drugs don’t need to outcompete substrate
• Precedents among infectious disease targets– HIV integrase & reverse transcriptase – hepatitis C virus NS5B polymerase– Bacillus anthracis edema factor
• Precedent among helminth targets: protein overactivation!– ivermectin increases opening of Glu-gated Cl
channels• Hard to predict
Conclusion• B. malayi iPGM has low druggability
– T. brucei iPGM may not be druggable either•“Druggability paradox” of target-based drug development
• Proteins are unsuitable for resource-intensive HTS unless considered druggable…
• …yet druggability is difficult to predict without HTS data.
Johnleonard.com; Wikipedia
Solutions to the druggability paradox?
• 1. Don’t be a chicken – just screen anyway! – high-risk (e.g., P. falciparum PK7 and MAPK2)
• 2. More critical evaluation of structures for druggability– Jedrzejas 2000: “The metabolic importance of iPGMs for some
bacteria, in particular Gram-positive bacteria, and the apparent absence of iPGMs in vertebrates make this class of enzyme an ideal target for novel antibacterial drugs.”
• 3. Let compounds tell you what the druggable targets are– a. look at precedents from other species
• some tRNA synthetases are druggable in bacteria & are now being targeted in parasites
• engineering of human kinases suggested druggability of parasite CDPKs
– b. pathway-based screening, then ID targets later– c. phenotypic screening, then ID targets later
“Worms in All the People”
• by The Anastomoses (1st-year UW med students)• to the tune of “Eleanor Rigby” (The Beatles)
youtube.com/watch?v=rYRFXswqIZo