Proton transfer activity of the reconstituted Mycobacterium tuberculosis MmpL3 is modulated by substrate mimics and inhibitors.

Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.

Transporters Involved in the Biogenesis and Functionalization of the Mycobacterial Cell Envelope.

The biology of mycobacteria is dominated by a complex cell envelope of unique composition and structure and of exceptionally low permeability. This cell envelope is the basis of many of the pathogenic features of mycobacteria and the site of susceptibility and resistance to many antibiotics and host defense mechanisms. This review is focused on the transporters that assemble and functionalize this complex structure. It highlights both the progress and the limits of our understanding of how (lipo)polysaccharides, (glyco)lipids, and other bacterial secretion products are translocated across the different layers of the cell envelope to their final extra-cytoplasmic location. It further describes some of the unique strategies evolved by mycobacteria to import nutrients and other products through this highly impermeable barrier.

Structural Determinants of Indole-2-carboxamides: Identification of Lead Acetamides with Pan Antimycobacterial Activity.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death in developing countries. Non-tuberculous mycobacteria (NTM) infections are rising and prey upon patients with structural lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. All mycobacterial infections require lengthy treatment regimens with undesirable side effects. Therefore, new antimycobacterial compounds with novel mechanisms of action are urgently needed. Published indole-2-carboxamides (IC) with suggested inhibition of the essential transporter MmpL3 showed good potency against whole-cell M.tb, yet had poor aqueous solubility. This project focused on retaining the required MmpL3 inhibitory pharmacophore and increasing the molecular heteroatom percentage by reducing lipophilic atoms. We evaluated pyrrole, mandelic acid, imidazole, and acetamide functional groups coupled to lipophilic head groups, where lead acetamide-based compounds maintained high potency against mycobacterial pathogens, had improved in vitro ADME profiles over their indole-2-carboxamide analogs, were non-cytotoxic, and were determined to be MmpL3 inhibitors.

Microbiological profile, preclinical pharmacokinetics and efficacy of CRS0393, a novel antimycobacterial agent targeting MmpL3.

The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 µg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.

The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria.

Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.

Direct Inhibition of MmpL3 by Novel Antitubercular Compounds.

MmpL3, an essential transporter involved in the export of mycolic acids, is the proposed target of a number of antimycobacterial inhibitors under development. Whether MmpL3 serves as the direct target of these compounds, however, has been called into question after the discovery that some of them dissipated the proton motive force from which MmpL transporters derive their energy. Using a combination of in vitro and whole-cell-based approaches, we here provide evidence that five structurally distinct MmpL3 inhibitor series, three of which impact proton motive force in Mycobacterium tuberculosis, directly interact with MmpL3. Medium- to high-throughput assays based on these approaches were developed to facilitate the future screening and optimization of MmpL3 inhibitors. The promiscuity of MmpL3 as a drug target and the mechanisms through which missense mutations located in a transmembrane region of this transporter may confer cross-resistance to a variety of chemical scaffolds are discussed in light of the exquisite vulnerability of MmpL3, its apparent mechanisms of interaction with inhibitors, and evidence of conformational changes induced both by the inhibitors and one of the most commonly identified resistance mutations in MmpL3.