Increasing rates of
drug-resistant Gram-negative (GN)
infections, combined with a lack of new GN-effective
antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial
therapies. Therefore, we sought to identify novel
antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N-(phenyl)
thioacetamide-linked 1,2,3-triazoles (TAT), that target
cysteine synthase A (CysK), an
enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to
folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN
antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-
l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these
antimetabolites impose substantial disruption of key metabolic networks beyond
cysteine biosynthesis. This study highlights the potential of
antimetabolite drug discovery as a promising approach to the discovery of novel GN
antibiotics and the pharmacological promise of TAT CysK probes.