Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b–2d, 3a–3d, and 4a–4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a–4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 16–64 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.     

GC/MS analysis and potential synergistic effect of mandarin and marjoram oils on Helicobacter pylori

Helicobacter pylori can cause chronic gastritis, peptic ulcer, and gastric carcinoma. This study compares chemical composition and anti-H. pylori activity of mandarin leaves and marjoram herb essential oils, and their combined oil. GC/MS analysis of mandarin oil revealed six compounds (100% identified), mainly methyl-N-methyl anthranilate (89.93%), and 13 compounds (93.52% identified) of marjoram oil, mainly trans-sabinene hydrate (36.11%), terpinen-4-ol (17.97%), linalyl acetate (9.18%), and caryophyllene oxide (8.25%)). Marjoram oil (MIC = 11.40 µg/mL) demonstrated higher activity than mandarin oil (MIC = 31.25 µg/mL). The combined oil showed a synergistic effect at MIC of 1.95 µg/mL (same as clarithromycin). In-silico molecular docking on H. pylori urease, CagA, pharmacokinetic and toxicity studies were performed on major compounds from both oils. The best scores were for caryophyllene oxide then linalyl acetate and methyl-N-methyl anthranilate. Compounds revealed high safety and desirable properties. The combined oil can be an excellent candidate to manage H. pylori.     

1,3-Azoles from ortho-naphthoquinones: Synthesis of aryl substituted imidazoles and oxazoles and their potent activity against Mycobacterium tuberculosis

Twenty-three naphthoimidazoles and six naphthoxazoles were synthesised and evaluated against susceptible and rifampicin- and isoniazid-resistant strains of Mycobacterium tuberculosis. Among all the compounds evaluated, fourteen presented MIC values in the range of 0.78 to 6.25 μg/mL against susceptible and resistant strains of M. tuberculosis. Five structures were solved by X-ray crystallographic analysis. These substances are promising antimycobacterial prototypes.     

Synthesis and in vitro antitubercular activity of a series of quinoline derivatives

A series of 33 quinoline derivatives have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H₃₇Rv using the Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 5e and 5f exhibited a significant activity at 6.25 and 3.12 μg/mL, respectively, when compared with first line drugs such as ethambutol and could be a good starting point to develop new lead compounds in the fight against multi-drug resistant tuberculosis.     

Synthesis and antitubercular activity of 7-chloro-4-quinolinylhydrazones derivatives

A series of twenty-one 7-chloro-4-quinolinylhydrazones (3a–u) have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H₃₇Rv. The compounds 3f, 3i and 3o were non-cytotoxic and exhibited an important minimum inhibitory concentration (MIC) activity (2.5 μg/mL), which can be compared with that of the first line drugs, ethambutol (3.12 μg/mL) and rifampicin (2.0 μg/mL). These results can be considered an important start point for the rational design of new leads for anti-TB compounds.     

Platensimycin Activity against Mycobacterial β-Ketoacyl-ACP Synthases

Background

There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.

Methods and Findings

We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 µg/ml) and against Mycobacterium tuberculosis (MIC = 12 µg/ml). Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against β-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 µg/ml, to 30 and 124 µg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.

Significance

Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.     

Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism ⁹³⁷ACC>⁹³⁷GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.     

Synthesis,characterization and in vitro biological evaluation of some novel 1,3,5-triazine–Schiff base conjugates as potential antimycobacterial agents

A series of some novel 1,3,5-triazine–Schiff base conjugates (1–32) have been synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 4 (4-Methoxy-6-methyl-N-(3,4,5-trimethoxybenzylidene)-1,3,5-triazin-2-amine), 11 (4-Methoxy-6-methyl-N-(2-hydroxy-3-bromo-5-chloro-benzylidene)-1,3,5-triazin-2-amine) and 24 (4-Methoxy-6-methyl-N-(1-(2,5-dihydroxyphenyl)ethylidene)-1,3,5-triazin-2-amine) exhibited a significant activity at 3.125, 6.25 and 6.25 μg/mL, respectively, when compared with the antitubercular drugs such as ethambutol (3.125 μg/mL), pyrazinamide (6.25 μg/mL) and streptomycin (6.25 μg/mL) and it could be a potential starting point to develop new lead compounds in the fight against Mycobacterium tuberculosis H37Rv.     

Synthesis and screening of (E)-1-(β-D-galactopyranosyl)-4-(aryl)but-3-ene-2-one against Mycobacterium tuberculosis

A series of galactose-derived aryl enones were synthesised and screened against Mycobacterium tuberculosis H₃₇Rv. Preliminary results were promising with MIC values in the range 1.56-12.5 μg/mL.     

Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the β-Ketoacyl-ACP Synthase mtFabH

Background

Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world''s population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration.

