Reversed isoniazids: Design,synthesis and evaluation against Mycobacterium tuberculosis

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a–c, 7 and 8; H37Rv-MIC₉₉ ≤1.25?µM, R5401-MIC₉₉ ≤2.5?µM, X_61-MIC₉₉ ≤5?µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a–c and 3b (MIC₉₀ ≤13.42?µM) and inhibition of EB efflux demonstrated by these compounds are encouraging.     

Synthesis of new verapamil analogues and their evaluation in combination with rifampicin against Mycobacterium tuberculosis and molecular docking studies in the binding site of efflux protein Rv1258c

New verapamil analogues were synthesized and their inhibitory activities against Mycobacterium tuberculosis H37Rv determined in vitro alone and in combination with rifampicin (RIF). Some analogues showed comparable activity to verapamil and exhibited better synergies with RIF. Molecular docking studies of the binding sites of Rv1258c, a M. tuberculosis efflux protein previously implicated in intrinsic resistance to RIF, suggested a potential rationale for the superior synergistic interactions observed with some analogues.     

Design, synthesis and antimycobacterial evaluation of novel 3-substituted-N-aryl-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide analogues

In the present investigation, a series of 3-substituted-N-aryl-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide analogues were synthesized and were evaluated for antitubercular activity by two fold serial dilution technique. All the newly synthesized compounds showed moderate to high inhibitory activities against Mycobacterium tuberculosis H₃₇Rv and INH resistant M. tuberculosis. The compound N,3-bis(4-fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide (4c) was found to be the most promising compound active against M. tuberculosis H₃₇Rv and isoniazid resistant M. tuberculosis with minimum inhibitory concentration 0.78 μM.     

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.     

Synthesis and antimycobacterial evaluation of novel 5,6-dimethoxy-1-oxo-2,5-dihydro-1H-2-indenyl-5,4-substituted phenyl methanone analogues

In present investigation, a series of substituted phenyl-5,6-dimethoxy-1-oxo-2,5-dihydro-1H-2-indenylmethanone analogues were synthesized and were evaluated for antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv and INH resistant M. tuberculosis. All the newly synthesized compounds were showing moderate to high inhibitory activities. The compound 5,6-dimethoxy-1-oxo-2,5-dihydro-1H-2-indenyl-4-fluorophenylmethanone (5g) was found to be the most promising compounds active against M. tuberculosis H₃₇Rv and isoniazid (INH) resistant M. tuberculosis with Minimum inhibitory concentration 0.10 and 0.10 μM.     

In Vitro Activity of Rifampicin and Verapamil Combination in Multidrug-Resistant Mycobacterium tuberculosis

The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H₃₇Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H₃₇Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.     

A novel bi-functional chalcone inhibits multi-drug resistant Staphylococcus aureus and potentiates the activity of fluoroquinolones

Staphylococcus aureus is the leading cause of bacteraemia and the dwindling supply of effective antibacterials has exacerbated the problem of managing infections caused by this bacterium. Isoliquiritigenin (ISL) is a plant flavonoid that displays therapeutic potential against S. aureus. The present study identified a novel mannich base derivatives of ISL, IMRG4, active against Vancomycin intermediate S. aureus (VISA). IMRG4 damages the bacterial membranes causing membrane depolarization and permeabilization, as determined by loss of salt tolerance, flow cytometric analysis, propidium idodie and fluorescent microscopy. It reduces the intracellular invasion of HEK-293 cells by S. aureus and decreases the staphylococcal load in different organs of infected mice models. In addition to anti-staphylococcal activity, IMRG4 inhibits the multidrug efflux pump, NorA, which was determined by molecular docking and EtBr efflux assays. In combination, IMRG4 significantly reduces the MIC of norfloxacin for clinical strains of S. aureus including VISA. Development of resistance against IMRG4 alone and in combination with norfloxacin was low and IMRG4 prolongs the post-antibiotic effect of norfloxacin. These virtues combined with the low toxicity of IMRG4, assessed by MTT assay and haemolysis, makes it an ideal candidate to enter drug development pipeline against S. aureus.     

