Broad-Spectrum In Vitro Activity of Nα-Aroyl-N-Aryl-Phenylalanine Amides against Non-Tuberculous Mycobacteria and Comparative Analysis of RNA Polymerases.

This study investigates the in vitro activity of Nα-aroyl-N-aryl-phenylalanine amides (AAPs), previously identified as antimycobacterial RNA polymerase (RNAP) inhibitors, against a panel of 25 non-tuberculous mycobacteria (NTM). The compounds, including the hit compound MMV688845, were selected based on their structural diversity and previously described activity against mycobacteria. Bacterial strains, including the M. abscessus complex, M. avium complex, and other clinically relevant NTM, were cultured and subjected to growth inhibition assays. The results demonstrate significant activity against the most common NTM pathogens from the M. abscessus and M. avium complexes. Variations in activity were observed against other NTM species, with for instance M. ulcerans displaying high susceptibility and M. xenopi and M. simiae resistance to AAPs. Comparative analysis of RNAP ß and ß' subunits across mycobacterial species revealed strain-specific polymorphisms, providing insights into differential compound susceptibility. While conservation of target structures was observed, differences in compound activity suggested influences beyond drug-target interactions. This study highlights the potential of AAPs as effective antimycobacterial agents and emphasizes the complex interplay between compound structure, bacterial genetics, and in vitro activity.

Imidazopyridine Amides: Synthesis, Mycobacterium smegmatis CIII2CIV2 Supercomplex Binding, and In Vitro Antimycobacterial Activity.

Q203 (telacebec) is an imidazopyridine amide (IPA) targeting the respiratory CIII2CIV2 supercomplex of the mycobacterial electron transport chain (ETC). Aiming for a better understanding of the molecular mechanism of action of IPA, 27 analogues were prepared through a seven-step synthetic scheme. Oxygen consumption assay was designed to test the inhibition of purified Mycobacterium smegmatis CIII2CIV2 by these compounds. The assay results generally supported structure-activity relationship information obtained from the structure of M. smegmatis CIII2CIV2 bound to Q203. The IC50 of Q203 and compound 27 was 99 ± 32 and 441 ± 138 nM, respectively. All IPAs including Q203 showed no inhibition of mitochondrial ETC, proving their selectivity against mycobacteria. In vitro Mycobacterium tuberculosis growth inhibition and M. smegmatis CIII2CIV2 binding did not correlate perfectly. These observations suggest that further investigation into the mechanisms of resistance in different mycobacterial species is needed to understand the lack of the correlation pattern between CIII2CIV2 inhibition and cellular activity.

Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria.

Nα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide [MMV688845, Pathogen Box; Medicines for Malaria Venture; IUPAC: (2R)-N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide)] is a hit compound, which shows activity against Mycobacterium abscessus (MIC90 6.25-12.5 µM) and other mycobacteria. This work describes derivatization of MMV688845 by introducing a thiomorpholine moiety and the preparation of the corresponding sulfones and sulfoxides. The molecular structures of three analogs are confirmed by X-ray crystallography. Conservation of the essential R configuration during synthesis is proven by chiral HPLC for an exemplary compound. All analogs were characterized in a MIC assay against M. abscessus, Mycobacterium intracellulare, Mycobacterium smegmatis, and Mycobacterium tuberculosis. The sulfone derivatives exhibit lower MIC90 values (M. abscessus: 0.78 µM), and the sulfoxides show higher aqueous solubility than the hit compound. The most potent derivatives possess bactericidal activity (99% inactivation of M. abscessus at 12.5 µM), while they are not cytotoxic against mammalian cell lines.

In Vitro Profiling of the Synthetic RNA Polymerase Inhibitor MMV688845 against Mycobacterium abscessus.

In a library screen of tuberculosis-active compounds for anti-Mycobacterium abscessus activity, we previously identified the synthetic phenylalanine amide MMV688845. In Mycobacterium tuberculosis, this class was shown to target the RpoB subunit of RNA polymerase, engaging a binding site distinct from that of the rifamycins. Due to its bactericidal activity, rifampicin is a key drug for the treatment of tuberculosis (TB). However, this natural product shows poor potency against M. abscessus due to enzymatic modification, and its clinical use is limited. Here, we carried out in vitro microbiological profiling of MMV688845 to determine its attractiveness as a substrate for a chemistry optimization project. MMV688845 was broadly active against the M. abscessus complex, displayed bactericidal against M. abscessus in vitro, and in a macrophage infection model showed additivity with commonly used anti-M. abscessus antibiotics and synergy with macrolides. Analyses of spontaneous resistant mutants mapped resistance to RpoB, confirming that MMV688845 has retained its target in M. abscessus. Together with its chemical tractability, the presented microbiological profiling reveals MMV688845 as an attractive starting point for hit-to-lead development to improve potency and to identify a lead compound with demonstrated oral in vivo efficacy. IMPORTANCE Infections with nontuberculous mycobacteria are an increasing health problem, and only a few new drug classes show activity against these multidrug-resistant bacteria. Due to insufficient therapy options, the development of new drug leads is necessary and should be advanced. The lead compound MMV688845, a substance active against M. abscessus complex, was characterized in depth. In various assays, it showed activity against M. abscessus, synergy with other antibiotics, and bactericidal effects.

Structure of V-ATPase from citrus fruit.

We used the Legionella pneumophila effector SidK to affinity purify the endogenous vacuolar-type ATPases (V-ATPases) from lemon fruit. The preparation was sufficient for cryoelectron microscopy, allowing structure determination of the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum ΔpH of ∼3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared with yeast and mammalian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual configuration of transmembrane α helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.

Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203).

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.