[Show abstract][Hide abstract] ABSTRACT: We present here the novel ketolide RBx 14255, a semisynthetic macrolide derivative obtained by the derivatization of clarithromycin,
for its in vitro and in vivo activities against sensitive and macrolide-resistant Streptococcus pneumoniae. RBx 14255 showed excellent in vitro activity against macrolide-resistant S. pneumoniae, including an in-house-generated telithromycin-resistant strain (S. pneumoniae 3390 NDDR). RBx 14255 also showed potent protein synthesis inhibition against telithromycin-resistant S. pneumoniae 3390 NDDR. The binding affinity of RBx 14255 toward ribosomes was found to be more than that for other tested drugs. The
in vivo efficacy of RBx 14255 was determined in murine pulmonary infection induced by intranasal inoculation of S. pneumoniae ATCC 6303 and systemic infection with S. pneumoniae 3390 NDDR strains. The 50% effective dose (ED50) of RBx 14255 against S. pneumoniae ATCC 6303 in a murine pulmonary infection model was 3.12 mg/kg of body weight. In addition, RBx 14255 resulted in 100% survival
of mice with systemic infection caused by macrolide-resistant S. pneumoniae 3390 NDDR at 100 mg/kg four times daily (QID) and at 50 mg/kg QID. RBx 14255 showed favorable pharmacokinetic properties
that were comparable to those of telithromycin.
Preview · Article · Feb 2014 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an
immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean.
UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall
biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one
copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of
both enzymes. These enzymes had an apparent affinity constant (K
) for its substrate UDP-N-acetylglucosamine 36±5.2 and 17.8±2.5μM and for phosphoenolpyruvate 0.84±0.13μM and 0.45±0.07μM
for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC50 for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used
to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.
KeywordsMurA-UDP-N-acetylglucosamine-Enolpyruvyl-UDP-N-acetylglucosamine-Fosfomycin-High throughput screening
Full-text · Article · Sep 2010 · World Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new
investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics
study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically
inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners
the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively.
The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from
the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity
against the target pathogen.
[Show abstract][Hide abstract] ABSTRACT: Enzymes of the cytochrome P450 (CYP) superfamily are the major determinants of half-life and execute pharmacological effects of many therapeutic drugs. In new drug discovery research, recombinant (human) CYPs are also used for identifying active or inactive metabolites that could lead to increased potency or toxicity of a molecule. In addition, CYP inhibition by anticancer drugs might lead to adverse drug reactions, multiple-drug resistance, and drug-drug interactions. During the discovery and pre-clinical evaluation of a New Chemical Entity (NCE), large amounts of purified recombinant CYPs are required for studying metabolism and pharmacokinetic parameters. Therefore, present research efforts are focused to over-express these human CYPs in bacteria, yeast, insect and mammalian cells, followed by their purification on an industrial scale to facilitate identification of novel anticancer drugs. This review summarizes the merits and limitations of these expression systems for an optimized production of individual CYP isoforms, and their usefulness in the discovery and development of target-based, safe and efficacious NCEs for the treatment of cancer.
[Show abstract][Hide abstract] ABSTRACT: Decreased susceptibility of Neisseria meningitidis isolates to ciprofloxacin emerged from an outbreak in Delhi, India. Results of antimicrobial susceptibility testing of the meningococcal isolates to ciprofloxacin and further sequencing of DNA gyrase A quinolone-resistance-determining region confirmed the emergence of ciprofloxacin resistance in the outbreak.
Full-text · Article · Nov 2007 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP-area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with beta-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8-16 microg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 microg/mL to > 16 microg/mL, with a PAP-AUC ratio of > 1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and beta-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.
No preview · Article · Apr 2006 · International Journal of Antimicrobial Agents
[Show abstract][Hide abstract] ABSTRACT: Uracil-DNA glycosylase (Ung), a DNA repair enzyme, pioneers uracil excision repair pathway. Structural determinations and mutational analyses of the Ung class of proteins have greatly facilitated our understanding of the mechanism of uracil excision from DNA. More recently, a hybrid quantum-mechanical/molecular mechanical analysis revealed that while the histidine (H67 in EcoUng) of the GQDPYH motif (omega loop) in the active site pocket is important in positioning the reactants, it makes an unfavorable energetic contribution (penalty) in achieving the transition state intermediate. Mutational analysis of this histidine is unavailable from any of the Ung class of proteins. A complication in demonstrating negative role of a residue, especially when located within the active site pocket, is that the mutants with enhanced activity are rarely obtained. Interestingly, unlike the most Ung proteins, the H67 equivalent in the omega loop in mycobacterial Ung is represented by P67. Exploiting this natural diversity to maintain structural integrity of the active site, we transplanted an H67P mutation in EcoUng. Uracil inhibition assays and binding of a proteinaceous inhibitor, Ugi (a transition state substrate mimic), with the mutant (H67P) revealed that its active site pocket was not perturbed. The catalytic efficiency (Vmax/Km) of the mutant was similar to that of the wild type Ung. However, the mutant showed increased Km and Vmax. Together with the data from a double mutation H67P/G68T, these observations provide the first biochemical evidence for the proposed diverse roles of H67 in catalysis by Ung.
Full-text · Article · Aug 2004 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The structures of a new crystal form of free Escherichia coli uracil DNA glycosylase (UDG), containing four molecules in the asymmetric unit, and two forms of its complex with the proteinaceous inhibitor Ugi, containing two and four crystallographically independent complexes, have been determined. A comparison of these structures and the already known crystal structures containing UDG shows that the enzyme can be considered to be made up of two independently moving structural entities or domains. A detailed study of free and DNA-bound human enzyme strengthens this conclusion. The domains close upon binding to uracil-containing DNA, whereas they do not appear to do so upon binding to Ugi. The comparative study also shows that the mobility of the molecule involves the rigid-body movement of the domains superposed on flexibility within domains.
[Show abstract][Hide abstract] ABSTRACT: Single-stranded DNA-binding proteins play an important role in DNA replication, repair and recombination. The protein from Mycobacterium tuberculosis (MtSSB) is a tetramer with 164 amino-acid residues in each subunit. The protein readily crystallizes in space group P3(1)21 (or P3(2)21) at pH 7.4 under appropriate conditions. Under different conditions, but at the same pH, orthorhombic crystals belonging to space group I222 or I2(1)2(1)2(1) were obtained after several months. Similar orthorhombic crystals were obtained when protein samples stored for several months were used for crystallization. The orthorhombic crystals obtained in different experiments, though similar to one another, exhibited variations in unit-cell parameters, presumably on account of different extents of proteolytic cleavage of the C-terminal region. Molecular-replacement calculations using different search models did not yield the structure. As part of attempts to solve the structure using isomorphous replacement, a good mercury derivative of the trigonal crystal has been prepared.
No preview · Article · Mar 2002 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions. Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world's population and kills about 2-3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli. In this study, use of the yeast two-hybrid assay suggests that the ECO:SSB and the MTU:SSB are even capable of heterooligomerization. However, the MTU:SSB failed to complement a Deltassb strain of E. coli. The sequence comparison suggested that MTU:SSB contained a distinct C-terminal domain. The C-terminal domain of ECO:SSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MTU:SSB and ECO:SSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Deltassb strain of E.coli. These data allude to the occurrence of a 'cross talk' between the N- and the C-terminal domains of the SSBs for their in vivo function. Further, compared with those of the ECO:SSB, the secondary/tertiary interactions within MTU:SSB were found to be less susceptible to disruption by guanidinium hydrochloride. Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.
Full-text · Article · Nov 2000 · Nucleic Acids Research