Optimization of Atypical Antimycobacterial Carbapenem Antibiotics

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. β-Lactam antibiotics have not traditionally been a part of these treatment regimens, due to β-lactamase mediated resistance, and because some classes of β- 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 (or penicillin binding protein, PBP), the latter of which is the primary target of typically administered β-lactams. Ldts and PBPs are nonhomologous proteins, which in part, explains the apparent ineffectiveness of this drug class against mycobacterial species. This application involves design and synthesis of β-lactam-derived antibiotics (carbapenem, penem, cephalosporin) with substantial and unusual (atypical) structural alterations, and their evaluation against Mtb and Mab, with the objective of improving activity against mycobacterial Ldts relative to their PBP counterparts and commensal flora. Synthetic methodology is in place to achieve the highly unusual antibiotic structural modifications under investigation. Our preliminary data indicate that atypical β-lactam modifications can improve antibiotic stability to β-lactamases and result in antibiotics which are superior in potency to those commercially available for treatment of mycobacterial infections. A class of atypically-modified cephalosporin with unusual activity against dormant Mtb has been identified. The objective of this project is to devise a β-lactam-derived agent more ideally suited to treat mycobacterial infections. Specifically, we anticipate the delivery of an orally bioavailable agent, with long plasma half-life, improved β-lactamase stability, and better potency and specificity for the mycobacterial pathogen than current commercial β-lactam agents. It is envisioned that these studies will narrow the spectrum of the antibiotic, rendering it more suitable for the long- term administration characteristic of antimycobacterial regimens. Complementary genetic and biochemical strategies will help clarify the poorly understood physiological roles of paralogous Ldts in cell wall biosynthesis and in vivo pathogenesis. We will use mass spectrometric analyses to define the target profile of atypical β- lactams and determine the role of Ldts (by gene knockouts and CRISPRi knockdowns) for survival under stress conditions encountered during infection. These data will guide our design of novel β-lactam compounds tailored for optimal specificity, potency, and in vivo efficacy.