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Metzler K.,University of Saskatchewan | Drlica K.,Public Health Research Institute Center | Blondeau J.M.,University of Saskatchewan | Blondeau J.M.,Royal University
Journal of Antimicrobial Chemotherapy

Background: Previous work showed a higher prevalence of macrolide/azalide resistance in provinces of Canada where azithromycin was the major treatment for Streptococcus pneumoniae as compared with regions where clarithromycin was the dominant treatment. These data provided a way to test the mutant selection window hypothesis, which predicts that the serum drug concentration (AUC24) relative to the mutant prevention concentration (MPC) would be higher for clarithromycin than for azithromycin. Methods: The MIC and MPC were determined for 191 penicillin/macrolide-susceptible clinical isolates of S. pneumoniae with azithromycin, clarithromycin and erythromycin using agar plate assays. Results: The MIC50/90 (mg/L) and MPC50/90 (mg/L), respectively, were as follows: azithromycin 0.13/0.25 and 1/4; clarithromycin 0.031/0.063 and 0.13/0.5; erythromycin 0.063/0.13 and 0.25/2. We calculated from published pharmacokinetic values that the AUC24/MPC90 for azithromycin was 0.85; for clarithromycin it was 96, and for erythromycin base and estolate it was 4 and 10, respectively. Thus the AUC. 24/MPC. 90 was about 50 times higher for clarithromycin than for azithromycin. Conclusions: The elevated prevalence of azithromycin resistance may derive in part from a low value of AUC24/MPC90 and/or time above MPC, since previous work indicates that the number of prescriptions per person was similar in the geographical regions examined. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. Source

Rooklin D.W.,New York University | Lu M.,Public Health Research Institute Center | Zhang Y.,New York University
Journal of the American Chemical Society

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) represents a new family of apyrase enzymes that catalyze the hydrolysis of nucleotide di- and triphosphates, thereby modulating extracellular purinergic and pyrimidinergic signaling. Among well-characterized phosphoryl transfer enzymes, hSCAN-1 is unique not only in its unusual calcium-dependent activation, but also in its novel phosphate-binding motif. Its catalytic site does not utilize backbone amide groups to bind phosphate, as in the common P-loop, but contains a large cluster of acidic ionizable side chains. By employing a state-of-the-art computational approach, we have revealed a previously uncharacterized catalytic calcium-binding site in hSCAN-1, which elucidates the unusual calcium-dependence of its apyrase activity. In a high-order coordination shell, the newly identified calcium ion organizes the active site residues to mediate nucleotide binding, to orient the nucleophilic water, and to facilitate the phosphoryl transfer reaction. From ab initio QM/MM molecular dynamics simulations with umbrella sampling, we have characterized a reverse protonation catalytic mechanism for hSCAN-1 and determined its free energy reaction profile. Our results are consistent with available experimental studies and provide new detailed insight into the structure-function relationship of this novel calcium-activated phosphoryl transfer enzyme. © 2012 American Chemical Society. Source

Johnson L.M.,University of Wisconsin - Madison | Mortenson D.E.,University of Wisconsin - Madison | Yun H.G.,University of Wisconsin - Madison | Horne W.S.,University of Wisconsin - Madison | And 4 more authors.
Journal of the American Chemical Society

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains. © 2012 American Chemical Society. Source

Kim M.-J.,Cornell University | Wainwright H.C.,University of Cape Town | Locketz M.,University of Cape Town | Bekker L.-G.,University of Cape Town | And 12 more authors.
EMBO Molecular Medicine

The progression of human tuberculosis (TB) to active disease and transmission involves the development of a caseous granuloma that cavitates and releases infectious Mycobacterium tuberculosis bacilli. In the current study, we exploited genome-wide microarray analysis to determine that genes for lipid sequestration and metabolism were highly expressed in caseous TB granulomas. Immunohistological analysis of these granulomas confirmed the disproportionate abundance of the proteins involved in lipid metabolism in cells surrounding the caseum; namely, adipophilin, acyl-CoA synthetase long-chain family member 1 and saposin C. Biochemical analysis of the lipid species within the caseum identified cholesterol, cholesteryl esters, triacylglycerols and lactosylceramide, which implicated low-density lipoprotein-derived lipids as the most likely source. M. tuberculosis infection in vitro induced lipid droplet formation in murine and human macrophages. Furthermore, the M. tuberculosis cell wall lipid, trehalose dimycolate, induced a strong granulomatous response in mice, which was accompanied by foam cell formation. These results provide molecular and biochemical evidence that the development of the human TB granuloma to caseation correlates with pathogen-mediated dysregulation of host lipid metabolism. © 2010 EMBO Molecular Medicine. Source

Sarathy J.,Novartis | Sarathy J.,Public Health Research Institute Center | Dartois V.,Novartis | Dartois V.,Public Health Research Institute Center | And 4 more authors.
Antimicrobial Agents and Chemotherapy

During active tuberculosis a spectrum of physiologically different Mycobacterium tuberculosis bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down M. tuberculosis from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating M. tuberculosis. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant M. tuberculosis. Copyright © 2013, American Society for Microbiology. All Rights Reserved. Source

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