An oral presentation of research published in PLoS Neglected Tropical Diseases (http://www.ncbi.nlm.nih.gov/pubmed/24416464).

1. 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
1 2 3 4 5 6 7 8 9 10 11 12
A NO
B
C
D
E
F
G
H
NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO
NO
NO
NO NO
NO NO
NO
NO
NO NO
NO NO
NO
NO
NO NO
NO NO
NO
NO
NO NO
NO NO
NO
NO
NO NO
1Division of Allergy & Infectious Diseases, University of Washington, Seattle, WA, USA
2Genzyme Corporation, Waltham, MA, USA
3The National Center for Drug Screening, Shanghai, China
4Division of Parasitology, New England Biolabs, Ipswich, MA, USA

2. 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.)

3. Lymphatic filariasis
• Caused by parasitic nematodes
– Wuchereria bancrofti
– Brugia malayi
– Brugia timori
• 120 million infections
science.smith.edu/departments/Biology/SWILLIAM/

4. 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)

5. Cofactor-independent
phosphoglycerate mutase (iPGM)
• Part of glycolysis and gluconeogenesis
biocadmin.otago.ac.nz

6. 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

7. 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

8. HTS of iPGM at 2 sites
Genzyme (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

9. HTS assay strategy
Read absorbance
at 340 nm in 384-
well plates

10. Genzyme HTS/follow-up

11. Selective inhibitors of B. malayi iPGM
Compound ID Structure
B. malayi
iPGM, IC50
H. sapiens (or *P.
falciparum)
dPGM, IC50
C. elegans
iPGM, IC50
C. elegans
larvae, LC50
3
O O
O
C
H
Genzyme-1 23.8 μM >30 μM 6.6 μM >25 μM
N
S
O
N
+
Genzyme-2 10.4 μM >30 μM 6.7 μM >25 μM
S
H C
3
S
N
O
Genzyme-3 F F
22.5 μM >30 μM 6.1 μM >25 μM
F F
F
Br
F
NCDS-1 38.3 μM >200 μM* N.D. N.D.

12. Why were there so few hits?
• problems with assay performance?
– Z’-factors > 0.5

13. 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

14. 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?

15. 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

16. 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

17. 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

18. 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

19. “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