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Journal of Biomolecular Screening
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Development of a Coupled VanA/VanX Assay: Screening for Inhibitors of Glycopeptide Resistance

Steven D. Pratt

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Xiaoling Xuei

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Alexander C. Mackinnon

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Angela M. Nilius

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Dena M. Hensey-Rudloff

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Ping Zhong

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Leonard Katz

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064

Resistance in Enterococcus faecium to the glycopeptide antibiotics vancomycin and teicoplanin is encoded by five genes: vanR, vanS, vanH, vanA, and vanX.1 The mechanism of resistance involves replacement of the dipeptide D-Ala-D-Ala, destined for the peptidoglycan layer with the depsipeptide D-Ala-D-lactate. This alteration lowers the binding affinity of vancomycin for the bacterial cell wall by a factor of 1000. The functions of VanA and VanX are the ligation of D-Ala and D-lactate, and the hydrolysis of D-Ala-D-Ala, respectively. We report here the overexpression of both genes as well as the D-Ala-D-Ala ligase (Ddl) from Enterococcus faecium, development of a coupled assay and several inhibitors obtained by high-throughput screening (HTS). All genes were expressed in E. coli by translational coupling to kdsB, the CMP-KDO synthetase gene, under control of a modified lac promoter. The coupled VanA/VanX assay employs colorimetric detection of inorganic phosphate (Pi) released in the VanA ligation reaction, with the VanX dipeptidase activity providing the D-Ala substrate for VanA. A secondary VanX assay uses cadmium-ninhydrin calorimetric detection of free amino acid released by the dipeptidase activity of the enzyme on D-Ala-D-Ala. We have also developed an assay using Ddl ligase. Over 250,000 compounds have been screened to date using the coupled assay.

Journal of Biomolecular Screening, Vol. 2, No. 4, 241-247 (1997)
DOI: 10.1177/108705719700200409
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