Methodology/Principal Findings

Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM''s novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H₃₇R_v and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H₃₇R_v with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC₅₀ of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism.

Conclusions/Significance

These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.     

Synthesis and evaluation of antitubercular activity of glycosyl thio- and sulfonyl acetamide derivatives

A series of glycosyl thioacetamide and glycosyl sulfonyl acetamide derivatives have been prepared following a convenient reaction protocol and evaluated for their antitubercular activity against Mycobacterium tuberculosis H₃₇Rv. Amongst 32 compounds evaluated 3 compounds were effective in inhibiting mycobacterial growth at MIC of 6.25 μg/mL, 6 compounds at MIC of 3.125 μg/mL and 1 compound at MIC of 1.56 μg/mL. All active compounds were found nontoxic in Vero cell lines and mice bone marrow macrophages.     

Amino ether analogues of 4,4′-dihydroxy-3-methoxy-6,7′-cyclolignan and their activity against drug-resistant bacteria

Larrea tridentata antibacterial lignan 4,4′-dihydroxy-3-methoxy-6,7′-cyclolignan (1) was derivatized to obtain eleven new amino ether derivatives (2 A-12 C). The structural elucidation of compounds was performed by analysis of 1D- and 2D NMR spectral data and HRESIMS. The antibacterial activity of compounds was determined against nine drug-resistant bacteria and two strains of Mycobacterium tuberculosis (sensitive ATCC 27294 H37Rv and drug-resistant G122). Results showed that all derivatives were devoid of activity towards six gram-negative clinical isolates assayed. However, seven derivatives displayed antibacterial activity against three gram-positive drug-resistant bacteria. Further, enhancement of antibacterial activity was only observed for the compounds 2 A and 10 C-12 C (MIC of 12.5 µg/mL) which were two-fold more active than the starting material 1 against vancomycin-resistant Enterococcus faecium. All derivatives, except compound 9 B, showed antitubercular activity against both M. tuberculosis strains. Interestingly, all the compounds, except for 2 A and 11 A, were more active than the starting material 1 (MIC of 50 µg/mL). Compound 4 C was the only compound as active as the positive control ethambutol against the drug-resistant strain M. tuberculosis G122 (MIC of 6.25 µg/mL). In addition, the derivative 7 C was the most active compound against the sensitive strain M. tuberculosis H37Rv (MIC of 6.25 µg/mL)     

A click chemistry approach to pleuromutilin derivatives,evaluation of anti-MRSA activity and elucidation of binding mode by surface plasmon resonance and molecular docking

Novel series of pleuromutilin analogs containing substituted 1,2,3-triazole moieties were designed, synthesised and assessed for their in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Initially, the in vitro antibacterial activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300, ATCC 29213, AD3, and 144) were tested by the broth dilution method. Most of the synthesised pleuromutilin analogs displayed potent activities. Among them, compounds 50, 62, and 64 (MIC = 0.5∼1 µg/mL) showed the most effective antibacterial activity and their anti-MRSA activity were further studied by the time-killing kinetics approach. Binding mode investigations by surface plasmon resonance (SPR) with 50S ribosome revealed that the selected compounds all showed obvious affinity for 50S ribosome (K_D = 2.32 × 10⁻⁸∼5.10 × 10⁻⁵ M). Subsequently, the binding of compounds 50 and 64 to the 50S ribosome was further investigated by molecular modelling. Compound 50 had a superior docking mode with 50S ribosome, and the binding free energy of compound 50 was calculated to be −12.0 kcal/mol.     

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background

Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.

Methodology/Principal Findings

Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC₅₀ 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC₅₀. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.

Conclusions/Significance

NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.     

A Trisubstituted Benzimidazole Cell Division Inhibitor with Efficacy against Mycobacterium tuberculosis

Trisubstituted benzimidazoles have demonstrated potency against Gram-positive and Gram-negative bacterial pathogens. Previously, a library of novel trisubstituted benzimidazoles was constructed for high throughput screening, and compounds were identified that exhibited potency against M. tuberculosis H37Rv and clinical isolates, and were not toxic to Vero cells. A new series of 2-cyclohexyl-5-acylamino-6-N, N-dimethylaminobenzimidazoles derivatives has been developed based on SAR studies. Screening identified compounds with potency against M. tuberculosis. A lead compound from this series, SB-P17G-A20, was discovered to have an MIC of 0.16 µg/mL and demonstrated efficacy in the TB murine acute model of infection based on the reduction of bacterial load in the lungs and spleen by 1.73±0.24 Log₁₀ CFU and 2.68±Log₁₀ CFU, respectively, when delivered at 50 mg/kg by intraperitoneal injection (IP) twice daily (bid). The activity of SB-P17G-A20 was determined to be concentration dependent and to have excellent stability in mouse and human plasma, and liver microsomes. Together, these studies demonstrate that SB-P17G-A20 has potency against M. tuberculosis clinical strains with varying susceptibility and efficacy in animal models of infection, and that trisubstituted benzimidazoles continue to be a platform for the development of novel inhibitors with efficacy.     

