Predicting the in vivo mechanism of action for drug leads using NMR metabolomics.
ABSTRACT New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the in vivo mechanism of action and efficacy of novel drug leads. Toward this end, we describe an unbiased approach to predict in vivo mechanisms of action from NMR metabolomics data. Mycobacterium smegmatis, a non-pathogenic model organism for Mycobacterium tuberculosis, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the M. smegmatis metabolome resulted in distinct clustering patterns correlating with in vivo drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity.
Article: Effect of chloramphenicol, erythromycin, moxifloxacin, penicillin and tetracycline concentration on the recovery of resistant mutants of Mycobacterium smegmatis and Staphylococcus aureus.[show abstract] [hide abstract]
ABSTRACT: The effect of antimicrobial concentration on colony-forming ability of resistant mutant subpopulations of Mycobacterium smegmatis and Staphylococcus aureus was measured for chloramphenicol, erythromycin, moxifloxacin, penicillin and tetracycline. The relationship between drug concentration and the recovery of mutant colonies was distinct for each bacterium-antimicrobial combination; however, in each case application of large numbers of cells to drug-containing agar plates revealed a progressive reduction in mutant recovery as antimicrobial concentration increased. The minimal concentration that allowed no mutant recovery from more than 10(10) input cells was measured to estimate the minimum inhibitory concentration (MIC) of the least susceptible, single-step mutant subpopulation, a parameter also called the mutant prevention concentration (MPC). These data expand the number of antimicrobial-bacterial combinations for which a mutant selection window can be measured.Journal of Antimicrobial Chemotherapy 08/2003; 52(1):61-4. · 5.07 Impact Factor
Article: Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics.[show abstract] [hide abstract]
ABSTRACT: Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major beta-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most beta-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based beta-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from beta-lactamase deletion mutants) that are hypersusceptible to beta-lactam antibiotics might reveal novel genes involved with other mechanisms of beta-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine beta-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode beta-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.Journal of Bacteriology 04/2005; 187(6):1892-900. · 3.83 Impact Factor
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ABSTRACT: A simple, robust assay system which can be used to screen for inhibitors of mycobacterial growth has been developed. A strain of the rapidly growing saprophyte Mycobacterium aurum is used as the test organism. Inhibition of its growth is highly predictive of activity against Mycobacterium tuberculosis, which cannot itself be used in screening because of its growth characteristics and highly infectious nature. The viability of M. aurum in the presence of a test sample is monitored by measuring the uptake of radiolabelled uracil into the cells. In a microtiter plate format, the screen has the potential for testing several thousand samples per day.Antimicrobial Agents and Chemotherapy 11/1995; 39(10):2235-8. · 4.84 Impact Factor