Abstract
Tuberculosis is the leading infectious killer worldwide, with two billion infections and 1.25 million annual deaths. Antibiotic resistance drives prolonged, failure-prone treatments, underscoring the need for drugs with new mechanisms. Mycobacterial growth signaling pathways are promising targets. The Mycobacterium tuberculosis kinase PknB and phosphatase PstP are essential for growth and virulence. PknB inhibition sensitizes mycobacteria to β-lactams, and we hypothesized that concurrent PstP inhibition would enhance this effect. High-throughput screening identified 126 biochemical PstP inhibitors, four of which showed appreciable growth inhibition. These inhibitors acted additively with PknB inhibitors and displayed enhanced additive effects with β-lactams. PstP inhibition alone was bacteriostatic but antagonized β-lactam activity, consistent with on-target activity. PstP inhibition was additive with kinase inhibition, and inhibition of PstP and PknB with the addition of a β-lactam showed enhanced activity, with some treatment combinations even becoming bactericidal. PstP inhibitors showed low cytotoxicity, low apoptotic activity, and minimal host-cell phosphoprotein effects. Combinatorial treatments have IC(90) values below individual toxicities, suggesting that effective concentrations of these hits with other inhibitors could be effective at therapeutically relevant concentrations, suggesting high potential for further development and optimization. These represent the first PstP inhibitors with microbiologic activity and demonstrate additive inhibition between β-lactams and kinase/phosphatase targeting. Further development of compounds targeting this pathway may produce an effective tripartite therapy.