The Rationale for Using Rifabutin in the Treatment of MDR and XDR Tuberculosis Outbreaks

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.     

In vitro anti-TB properties,in silico target validation,molecular docking and dynamics studies of substituted 1,2,4-oxadiazole analogues against Mycobacterium tuberculosis

The alarming increase in multi- and extensively drug-resistant (MDR and XDR) strains of Mycobacterium tuberculosis (MTB) has triggered the scientific community to search for novel, effective, and safer therapeutics. To this end, a series of 3,5-disubstituted-1,2,4-oxadiazole derivatives (3a–3i) were tested against H37Rv, MDR and XDR strains of MTB. Of which, compound 3a with para-trifluorophenyl substituted oxadiazole showed excellent activity against the susceptible H37Rv and MDR-MTB strain with a MIC values of 8 and 16 µg/ml, respectively.To understand the mechanism of action of these compounds (3a–3i) and identify their putative drug target, molecular docking and dynamics studies were employed against a panel of 20 mycobacterial enzymes reported to be essential for mycobacterial growth and survival. These computational studies revealed polyketide synthase (Pks13) enzyme as the putative target. Moreover, in silico ADMET predictions showed satisfactory properties for these compounds, collectively, making them, particularly compound 3a, promising leads worthy of further optimisation.     

Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis,in vitro,kinetic and docking studies

In the present study, a series of azo derivatives (TR-1 to TR-9) have been synthesised via the diazo-coupling approach between substituted aromatic amines with phenol or naphthol derivatives. The compounds were evaluated for their therapeutic applications against alpha-glucosidase (anti-diabetic) and pathogenic bacterial strains E. coli (gram-negative), S. aureus (gram-positive), S. aureus (gram-positive) drug-resistant strain, P. aeruginosa (gram-negative), P. aeruginosa (gram-negative) drug-resistant strain and P. vulgaris (gram-negative). The IC₅₀ (µg/mL) of TR-1 was found to be most effective (15.70 ± 1.3 µg/mL) compared to the reference drug acarbose (21.59 ± 1.5 µg/mL), hence, it was further selected for the kinetic studies in order to illustrate the mechanism of inhibition. The enzyme inhibitory kinetics and mode of binding for the most active inhibitor (TR-1) was performed which showed that the compound is a non-competitive inhibitor and effectively inhibits the target enzyme by binding to its binuclear active site reversibly.     

Synthesis and antimycobacterial evaluation of pyrazinamide derivatives with benzylamino substitution

A series of 19 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro whole cell antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two types of Mycobacterium avium. The series consisted of 3-(benzylamino)-5-cyanopyrazine-2-carboxamides and 3-(benzylamino)pyrazine-2,5-dicarbonitriles with various substituents on the phenyl ring. RP-HPLC method was used to determine the lipophilicity of the prepared compounds. Nine compounds exerted similar or better activity against Mycobacterium tuberculosis compared to pyrazinamide (MIC = 6.25–12.5 μg/mL). 3-(Benzylamino)pyrazine-2,5-dicarbonitrile inhibited all of the tested mycobacterial strains with MIC within the range 12.5–25 μg/mL. Although not the most active, 4-NH₂ substituted compounds possessed the lowest in vitro cytotoxicity (hepatotoxicity), leading to selectivity index SI = 5.5 and SI >21.     

An atom economic synthesis and antitubercular evaluation of novel spiro-cyclohexanones

The 1,3-dipolar cycloaddition of azomethine ylides derived from acenaphthenequinone and α-amino acids viz. sarcosine, phenylglycine, 1,3-thiazolane-4-carboxylic acid and proline to a series of 2,6-bis[(E)-arylmethylidene]cyclohexanones afforded novel spiro-heterocycles chemo-, regio- and stereoselectively in quantitative yields. These compounds were screened for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB) using agar dilution method. Two compounds, 4-(2,4-dichlorophenyl)-5-phenylpyrrolo(spiro[2.2″]acenaphthene-1″-one)spiro[3.2′]-6′-(2,4-dichlorophenylmethylidene)cyclohexanone (4i) and spiro[5.2″]acenaphthene-1″-onespiro[6.2′]-6′-(2,4-dichlorophenylmethylidene)cyclohexanone-7-(2,4-dichlorophenyl)tetrahydro-1H-pyrrolo[1,2-c][1,3]thiazole (5i) display maximum activity in vitro with a MIC value of 0.40 μg/mL against MTB and were 4 and 15.6 times more potent than ethambutol and pyrazinamide, respectively.