A high-throughput target-based screening approach for the identification and assessment of Mycobacterium tuberculosis mycothione reductase inhibitors.

The World Health Organization's goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens. IMPORTANCE: The growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Design, Synthesis, and Antimycobacterial Evaluation of Novel Tetrahydroisoquinoline Hydrazide Analogs.

A series of novel 2-substituted-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carbohydrazide were designed, synthesized and structures were confirmed by analytical methods, viz., 1 H-NMR, 13 C-NMR and Mass spectrometry. Synthesized derivatives were evaluated for their anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb) H37Ra. Among all the evaluated compounds, 10A25 containing biphenyl moiety exhibited significant inhibition with IC50 4.7 µM. 10A19, with an electron-withdrawing Iodo group in the ortho position of the phenyl exhibited significant anti-tubercular activity with IC50 8.8 µM. IC50 values of the remaining compounds ranged from 9.2 to 73.6 µM. Molecular docking study of the significantly active compound 10A25 was performed to determine the putative binding position of the test ligand at the active site of the selected target proteins Mycobacterium tuberculosis enoyl reductase (InhA) PDB - 4TZK and peptide deformylase PDB - 3E3U. A suitable single crystal for one of the active compounds, 10A12, was generated and analysed to further confirm the structure of the compounds.

Design, synthesis and biological evaluation of benzo-[d]-imidazo-[2,1-b]-thiazole and imidazo-[2,1-b]-thiazole carboxamide triazole derivatives as antimycobacterial agents.

In the search for new anti-mycobacterial agents, we revealed the importance of imidazo-[2,1-b]-thiazole and benzo-[d]-imidazo-[2,1-b]-thiazole carboxamide derivatives. We designed, in silico ADMET predicted and synthesized four series of novel imidazo-[2,1-b]-thiazole and benzo-[d]-imidazo-[2,1-b]-thiazole carboxamide analogues in combination with piperazine and various 1,2,3 triazoles. All the synthesized derivatives were characterized by 1H NMR, 13C NMR, HPLC and MS spectral analysis and evaluated for in vitro antitubercular activity. The most active benzo-[d]-imidazo-[2,1-b]-thiazole derivative IT10, carrying a 4-nitro phenyl moiety, displayed IC90 of 7.05 µM and IC50 of 2.32 µM against Mycobacterium tuberculosis (Mtb) H37Ra, while no acute cellular toxicity was observed (>128 µM) towards the MRC-5 lung fibroblast cell line. Another benzo-[d]-imidazo-[2,1-b]-thiazole compound, IT06, which possesses a 2,4-dichloro phenyl moiety, also showed significant activity with IC50 2.03 µM and IC90 15.22 µM against the tested strain of Mtb. Furthermore, the selected hits showed no activity towards a panel of non-tuberculous mycobacteria (NTM), thus suggesting a selective inhibition of Mtb by the tested imidazo-[2,1-b]-thiazole derivatives over the selected panel of NTM. Molecular docking and dynamics studies were also carried out for the most active compounds IT06 and IT10 in order to understand the putative binding pattern, as well as stability of the protein-ligand complex, against the selected target Pantothenate synthetase of Mtb.

Design, Synthesis and Biological Evaluation of Novel Spiro-[Chroman-2,4'-Piperidin]-4-One Analogs as Anti-Tubercular Agents.

A series of novel spiro-[chromane-2,4'-piperidin]-4(3H)-one derivatives were designed, synthesized and structures were confirmed by analytical methods, viz., 1 H-NMR, 13 C-NMR and mass spectrometry. The synthetic derivatives were evaluated for their anti-tuberculosis (anti-TB) activity against Mycobacterium tuberculosis (Mtb) strain H37Ra. Among all the evaluated Compounds, PS08 exhibited significant inhibition with MIC value of 3.72 µM while MIC values of the remaining Compounds ranged from 7.68 to 230.42 µM in comparison to the standard drug INH (MIC 0.09 µM). The two most active Compounds however showed acute cytotoxicity towards the human MRC-5 lung fibroblast cell lines. The in silico ADMET profiles of the titled Compounds were predicted and found within the prescribed limits of the Lipinski and Jorgenson rules. Molecular docking study of the notably active Compound (PS08) was also carried out after performing validation in order to understand the putative binding position of the test ligand at the active site of selected target protein Mtb tyrosine phosphatase (PtpB).

Correction: Seeking potent anti-tubercular agents: design and synthesis of substituted-N-(6-(4-(pyrazine-2-carbonyl)piperazine/homopiperazine-1-yl)pyridin-3-yl)benzamide derivatives as anti-tubercular agents.

[This corrects the article DOI: 10.1039/D0RA01348J.].

The synthesis and in vitro biological evaluation of novel fluorinated tetrahydrobenzo[j]phenanthridine-7,12-diones against Mycobacterium tuberculosis.

Tuberculosis (TB) still has a major impact on public health. In order to efficiently eradicate this life-threatening disease, the exploration of novel anti-TB drugs is of paramount importance. As part of our program to design new 2-azaanthraquinones with anti-mycobacterial activity, various "out-of-plane" tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. In this study, the scaffold of the most promising hits was further optimized in an attempt to improve the bioactivity and to decrease enzymatic degradation. The rudiment bio-evaluation of a small library of fluorinated tetrahydrobenzo[j]phenanthridine-7,12-dione derivatives indicated no significant improvement of the bio-activity against intracellular and extracellular Mycobacterium tuberculosis (Mtb). Though, the derivatives showed an acceptable toxicity against J774A.1 macrophages and early signs of genotoxicity were absent. All derivatives showed to be metabolic stabile in the presence of both phase I and phase II murine or human microsomes. Finally, the onset of reactive oxygen species within Mtb after exposure to the derivatives was measured by electron paramagnetic resonance (EPR). Results showed that the most promising fluorinated derivative is still a possible candidate for the subversive inhibition of mycothione reductase.