Search

Infections due to Gram-negative bacteria are increasingly dangerous due to the spread of multi-drug resistant strains, emphasizing the urgent need for new antibiotics with alternative modes of action. We have previously identified a novel class of antibacterial agents, thioacetamide-triazoles, using an antifolate targeted screen and determined their mode of action which is dependent on activation by cysteine synthase A. Herein, we report a detailed examination of the anti- E. coli structure-activity relationship of the thioacetamide-triazoles. Analogs of the initial hit compounds were synthesized to study the contribution of the aryl, thioacetamide, and triazole sections. A clear structure-activity relationship was observed generating compounds with excellent inhibition values. Substitutions to the aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems. Substitutions to the central thioacetamide linker section were more nuanced; the introduction of a methyl branch to the thioacetamide linker substantially decreased antibacterial activity, but the isomeric propionamide and N -benzamide systems retained activity. Changes to the triazole portion of the molecule dramatically decreased the antibacterial activity, further indicating that 1,2,3-triazole is critical for potency. From these studies, we have identified new lead compounds with desirable in-vitro ADME properties and in-vivo pharmacokinetic properties.

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.