Sreevalli Sharma

Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates

ABSTRACT Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

Bactericidal Activity and Mechanism of Action of AZD5847, a Novel Oxazolidinone for Treatment of Tuberculosis

ABSTRACT Treatment of tuberculosis (TB) is impaired by the long duration and complexity of therapy and the rising incidence of drug resistance. There is an urgent need for new agents with improved efficacy, safety, and compatibility with combination chemotherapies. Oxazolidinones offer a potential new class of TB drugs, and linezolid—the only currently approved oxazolidinone—has proven highly effective against extensively drug-resistant (XDR) TB in experimental trials. However, widespread use of linezolid is prohibited by its significant toxicities. AZD5847, a novel oxazolidinone, demonstrates improved in vitro bactericidal activity against both extracellular and intracellular M. tuberculosis compared to that of linezolid. Killing kinetics in broth media and in macrophages indicate that the rate and extent of kill obtained with AZD5847 are superior to those obtained with linezolid. Moreover, the efficacy of AZD5847 was additive when tested along with a variety of conventional TB agents, indicating that AZD5847 may function well in combination therapies. AZD5847 appears to function similarly to linezolid through impairment of the mycobacterial 50S ribosomal subunit. Future studies should be undertaken to further characterize the pharmacodynamics and pharmacokinetics of AZD5847 in both in vitro and animal models as well is in human clinical trials.

Aminopyrazinamides: Novel and Specific GyrB Inhibitors that Kill Replicating and Nonreplicating Mycobacterium tuberculosis

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

Rv1218c, an ABC Transporter of Mycobacterium tuberculosis with Implications in Drug Discovery

ABSTRACT Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed.

Thiazolopyridine Ureas as Novel Antitubercular Agents Acting through Inhibition of DNA Gyrase B.

A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 μM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.

In Vitro and In Vivo Efficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

ABSTRACT Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

In Vitro and In Vivo Activities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

ABSTRACT Oxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starved Mycobacterium tuberculosis strain 18b. The in vitro drug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection with M. tuberculosis 18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.

1,4-Azaindole, a Potential Drug Candidate for Treatment of Tuberculosis

ABSTRACT New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.

Thiazolopyridone ureas as DNA gyrase B inhibitors: optimization of antitubercular activity and efficacy.

Scaffold hopping from the thiazolopyridine ureas led to thiazolopyridone ureas with potent antitubercular activity acting through inhibition of DNA GyrB ATPase activity. Structural diversity was introduced, by extension of substituents from the thiazolopyridone N-4 position, to access hydrophobic interactions in the ribose pocket of the ATP binding region of GyrB. Further optimization of hydrogen bond interactions with arginines in site-2 of GyrB active site pocket led to potent inhibition of the enzyme (IC50 2 nM) along with potent cellular activity (MIC=0.1 μM) against Mycobacterium tuberculosis (Mtb). Efficacy was demonstrated in an acute mouse model of tuberculosis on oral administration.

Mechanisms involved in attenuated cardio-protective role of ischemic preconditioning in metabolic disorders.

Myocardial infarction is a pathological state which occurs due to severe abrogation of the blood supply (ischemia) to a part of heart, which can cause myocardial damage. The short intermittent cycles of sub-lethal ischemia and reperfusion has shown to improve the tolerance of the myocardium against subsequent prolonged ischemia/reperfusion (I/R)-induced injury, which is known as ischemic preconditioning (IPC). Although, IPC-induced cardioprotection is well demonstrated in various species, including human beings, accumulated evidence clearly suggests critical abrogation of the beneficial effects of IPC in diabetes mellitus, hyperlipidemia and hyperhomocysteinemia. Various factors are involved in the attenuation of the cardioprotective effect of preconditioning, such as the reduced release of calcitonin gene-related peptide (CGRP), the over-expression of glycogen synthase kinase-3β (GSK-3β) and phosphatase and tensin homolog (PTEN), impairment of mito-KATP channels, the consequent opening of mitochondrial permeability transition pore (MPTP), etc. In this review, we have critically discussed the various signaling pathways involved in abrogated preconditioning in chronic diabetes mellitus, hyperlipidemia and hyperhomocysteinemia. We have also focused on the involvement of PTEN in abrogated preconditioning and the significance of PTEN inhibitors.