A coupled spectrophotometric assay for L-cysteine:: 1-D-myo-inosityl 2-amino-2-deoxy-α-D-glucopyranoside ligase and its application for inhibitor screening

Most actinomycetes. including Mycobacterium tuberculosis, do not produce glutathione but make an alternative thiol, mycothiol, which has functions similar to those of glutathione. A key step in mycothiol biosynthesis is the ATP-dependent ligation of Cys to GlcN-Ins catalyzed by MshC to produce Cys-GlcN-Ins, AMP, and PPi. MshC is essential for growth of M. tuberculosis and is therefore a potential target for drugs directed against tuberculosis. A coupled-enzyme assay for MshC was developed using pyrophosphatase to convert pyrophosphate to phosphate and spectrophotometric detection of the latter via the phosphomolybdate complex with malachite green. The assay was readily adapted for use in a 96-well microtiter plate format. A secondary high-performance liquid chromatography assay measuring Cys-GlcN-Ins production was used to validate potential hits. Preliminary testing on a library of 2024 compounds predicted to inhibit ATP-dependent enzymes identified many promiscuous and pyrophosphatase inhibitors of MshC and a single validated inhibitor with IC50 similar to 100 mu M. (c) 2006 Elsevier Inc. All rights reserved.