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

Salmonellosis, including typhoid and paratyphoid fever, causes significant diarrheal disease worldwide underscoring the urgent need for effective treatments in the face of rising 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. Our research has developed AR-12 analogs for pre-clinical development, focusing on enhancing potency and decreasing cytotoxicity. This involved systematic optimization of various points of diversity and the pyrazole core, resulting in significant improvements in combating resistant intracellular S. Typhimurium. Primary screening of 81 AR-12 analogs assessed changes in intracellular S. Typhimurium burden (IC50), host cell viability (LC50), and direct effects on planktonic S. Typhimurium (MIC50), calculating selectivity (LC50/IC50) to determine host-directed potency versus cytotoxicity. Of the full library, only seven affected planktonic Salmonella growth below 20 {micro}M, suggesting host-directed activity in most of the compounds. Further, 38 analogs were found to be both more potent and less cytotoxic than parent compound AR-12, while only three were less potent and more cytotoxic. 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-12s 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 cells vesicle-mediated transport system.