Bacterial RNA polymerase (RNAP) is an established target for broad-spectrum antibacterial therapy, including in Mycobacterium tuberculosis (Mtb). Rifampicin (RIF) is a natural product inhibitor of RNAP and a cornerstone component of the WHO-recommended, first-line, short-course, 6-month, standard-of-care (SoC) TB regimen of RIF, isoniazid, pyrazinamide, and ethambutol (RHZE). RIF is facing growing drug resistance development in the clinic, including multiple-drug resistance (MDR-TB), defined as tuberculosis caused by an organism that is resistant to both RIF and isoniazid. RNAP is essential for bacterial growth, making its inhibition a lethal event, including under non-replicating conditions, which is also the most likely reason that RIF is the most sterilizing agent in the SoC regimen that is credited for reducing the duration of therapy from 18-24 down to 9-12 months. This is also the reason that therapy for MDR-TB, where RIF is obsolete, requires treatment durations of 18-24 months, with less efficacious, and poorly tolerated drugs and often with inferior cure rates. Since RIF is key in the short-course, 6-month duration of the modern first-line regimen for drug-susceptible TB, loss of RIF susceptibility, as in MDR-TB, mandates the extended therapy of 18 or more months with second-line drugs and that is associated with poorer outcomes. Restoring even a portion of the treatment-shortening capacity of RIF could dramatically shorten the duration of MDR/XDR-TB treatment, reduce the selection of additional second- line resistance and greatly improve outcomes. There is therefore an urgent need for novel anti-TB agents that inhibit Mtb RNAP and produce the same therapeutic efficacy as RIF, but with novel binding sites on Mtb RNAP that are distinct from the RIF binding site to avoid cross-resistance to allow efficacy against MDR/XDR-TB. We have identified a series of small molecules that are non-rifamycin, non-RIF cross-resistant, RNAP inhibitors that bind at a non-overlapping site with RIF on Mtb RNAP. The proposed project in collaboration with Cores A, B, and C will deliver a safe drug development candidate with potent activity against both replicating and non- replicating Mtb. The resulting candidate will have demonstrated sterilizing efficacy within novel 3- and 4-drug, proteostasis-disrupting combinations that produce relapse-free cure in mouse Mtb infection models with durations shorter than the SoC regimen, including against MDR/XDR-TB, with the propensity to suppress resistance development. The other drug candidates in the combination will emerge from accompanying Projects 1 and 2 (modulators of ClpC1 and Clp P1/P2), and a safer oxazolidinone currently undergoing IND- enabling toxicity studies (Ribosome inhibitor) as described in Project 4.
