Cephem-Pyrazinoic Acid Conjugates: Circumventing Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. ß-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of ß-lactamases that destroy the electrophilic ß-lactam warhead. We have developed novel ß-lactam conjugates, which exploit this inherent ß-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the ß-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.

Synthesis and biological evaluation of orally active prodrugs and analogs of para-aminosalicylic acid (PAS).

Tuberculosis (TB) is one of the world's most deadly infectious diseases resulting in nearly 1.3 million deaths annually and infecting nearly one-quarter of the population. para-Aminosalicylic acid (PAS), an important second-line agent for treating drug-resistant Mycobacterium tuberculosis, has moderate bioavailability and rapid clearance that necessitate high daily doses of up to 12 g per day, which in turn causes severe gastrointestinal disturbances presumably by disruption of gut microbiota and host epithelial cells. We first synthesized a series of alkyl, acyloxy and alkyloxycarbonyloxyalkyl ester prodrugs to increase the oral bioavailability and thereby prevent intestinal accumulation as well as undesirable bioactivation by the gut microbiome to non-natural folate species that exhibit cytotoxicity. The pivoxyl prodrug of PAS was superior to all of the prodrugs examined and showed nearly quantitative absorption. While the conceptually simple prodrug approach improved the oral bioavailability of PAS, it did not address the intrinsic rapid clearance of PAS mediated by N-acetyltransferase-1 (NAT-1). Thus, we next modified the PAS scaffold to reduce NAT-1 catalyzed inactivation by introduction of groups to sterically block N-acetylation and fluorination of the aryl ring of PAS to attenuate N-acetylation by electronically deactivating the para-amino group. Among the mono-fluorinated analogs prepared, 5-fluoro-PAS, exhibited the best activity and an 11-fold decreased rate of inactivation by NAT-1 that translated to a 5-fold improved exposure as measured by area-under-the-curve (AUC) following oral dosing to CD-1 mice. The pivoxyl prodrug and fluorination at the 5-position of PAS address the primary limitations of PAS and have the potential to revitalize this second-line TB drug.

Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry.

Precision antimicrobials aim to kill pathogens without damaging commensal bacteria in the host, and thereby cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of a synthetic host defence peptide that targets a specific pathogen by mimicking key molecular features of the pathogen's channel-forming membrane proteins. By exploiting physical and structural vulnerabilities within the pathogen's cellular envelope, we designed a peptide sequence that undergoes instructed tryptophan-zippered assembly within the mycolic acid-rich outer membrane of Mycobacterium tuberculosis to specifically kill the pathogen without collateral toxicity towards lung commensal bacteria or host tissue. These mycomembrane-templated assemblies elicit rapid mycobactericidal activity and enhance the potency of antibiotics by improving their otherwise poor diffusion across the rigid M. tuberculosis envelope with respect to agents that exploit transmembrane protein channels for antimycobacterial activity. This biomimetic strategy may aid the design of other narrow-spectrum antimicrobial peptides.

Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis.

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

Investigation of ( S)-(-)-Acidomycin: A Selective Antimycobacterial Natural Product That Inhibits Biotin Synthase.

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of ( S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 µM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 µM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a Ki of approximately 1 µM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5'-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis.

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as, para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter, designated MetM, play crucial roles in this process. Disruption of metM by transposon insertion resulted in a ≥30-fold decrease in uptake of methionine in M. bovis BCG, indicating that metM is the major facilitator of methionine transport. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.