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.

Expanding the squaramide library as mycobacterial ATP synthase inhibitors: Innovative synthetic pathway and biological evaluation.

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.

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.

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

Novel heterocyclic hydroxamates as inhibitors of the mycobacterial zinc metalloprotease Zmp1 to probe its mechanism of function.

Mycobacterial zinc metalloprotease-1 (Zmp1) is an essential enzyme for intracellular survival and pathogenicity of Mycobacterium tuberculosis. However, the exact mechanism of function of this enzyme remains unclear. This paper examines the effect of novel organic molecules on the inhibition of Zmp1. We followed our previous results and synthesised three libraries of new hydroxamates. All compounds were studied for their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis by MALDI-TOF MS. Furthermore, a macrophage infection assay was performed to evaluate intracellular antimycobacterial activity. In the whole-cell assay, no direct activity of synthesised heterocyclic hydroxamates was observed against Mycobacterium tuberculosis and Mycobacterium bovis. No acute cellular toxicity was observed against the murine RAW 264.7 macrophage cell line and human MRC-5 lung fibroblast cell line. However, thiazolidinediones 2 showed the dose-dependent inhibition of intracellular survival of Mycobacterium tuberculosis H37Ra. The inhibition was structure-dependent, with the most active derivative 2f inducing an 83.2% reduction of bacterial survival within the macrophage host cell. The promising biological activity confirmed thiazolidinediones 2 as Zmp1 inhibitors that can be used as tool compounds for further exploration of the role of Zmp1 for in vivo pathogenicity. In the long run, thiazolidinediones 2 show the potential to act as a scaffold for Zmp1 inhibitors to target intracellular Mtb as a novel tuberculosis treatment strategy.

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.

Inhibition of Bacterial Adhesion and Biofilm Formation by Seed-Derived Ethanol Extracts from Persea americana Mill.

The increase in antibiotic resistance demands innovative strategies to combat microorganisms. The current study evaluated the antibacterial and antivirulence effects of ethanol extracts from Persea americana seeds obtained by the Soxhlet (SE) and maceration (MaE) methods. The UHPLC-DAD-QTOF analysis showed mainly the presence of polyphenols and neolignan. Ethanol extracts were not cytotoxic to mammalian cells (CC50 > 500 µg/mL) and displayed a moderate antibacterial activity against Pseudomonas aeruginosa (IC50 = 87 and 187 µg/mL) and Staphylococcus aureus (IC50 = 144 and 159 µg/mL). Interestingly, no antibacterial activity was found against Escherichia coli. SE and MaE extracts were also able to significantly reduce the bacterial adhesion to A549 lung epithelial cells. Additionally, both extracts inhibited the biofilm growth at 24 h and facilitated the release of internal cell components in P. aeruginosa, which might be associated with cell membrane destabilization. Real-time PCR and agarose electrophoresis gel analysis indicated that avocado seed ethanol extracts (64 µg/mL) downregulated virulence-related factors such as mexT and lasA genes. Our results support the potential of bioproducts from P. americana seeds as anti-adhesive and anti-biofilm agents.

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).

Differential Induction Pattern Towards Classically Activated Macrophages in Response to an Immunomodulatory Extract from Pleurotus ostreatus Mycelium.

Pleurotus ostreatus mushroom preparations have been investigated because of their ability to modulate the immune function. However, there is still no consensus regarding the activation and polarizing effect on macrophages by Pleurotus-derived bioproducts. This study examined the immune-activating effect of a mycelium-derived P. ostreatus aqueous extract (HW-Pm) on macrophage functions, by means of the determination of nitric oxide (NO) production, the mRNA expression of inducible nitric oxide synthase (iNOS), Arginase-1 and FIZZ and the cytokine levels. The phagocytic activity and the activation of NF-κB in U937 reporter cells were also investigated. No cytotoxicity was observed in macrophages treated with HW-Pm (IC50 > 1024 µg/mL) by the resazurin test. HW-Pm induced high levels of NO production and iNOS expression in macrophages. In contrast, HW-Pm did not induce Arginase-1 and FIZZ mRNA expressions. The mushroom extract increased TNF-α and IL-6 production and the phagocytic function in murine macrophages. It also stimulated the activation of the NF-κB promoter. The P. ostreatus mycelium extract has a potential application as a natural immune-enhancing agent, by targeting macrophage activation towards the classically activated subset and stimulating macrophage-mediated innate immune responses.