Characterization of an interplay between a Mycobacterium?tuberculosis MazF homolog,Rv1495 and its sole DNA topoisomerase I

The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.     

Identification and synthesis of novel inhibitors of mycobacterium ATP synthase

A non-diaryl quinoline scaffold 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one was identified by screening of diverse set of compounds against M. smegmatis ATP synthase. Herein, we disclose our efforts to develop the structure activity relationship against Mycobacterium tuberculosis (Mtb.H37Rv strain) around the identified hit 1. A scaffold hopping approach was used to identify compounds 14a, 14b and 24a with improved activity against MTb.H37Rv.     

Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present in Mycobacterium fortuitum and Mycobacterium tuberculosis

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.     

Tannic acid affects the phenotype of Staphylococcus aureus resistant to tetracycline and erythromycin by inhibition of efflux pumps

The widespread use of antibiotics created selective pressure for the emergence of strains that would persist despite antibiotic toxicity. The bacterial resistance mechanisms are several, with efflux pumps being one of the main ones. These pumps are membrane proteins with the function of removing antibiotics from the cell cytoplasm. Due to this importance, the aim of this work was to evaluate the inhibitory effect of tannic acid against efflux pumps expressed by the Staphylococcus aureus RN4220 and IS-58 strains. The efflux pump inhibition was assayed using a sub-inhibitory concentration of efflux pump standard inhibitors and tannic acid (MIC/8), observing their capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due the possible inhibitory effect of these substances. The MICs of EtBr and antibiotics were significantly different in the presence of tannic acid, indicating the inhibitory effect of this product against efflux pumps of both strains. These results indicate the possible usage of tannic acid as an inhibitor and an adjuvant in the antibiotic therapy against multidrug resistant bacteria (MDR).     

Enhancing activity of antibiotics against Staphylococcus aureus: Zanthoxylum capense constituents and derivatives

Six compounds (1–6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7–13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1–13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1–13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8−11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8, 11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of efflux pumps mechanism.These results suggested that Z. capense constituents may be valuable as leads for restoring antibiotic activity against MRSA strains.     

Characterization of a chromosomal toxin-antitoxin, Rv1102c-Rv1103c system in Mycobacterium tuberculosis

Toxin-antitoxin systems, ubiquitous in prokaryotic genomes, have been proposed to play an important role in several stress responses. While Mycobacterium tuberculosis contains more than 80 putative TA loci, the roles they play in this pathogen are yet to be studied. Here, we characterize a chromosomal Rv1102c-Rv1103c TA system in M. tuberculosis. We found that the Rv1102c toxin interacts with the Rv1103c antitoxin in a pull-down assay and the yeast two-hybrid system. Rv1102c cleaved the era mRNA in Escherichia coli, and cleavage was inhibited by co-expression of Rv1103c. Heterologous expression of Rv1102c led to growth arrest in E. coli, which was fully recovered only when Rv1103c was co-expressed in cis with Rv1102c, suggesting that the production and assembly of Rv1102c and Rv1103c are tightly linked. Our additional results indicate that translational coupling of the Rv1102c and Rv1103c genes is important for Rv1102c-Rv1103c binding. Finally, we discovered that the expression of Rv1102c induced growth arrest and increased the level of persister cells in Mycobacterium smegmatis. These results suggest that the Rv1102c-Rv1103c TA system could play a role in M. tuberculosis pathogenesis via generating bacilli that survive in the face of multidrug therapy.     

Studies on substituted benzo[h]quinazolines,benzo[g]indazoles,pyrazoles, 2,6-diarylpyridines as anti-tubercular agents

Various substituted 5,6-dihydro-8-methoxybenzo[h]quinazolin-2-amine, 1-(3-(4-alkoxyphenyl)-7-methoxy-3,3a,4,5-tetrahydro-2H benzo[g]indazol-2-yl)ethanone, pyrazole and 2,6-diarylpyridine derivatives have been synthesized in good yields by an efficient methodology. The synthesized compounds (4–23) were evaluated for their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain. Compounds 6a, 6c, 8a, 19a and 19e exhibited significant anti-tubercular activity at MIC values 50, 100, 50, 25 and 100 μM concentration. In vitro cytotoxicity data using THP-1 cells indicated that most active compound 19a is safe as its MIC value is much lower than the cytotoxic value.     

