Novel compounds derived from AR-12 that demonstrate host-directed clearance of intracellular Salmonella enterica Serovar Typhimurium.

Salmonella infections, including typhoid and paratyphoid fevers, impose a substantial burden in low-income countries, contributing to significant morbidity and mortality associated with enteric and systemic infections. Developing effective treatments for Salmonella infections requires further consideration, especially due to widespread antibiotic resistance. Host-targeted therapies, such as AR-12, offer a promising solution to combat drug resistance. AR-12 has demonstrated broad-spectrum antimicrobial activity against various bacterial pathogens, including Salmonella enterica serovar Typhimurium, S. Typhi, Francisella tularensis, and F. novicida, as well as protozoan parasites and fungal pathogens. To expand its HDT potential against S. Typhimurium, we conducted a medicinal chemistry campaign using AR-12 as a scaffold with systematic optimization of various points of diversity and the pyrazole core to develop analogs informed by structure-activity relationship. This work led to the development of 81 AR-12 analogs. Primary screening identified 38 analogs that are both more potent and less cytotoxic than parent compound AR-12, while only three were less potent and more cytotoxic. Further, only seven compounds affected planktonic Salmonella growth below 20 μM, suggesting host-directed activity in most of the compounds. Twelve analogs were chosen for secondary screening in MDR S. Typhimurium. Compounds 372, 373, and 378 demonstrated remarkable selectivity, with values exceeding 1500 for both susceptible and MDR S. Typhimurium, compared to AR-12's selectivity of around 20. This approximately 100-fold improvement, coupled with improved potency against intracellular Salmonella, suggests these analogs have significantly greater host-directed activity than direct antibacterial effects. Proteomic analysis for the two most potent compounds, 341 and 370 revealed enrichment of vesicle-mediated transport proteins, specifically with respect to retrograde transport at the trans-Golgi-network and intra-Golgi traffic. These results suggest that the analogs reduce intracellular S. Typhimurium replication by disrupting its exploitation of the host cell's vesicle-mediated transport system.