Synthesis and structure-activity studies of novel anhydrohexitol-based Leucyl-tRNA synthetase inhibitors.

Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of antimicrobials. This enzyme catalyzes the formation of charged tRNALeu molecules, an essential substrate for protein translation. In the first step of catalysis LeuRS activates leucine using ATP, forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the aminoacyl-tRNA synthetase family but, to date, they have demonstrated poor antibacterial activity. We synthesized a small series of 1,5-anhydrohexitol-based analogues coupled to a variety of triazoles and performed detailed structure-activity relationship studies with bacterial LeuRS. In an in vitro assay, Kiapp values in the nanomolar range were demonstrated. Inhibitory activity differences between the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray crystallographic studies of N. gonorrhoeae LeuRS in complex with all the inhibitors highlighted the crucial interactions defining their relative enzyme inhibitory activities. We further examined their in vitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak antibacterial activity against M. tuberculosis was detected, the extensive structural data which were obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic development.

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.

Pyrazinamide is an important first-line drug used in shortening TB therapy. In our current work, a series of novel substituted-N-(6-(4-(pyrazine-2-carbonyl)piperazine/homopiperazine-1-yl)pyridin-3-yl)benzamide derivatives were designed, synthesized, and evaluated for their anti-tubercular activity against Mycobacterium tuberculosis H37Ra. Among the tested compounds, five compounds (6a, 6e, 6h, 6j and 6k) from Series-I and one compound (7e) from Series-II exhibited significant activity against Mycobacterium tuberculosis H37Ra with 50% inhibitory concentrations (IC50) ranging from 1.35 to 2.18 µM. To evaluate the efficacy of these compounds, we examined their IC90 values. Five of the most active compounds were found to be more active with IC90s ranging from 3.73 to 4.00 µM and one compound (6e) showed an IC90 of 40.32 µM. Moreover, single crystals were developed for 6d, 6f and 6n. In addition, most active compounds were evaluated for their cytotoxicity on HEK-293 (human embryonic kidney) cells. Our results indicate that the compounds are nontoxic to human cells. The molecular interactions of the derivatised conjugates in docking studies reveal their suitability for further development.

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.].

Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor Zmp1.

Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the intracellular growth of Mycobacterium tuberculosis at a concentration of 100 and 10 µM. The thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated library.

1-(Piperidin-3-yl)thymine amides as inhibitors of M. tuberculosis thymidylate kinase.

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable MtbTMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3. Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC50=35 µM). This finding opens avenues for future modifications.

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.

Opportunities for Overcoming Mycobacterium tuberculosis Drug Resistance: Emerging Mycobacterial Targets and Host-Directed Therapy.

The ever-increasing incidence of drug-resistant Mycobacterium tuberculosis infections has invigorated the focus on the discovery and development of novel treatment options. The discovery and investigation of essential mycobacterial targets is of utmost importance. In addition to the discovery of novel targets, focusing on non-lethal pathways and the use of host-directed therapies has gained interest. These adjunctive treatment options could not only lead to increased antibiotic susceptibility of Mycobacterium tuberculosis, but also have the potential to avoid the emergence of drug resistance. Host-directed therapies, on the other hand, can also reduce the associated lung pathology and improve disease outcome. This review will provide an outline of recent opportunities.