Mycobacterium tuberculosis Rv3402c Enhances Mycobacterial Survival within Macrophages and Modulates the Host Pro-Inflammatory Cytokines Production via NF-Kappa B/ERK/p38 Signaling

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H₂O₂ exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.     

A Nano-MgO and Ionic Liquid-Catalyzed ‘Green’ Synthesis Protocol for the Development of Adamantyl-Imidazolo-Thiadiazoles as Anti-Tuberculosis Agents Targeting Sterol 14α-Demethylase (CYP51)

In this work, we describe the ‘green’ synthesis of novel 6-(adamantan-1-yl)-2-substituted-imidazo[2,1-b][1,3,4]thiadiazoles (AITs) by ring formation reactions using 1-(adamantan-1-yl)-2-bromoethanone and 5-alkyl/aryl-2-amino1,3,4-thiadiazoles on a nano material base in ionic liquid media. Given the established activity of imidazothiadiazoles against M. tuberculosis, we next examined the anti-TB activity of AITs against the H₃₇Rv strain using Alamar blue assay. Among the tested compounds 6-(adamantan-1-yl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (3f) showed potent inhibitory activity towards M. tuberculosis with an MIC value of 8.5 μM. The inhibitory effect of this molecule against M. tuberculosis was comparable to the standard drugs such as Pyrazinamide, Streptomycin, and Ciprofloxacin drugs. Mechanistically, an in silico analysis predicted sterol 14α-demethylase (CYP51) as the likely target and experimental activity of 3f in this system corroborated the in silico target prediction. In summary, we herein report the synthesis and biological evaluation of novel AITs against M. tuberculosis that likely target CYP51 to induce their antimycobacterial activity.     

Trichoderins,novel aminolipopeptides from a marine sponge-derived Trichoderma sp., are active against dormant mycobacteria

Three new aminolipopeptides, designated trichoderins A (1), A1 (2), and B (3), were isolated from a culture of marine sponge-derived fungus of Trichoderma sp. as anti-mycobacterial substances with activity against active and dormant bacilli. The chemical structures of trichoderins were determined on the basis of spectroscopic study. Trichoderins showed potent anti-mycobacterial activity against Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis H37Rv under standard aerobic growth conditions as well as dormancy-inducing hypoxic conditions, with MIC values in the range of 0.02–2.0 μg/mL.     

Secondary metabolites from the leaves of the medicinal plant goldenseal (Hydrastis canadensis)

The study presented herein constitutes an extensive investigation of constituents in Hydrastis canadensis L. (Ranunculaceae) leaves. It describes the isolation and identification of two previously unknown compounds, 3,4-dimethoxy-2-(methoxycarbonyl)benzoic acid (1) and 3,5,3′-trihydroxy-7,4′-dimethoxy-6,8-C-dimethyl-flavone (2), along with the known compounds (±)-chilenine (3), (2R)-5,4′-dihydroxy-6-C-methyl-7-methoxy-flavanone (4), 5,4′-dihydroxy-6,8-di-C-methyl-7-methoxy-flavanone (5), noroxyhydrastinine (6), oxyhydrastinine (7) and 4′,5′-dimethoxy-4-methyl-3′-oxo-(1,2,5,6-tetrahydro-4H-1,3-dioxolo-[4′,5′:4,5]-benzo[1,2-e]-1,2-oxazocin)-2-spiro-1′-phtalan (8). Compounds 3–8 have been reported from other sources, but this is the first report of their presence in H. canadensis extracts. A mass spectrometry based assay was employed to demonstrate bacterial efflux pump inhibitory activity against Staphylococcus aureus for 2, with an IC₅₀ value of 180 ± 6 μM. This activity in addition to that of other bioactive compounds such as flavonoids and alkaloids, may explain the purported efficacy of H. canadensis for treatment of bacterial infections. Finally, this report includes high mass accuracy fragmentation spectra for all compounds investigated herein which were uploaded into the Global Natural Products Social molecular networking library and can be used to facilitate their future identification in H. canadensis or other botanicals.