Discovery and characterization of the antimetabolite action of thioacetamide-linked 1,2,3-triazoles as disruptors of cysteine biosynthesis in Gram-negative bacteria

Increasing rates of drug-resistant Gram-negative (GN) infections combined with a lack of new GN-effective antibiotic classes is driving the need for the discovery of new agents. As bacterial metabolism represents an underutilized mechanism targeted in current antimicrobial therapies, we sought to identify novel antimetabolites and explore the specific impacts of these inhibitors on key pathways in bacterial metabolism. This study describes the successful application of this approach to discover 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 revealed 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. Mid-log preparations of E. coli K12 MG1655 were treated with an antimicrobial thioacetamide-linked 1,2,3-triazole inhibitor to investigate the associated transcriptional response. The testing was performed in two M9-based medias with either magnesium sulfate or taurine representing the sulfur source. The experiment was performed in triplicate.