Exploiting a multiplex HTS assay to identify novel boron-based inhibitors of mycobacterial class II aminoacyl-tRNA synthetases - In 2020 alone, there were 10 million cases and 1.5 million deaths worldwide attributable to Mycobacterium tuberculosis (Mtb) infection. The emergence of strains resistant to isoniazid and rifampicin – the two most effective first-line drugs used for treatment – is of great concern. Therefore, there is a pressing need for discovery of novel inhibitory molecules that are effective against Mtb. To address this global health threat, our laboratories at the University of Central Florida, in collaboration with AN2Therapeutics (formerly Anacor Pharmaceuticals), aim to develop novel boron-containing antibiotics targeting Mtb. In recent years, boron chemistry has proven to be remarkably useful for generating novel lead compounds and commercial drugs for important diseases, especially in the antimicrobial and antiparasitic space. Aminoacyl-tRNA synthetases (aaRSs) are efficacious targets due to their essential role in protein synthesis. aaRSs form the aminoacyl-tRNA (aa-tRNA) used by ribosomes to decode mRNA. Pioneering work at AN2Therapeutics led to the development of a class of boron-containing compounds (BCCs) that inhibit the LeuRS from various pathogens. These compounds include the FDA-approved drug tavaborole, two clinically- relevant inhibitors Epetraborole and Ganfeborole (GSK656), and a candidate drug for African sleeping sickness, Acoziborole. These BCCs utilize a tRNA trapping mechanism, involving a BCC-tRNA adduct bound to the enzyme, which ultimately blocks LeuRS activity and protein synthesis. To overcome the limitations of traditional approaches, we have developed an innovative continuous multiplexed high-throughput screening assay to target up to four aaRSs simultaneously. This approach enables the development of multi-target drugs capable of inhibiting multiple enzymes. Such inhibitors hold great promise in circumventing emerging drug resistance phenotypes, a major challenge in tuberculosis (TB) treatment. Our specific aims include: Primary screening of the AN2Therapeutics BCC library using 1) a multi-aaRS high-throughput assay and 2) an Mtb whole-cell assay. This approach delves into the unexplored potential of class II aaRSs from Mtb as drug targets. Effectiveness of hits will be assessed against live Mtb, and hits will be counterscreened against human cytoplasmic and mitochondrial aaRSs to allow triage of compounds with insufficient species specificity. Microbiological and mechanistic characterization of HTS hits. Top hits will be prioritized based on their low cytotoxicity towards human cells and minimal resistance frequencies in Mtb. Sequencing analysis will help identify resistance mechanisms and confirm drug targets. The potency of hit compounds will be assessed in dormant and intramacrophage Mtb assays, reflecting the challenging conditions encountered during TB infection. Additionally, their activity against various Mycobacterium species and multidrug-resistant Mtb strains will be evaluated to ensure broad-spectrum effectiveness.