Optimization and Characterization of a Galleria mellonella Larval Infection Model for Virulence Studies and the Evaluation of Therapeutics Against Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Infection is linked to high morbidity and mortality rates and antibiotic resistance within this pathogen is on the rise. Therefore, there is a need for novel antimicrobial therapies. To lower the time and costs of the drug discovery process, alternative in vivo models should be considered. As such, Galleria mellonella larvae can be of great value. The larval immunity consisting of several types of haemocytes is remarkably similar to the human innate immune system. Furthermore, these larvae don't require specific housing, are cheap and are easy to handle. In this study, the use of a G. mellonella infection model to study early pneumococcal infections and treatment is proposed. Firstly, the fitness of this model to study pneumococcal virulence factors is confirmed using streptococcal strains TIGR4, ATCC®49619, D39 and its capsule-deficient counterpart R6 at different inoculum sizes. The streptococcal polysaccharide capsule is considered the most important virulence factor without which streptococci are unable to sustain an in vivo infection. Kaplan-Meier survival curves showed indeed a higher larval survival after infection with streptococcal strain R6 compared to strain D39. Then, the infection was characterized by determining the number of haemocytes, production of oxygen free radicals and bacterial burden at several time points during the course of infection. Lastly, treatment of infected larvae with the standard antibiotics amoxicillin and moxifloxacin was evaluated. Treatment has proven to have a positive outcome on the course of infection, depending on the administered dosage. These data imply that G. mellonella larvae can be used to evaluate antimicrobial therapies against S. pneumoniae, apart from using the larval model to study streptococcal properties. The in-depth knowledge acquired regarding this model, makes it more suitable for use in future research.

Synthesis and antitubercular activity of 1- and 3-substituted benzo[g]isoquinoline-5,10-diones.

In this study, a small library of twenty benzo[g]isoquinoline-5,10-diones were synthesized in a novel straightforward approach, starting from 2-methyl-1,4-naphthoquinone (vitamin K). An intramolecular Heck reaction of a N-vinylacetamide was a crucial step in the synthetic route, at which the combination of cesium carbonate and a bulky, electron rich trialkylphosphine (tBuCy2P.HBF4) provided high 6-endo-trig selectivity. The anti-tubercular activity against Mycobacterium tuberculosis H37Ra and acute cytotoxicity against J774 A.1 macrophages were studied. From the structure activity relationship, it could be derived that in general the substitution of position 3 yielded analogs with a higher antitubercular potency. Among these, two analogs, 27a and 27b, showed remarkable activity with minimal inhibition concentrations of respectively 28.92 µM and 1.05 µM, and acute cytotoxic concentrations of >128 µM and 34.85 µM. In addition, the analogs and their possible metabolites were evaluated using a Vitotox™ assay to study the possibility of genotoxicity. Results indicated that none of the evaluated analogs and their possible metabolites showed early signs of genotoxicity.

Synthesis and in vitro investigation of halogenated 1,3-bis(4-nitrophenyl)triazenide salts as antitubercular compounds.

The diverse pharmacological properties of the diaryltriazenes have sparked the interest to investigate their potential to be repurposed as antitubercular drug candidates. In an attempt to improve the antitubercular activity of a previously constructed diaryltriazene library, eight new halogenated nitroaromatic triazenides were synthesized and underwent biological evaluation. The potency of the series was confirmed against the Mycobacterium tuberculosis lab strain H37Ra, and for the most potent derivative, we observed a minimal inhibitory concentration of 0.85 µm. The potency of the triazenide derivatives against M. tuberculosis H37Ra was found to be highly dependent on the nature of the halogenated phenyl substituent and less dependent on cationic species used for the preparation of the salts. Although the inhibitory concentration against J774A.1 macrophages was observed at 3.08 µm, the cellular toxicity was not mediated by the generation of nitroxide intermediate as confirmed by electron paramagnetic resonance spectroscopy, whereas no in vitro mutagenicity could be observed for the new halogenated nitroaromatic triazenides when a trifluoromethyl substituent was present on both the aryl moieties.