Figure 1.
Substrate and selected inhibitors of alanine racemase.
(A) alanine, (B) D-cycloserine, (C) o-carbamyl-D-serine, (D) L-alanine phosphonic acid, (E) fluoro- or chloro-vinyl glycine, and (F) fluoro-alanine.
Figure 2.
HTS assays for screening alanine racemase-specific inhibitors.
(A) Coupled alanine racemase assay. (B) L-alanine dehydrogenase coupling enzyme assay.
Figure 3.
Kinetics of alanine racemase and L-alanine dehydrogenase activities in HTS format.
(A) When converted to 384-well plate format, the alanine racemase reaction gives a linear increase in fluorescence over a 40-minute period (⧫) as compared to the background control without substrate (▴). (B) The L-alanine dehydrogenase reaction gives a linear increase in fluorescence over a 20-minute period (▪) as compared to the background control without substrate (⧫).
Figure 4.
(A) Results of positive and negative controls plotted for 367 representative wells of the alanine racemase assay. The positive control represents 100% inhibition (wells 1–176) and the negative control represents 0% inhibition in the presence of 1% DMSO (wells 177–367). (B) HTS data for one of the two replicates for the entire 53,000-compound screen, obtained from 138 384-well plates. The line indicates the 30% inhibition cut-off for hit selection. A similar distribution of positive and negative controls results were obtained for the coupling enzyme assay (data not shown). (C) Distribution of the 267 hits, with the hits categorized into 7 groups with respect to percent inhibition.
Table 1.
Chemical properties and activities of new alanine racemase inhibitors and cycloserine*.
Figure 5.
Electrospray ionization mass spectrometry analysis of enzyme-inhibitor interaction.
Alanine racemase (4 µM) and inhibitors or substrate (1 mM) mixtures were analyzed by ESMS. Arrowheads indicate the peaks corresponding to monomeric alanine racemase.