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

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

Simple and Rapid Method To Determine Antimycobacterial Potency of Compounds by Using Autoluminescent Mycobacterium tuberculosis

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (similar to 20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods

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

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

DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues

The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway producing the universally essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the cramped substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.De tre (3) första författarna delar förstaförfattarskapet.</p

Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of <i>Mycobacterium tuberculosis</i>

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of <i>M. tuberculosis</i> (Mtu) H37Rv. The series exhibited tangible structure–activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action

Benzimidazoles: Novel Mycobacterial Gyrase Inhibitors from Scaffold Morphing

Type II topoisomerases are well conserved across the bacterial species, and inhibition of DNA gyrase by fluoroquinolones has provided an attractive option for treatment of tuberculosis (TB). However, the emergence of fluoroquinolone-resistant strains of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) poses a threat for its sustainability. A scaffold hopping approach using the binding mode of novel bacterial topoisomerase inhibitors (NBTIs) led to the identification of a novel class of benzimidazoles as DNA gyrase inhibitors with potent anti-TB activity. Docking of benzimidazoles to a NBTI bound crystal structure suggested that this class of compound makes key contacts in the enzyme active site similar to the reported NBTIs. This observation was further confirmed through the measurement of DNA gyrase inhibition, and activity against <i>Mtb</i> strains harboring mutations that confer resistance to aminopiperidines based NBTIs and <i>Mtb</i> strains resistant to moxifloxacin. Structure–activity relationship modification at the C-7 position of the left-hand side ring provided further avenue to improve hERG selectivity for this chemical series that has been the major challenges for NBTIs

Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates

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-&beta;-naphthylamide (PA&beta;N). 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 PA&beta;N. 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.<br /