Abstract Mycobacterial infections, broadly including Mycobacterium tuberculosis (Mtb), and those caused by nontuberculous mycobacteria (NTM) like the fast-growing Mycobacterium abscessus (Mab), as well as the slow- growing Mycobacterium avium complex (MAC), represent some of the most clinically challenging and deadly infections of the 21st century. Treatments typically involve regimens of multiple antibiotics that are administered for lengthy periods of time, and poor outcomes are common. -Lactam antibiotics have not traditionally been a part of these treatment regimens, due to -lactamase mediated resistance, and because some classes of - lactams lack antimycobacterial activity. Significantly, for peptidoglycan crosslinking, mycobacterial species primarily use an alternate L,D-transpeptidase (Ldt) in place of the canonical D,D-transpeptidase (Ddt) (or penicillin binding protein, PBP), the latter of which is the primary target of typically administered -lactams. Ldts and Ddts are nonhomologous proteins which, in part, explains the apparent ineffectiveness of this drug class against mycobacterial species. This application involves design and synthesis of carbapenem antibiotics with substantial and unusual (atypical) structural alterations, in contrast to existing agents which differ only at the C2 position. Previously, we have demonstrated that these scaffold modifications can improve activity against Gram- negative pathogens, minimize efflux, and improve resistance to -lactamase-mediated hydrolysis. Preliminary studies for this project yielded an atypical carbapenem with superior potency compared to commercially available carbapenems against diverse Mtb and Mab clinical isolates. Unlike meropenem, this new atypical carbapenem maintained its potency against resistant mycobacterial pathogens in the absence of a combination -lactamase inhibitor, displaying up to 20-fold improvements over meropenem in activity against these resistant strains. Its excellent,broad spectrum antimycobacterial potency (MICs < 1 g/ml) was not diminished by carbapenemase producing mycobacterial species nor was activity further improved by combination with -lactamase inhibitors thus indicating -lactamase stability. Activity was also assessed against a diverse panel of nonmycobacterial species indicating the new antibiotic is beginning to demonstrate selectivity for the mycobacteria genus over other Gram-negative and Gram-positive species. We now plan to develop this initial discovery into an orally bioavailable clinically useful antimycobacterial agent and to further improve the antimycobacterial potency and specificity of this unique atypical carbapenem. Stability to carbapenemases will be an asset in an orally bioavailable agent since many carbapenemase inhibitors (e.g. avibactam) are not orally bioavailable. The scientific operations are guided by a highly qualified team including synthesis, microbiology, biochemistry, and structural biology. A thorough and rigorous array of analyses, including numerous in vitro and in vivo PK evaluations, will guide development.

Project Narrative The project involves the design, synthesis, and evaluation of new antibiotics to treat infections due mycobacterial pathogens, including both Mycobacterium tuberculosis as well as nontuberculous mycobacterial infections, such as Mycobacterium abscessus. These infections are extremely difficult to treat, requiring multidrug regimens that are administered over a lengthy period of time (months to years) often with poor clinical outcomes. We will develop newly discovered potent antimycobacterial agents with the objective of producing an orally bioavailable drug candidate with improved specificity for mycobacteria.