Antimicrobial Inhibitors of GlmU Acetyltransferase
amine extension of the pantothenate moiety of CoA and activity of these inhibitors was shown to be caused by inhi-
reaches to Ala-380 and Asn-377. The aromatic side chain on bition of GlmU acetyltransferase transferase. Although clin-
the sulfonamide is stabilized by -stacking with Trp-449. The ical drug candidates acting via GlmU are still a long time
importance of this interaction is illustrated by the W449G away, this work for the first time provides in vitro validation
mutant that confers resistance to compound 5 and by weaker of this enzyme activity as an antibacterial target.
activity against S. pneumoniae GlmU. S. pneumoniae GlmU
Acknowledgments—We thank the AstraZeneca R&D Boston clinical
activity could be measured only after the initial hit was
susceptibility group for MIC determinations and thank Rob Albert,
extended with larger groups in this region, thus providing addi-
Jason Breed, Amy Kutschke, Valerie Laganas, Stephania Livchak,
tional interactions which compensated for the absence of -
Kathy MacCormack, and Wei Yang for technical expertise with var-
interactions. Sequence analysis also suggested that S. aureus
ious parts of this study.
GlmU contains a shorter carboxyl-terminal end, lacking resi-
dues forming this part of the pocket (Fig. 6). This may explain
REFERENCES
why no activity was observed against S. aureus GlmU with this
scaffold.
1. Ho¨gberg, L. D., Heddini, A., and Cars, O. (2010) Trends Pharmacol. Sci.
31, 509–515
The compounds with improved biochemical potency dis-
played antimicrobial activity in a strain of H. influenzae
devoid of its main efflux pump. This activity was shown to be
consistent with inhibition of GlmU acetyltransferase in the
cell. Incorporation of N-acetylglucosamine is particularly
sensitive to compound 5, as can be expected when inhibiting
a key step in the synthesis of UDP-GlcNAc (Fig. 5). More
surprising was the increased sensitivity of acetate incorpo-
ration. We hypothesize this reflects inhibition of lipopoly-
saccharide synthesis. The LpxA protein, UDP-N-acetylglu-
cosamine acyltransferase, catalyzes the first step in lipid A
synthesis by transferring -hydroxymyristate from the acyl
transfer protein to UDP-GlcNAc (24). Inhibition of
UDP-GlcNAc synthesis could reduce turnover of the acyl-
acyl transfer protein pool and thus decrease incorporation of
acetic acid.
2. Bazan, J. A., and Martin, S. I. (2010) Drugs Today 46, 743–755
3. Silver, L. L. (2011) Clin. Microbiol. Rev. 24, 71–109
4. Mengin-Lecreulx, D., and van Heijenoort, J. (1994) J. Bacteriol. 176,
5788–5795
5. Mengin-Lecreulx, D., and van Heijenoort, J. (1993) J. Bacteriol. 175,
6150–6157
6. Mengin-Lecreulx, D., and van Heijenoort, J. (1996) J. Biol. Chem. 271,
32–39
7. Mochalkin, I., Lightle, S., Narasimhan, L., Bornemeier, D., Melnick, M.,
Vanderroest, S., and McDowell, L. (2008) Protein Sci. 17, 577–582
8. Pereira, M. P., Blanchard, J. E., Murphy, C., Roderick, S. L., and Brown,
E. D. (2009) Antimicrob. Agents Chemother. 53, 2306–2311
9. Sa´nchez, L., Pan, W., Vin˜as, M., and Nikaido, H. (1997) J. Bacteriol. 179,
6855–6857
10. Kreiswirth, B. N., Lo¨fdahl, S., Betley, M. J., O’Reilly, M., Schlievert, P. M.,
Bergdoll, M. S., and Novick, R. P. (1983) Nature 305, 709–712
11. Pioli, D., Hockney, R. C., Kara, B. V., and Bundell, K. R. (1999)
WO1999005297
12. Segel, I. H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equi-
librium and Steady-state Enzyme Systems, JohnWiley & Sons, Inc., New
York, NY
Antimicrobial activity against wild type strains of H. influ-
enzae and a strain of E. coli lacking its AcrB-TolC efflux
system is absent, suggesting poor permeation of the com-
pounds into the cell. This could be due to the polar nature of
the most potent compounds (logD7.4 of Ϫ0.5 and Ϫ0.3 for
compounds 4 and 5, respectively), arising from the carbox-
ylate moiety and resulting in poor membrane permeability.
Lowering the pH of the medium from 7.3 to 5.5 to increase
protonation and improve membrane permeability reduced
the MIC of compound 5 against E. coli tolC::Tn10 (data not
shown). This result suggests a path for improving the anti-
microbial activity of this series of GlmU acetyltransferase
inhibitors.
13. Clinical and Laboratory Standards Institute (2009) Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Ap-
proved Standard, 9th Ed., M07-A8, Vol. 29, no. 2. Clinical and Laboratory
Standards Institute, Wayne, PA
14. Saeed-Kothe, A., Yang, W., and Mills, S. D. (2004) Appl. Environ. Micro-
biol. 70, 4136–4143
15. Dalvit, C., Fogliatto, G., Stewart, A., Veronesi, M., and Stockman, B. (2001)
J. Biomol. NMR 21, 349–359
16. Scott, K. S., Keeler, J., Hwang, T. L., and Shaka, A. J. (1995) J. Am. Chem.
Soc. 117, 4199–4200
17. Buurman, E. T., Johnson, K. D., Kelly, R. K., and MacCormack, K. (2006)
Antimicrob. Agents Chemother. 50, 385–387
18. Hilliard, J. J., Goldschmidt, R. M., Licata, L., Baum, E. Z., and Bush, K.
(1999) Antimicrob. Agents Chemother. 43, 1693–1699
19. Gehring, A. M., Lees, W. J., Mindiola, D. J., Walsh, C. T., and Brown, E. D.
(1996) Biochemistry 35, 579–585
In summary, an earlier report (8) is confirmed here that
the GlmU acetyltransferase is “drug-able,” in that relatively
small, specific biochemical inhibitors can be found. Various
binding studies, including x-ray crystallography, comple-
mented enzymology studies that both guided iterative chem-
istry plans and explained the mostly Gram-negative spec-
trum of the biochemical activity. Compounds with single
digit nanomolar IC50s showed antimicrobial activity in
H. influenzae acrB::cat that could be measured in standard
20. Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and
Bourne, Y. (1999) EMBO J. 18, 4096–4107
21. Walker, J. E., Gay, N. J., Saraste, M., and Eberle, A. N. (1984) Biochem. J.
224, 799–815
22. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099–1108
23. Olsen, L. R., Vetting, M. W., and Roderick, S. L. (2007) Protein Sci. 16,
1230–1235
susceptibility assays (13). Importantly, this antimicrobial 24. Anderson, M. S., and Raetz, C. R. (1987) J. Biol. Chem. 262, 5159–5169
40742 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011