New High-Throughput Fluorimetric Assay for Discovering Inhibitors of UDP- N -Acetylmuramyl-l-Alanine: d-Glutamate (MurD) Ligase

Abstract

A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5′-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest™ assay kit from DiscoveRx. The novel assay has been validated by obtaining K _M,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC₅₀ value of 105 µM, was discovered. (Journal of Biomolecular Screening 2009:412-418)

Keywords

Mur ligases MurD inhibitors high-throughput screening fluorimetric assay cell wall biosynthesis

References

van Heijenoort J. : Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep 2001 ;18:503-519.

Green DW : The bacterial cell wall as a source of antibacterial targets. Expert Opin Ther Targets 2002;6:1-19.

Bertrand JA , Auger G. , Martin L. , Fanchon E. , Blanot D. , Le Beller D. , et al: Determination of the MurD mechanism through crystallographic analysis of enzyme complexes. J Mol Biol 1999 ;289:579-590.

Bouhss A. , Dementin S. , van Heijenoort J. , Parquet C. , Blanot D. : MurC and MurD synthetases of peptidoglycan biosynthesis: borohydride trapping of acyl-phosphate intermediates. Methods Enzymol 2002;354: 189-196.

Emanuele JJ Jr , Jin H. , Yanchunas J. Jr , Villafranca JJ : Evaluation of the kinetic mechanism of Escherichia coli uridine diphosphate-Nacetylmuramate:L-alanine ligase. Biochemistry 1997;36:7264-7271.

Falk PJ , Ervin KM , Volk KS , Ho HT : Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N-acetylmuramate:L-alanine ligase-catalyzed reaction. Biochemistry 1996 ;35:1417-1422.

Liger D. , Masson A. , Blanot D. , van Heijenoort J. , Parquet C. : Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli. Microb Drug Resist 1996;2:25-27.

Vaganay S. , Tanner ME , van Heijenoort J. , Blanot D. : Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli. Microb Drug Resist 1996;2:51-54.

Bouhss A. , Dementin S. , van Heijenoort J. , Parquet C. , Blanot D. : Formation of adenosine 5′-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli. FEBS Lett 1999;453:15-19.

10.

Kotnik M. , Humljan J. , Contreras-Martel C. , Oblak M. , Kristan K. , Herve M. , et al: Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase. J Mol Biol 2007;370:107-115.

11.

Walsh AW , Falk PJ , Thanassi J. , Discotto L. , Pucci MJ , Ho HT : Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria. J Bacteriol 1999 ;181:5395-5401.

12.

Bratkovič T. , Lunder M. , Urleb U. , Štrukelj B. : Peptide inhibitors of MurD and MurE, essential enzymes of bacterial cell wall biosynthesis . J Basic Microbiol 2008;48:202-206.

13.

Frlan R. , Kovač A. , Blanot D. , Gobec S. , Pečar S. , Obreza A. : Design and synthesis of novel N-benzylidensulfonohydrazide inhibitors of MurC and MurD as potential antibacterial agents. Molecules 2008;13:11-30.

14.

Deng G. , Gu RF , Marmor S. , Fisher SL , Jahic H. , Sanyal G. : Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis. J Pharm Biomed Anal 2003;35:817-828.

15.

Kiianitsa K. , Solinger JA , Heyer WD : NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal Biochem 2003;321:266-271.

16.

Charter NW , Kauffman L. , Singh R. , Eglen RM : A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5′-diphosphate accumulation. J Biomol Screen 2006;11:390-399.

17.

Humljan J. , Starčević S. , Štefanič Anderluh P. , Kocjan D. , Jenko B. , Urleb U. : Optimization of UDP-N-acetylmuramic acid synthesis. Pharmazie 2008;63:102-106.

18.

Reddy SG , Waddell ST , Kuo DW , Wong KK , Pompliano DL : Preparative enzymatic synthesis and characterization of the cytoplasmic intermediates of murein biosynthesis. J Am Chem Soc 1999 ;121:1175-1178.

19.

Humljan J. , Kotnik M. , Contreras-Martel C. , Blanot D. , Urleb U. , Dessen A. , et al: Novel naphthalene N-sulfonyl-D-glutamic acid derivatives as inhibitors of MurD, a key peptidoglycan biosynthesis enzyme. J Med Chem 2008;51:7486-7494.

20.

Bouhss A. , Dementin S. , Parquet C. , Mengin-Lecreulx D. , Bertrand JA , Le Beller D. , et al: Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine:D-glutamate ligase (MurD). Biochemistry 1999;38:12240-12247.

21.

McGovern SL , Caselli E. , Grigorieff N. , Shoichet BK : A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening . J Med Chem 2002;45:1712-1722.

22.

McGovern SL , Helfand BT , Feng B. , Shoichet BK : A specific mechanism of nonspecific inhibition . J Med Chem 2003;46:4265-4272.

23.

Ryan AJ , Gray NM , Lowe PN , Chung CW : Effect of detergent on "promiscuous" inhibitors. J Med Chem 2003;46:3448-3451.

24.

Zhang J-H. , Chung TDY , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

25.

Dementin S. , Bouhss A. , Auger G. , Parquet C. , Mengin-Lecreulx D. , Dideberg O. , et al: Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. Eur J Biochem 2001;268: 5800-5807.

26.

Pratviel-Sosa F. , Mengin-Lecreulx D. , van Heijenoort J. : Over-production, purification and properties of the uridine diphosphate N-acetylmuramoylL-alanine:D-glutamate ligase from Escherichia coli. Eur J Biochem 1991;202:1169-1176.

27.

Copeland RA : Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists. Hoboken, NJ: John Wiley, 2005.

28.

Anderson MS , Eveland SS , Onishi HR , Pompliano DL : Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion. Biochemistry 1996;35:16264-16269.

29.

Emanuele JJ , Jin H. , Jacobson BL , Chang CY , Einspahr HM , Villafranca JJ : Kinetic and crystallographic studies of Escherichia coli UDP-Nacetylmuramate:L-alanine ligase. Protein Sci 1996;5:2566-2574.

30.

Ehmann DE , Demeritt JE , Hull KG , Fisher SL : Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-Lalanine ligase. Biochim Biophys Acta 2004;1698:167-174.

31.

Kotnik M. , Stefanic Anderluh P. , Prezelj A. : Development of novel inhibitors targeting intracellular steps of peptidoglycan biosynthesis. Curr Pharm Des 2007;13:2283-2309.

32.

Payne DJ , Gwynn MN , Holmes DJ , Pompliano DL : Drugs for bad bugs: confronting the challenges of antibacterial discovery . Nat Rev Drug Discov 2007;6:29-40.