New techniques in antibiotic discovery and resistance: Raman spectroscopy.
Raman spectroscopy can play a role in both antibiotic discovery and understanding the molecular basis of resistance. A major challenge in drug development is to measure the population of the drug molecules inside a cell line and to follow the chemistry of their reactions with intracellular targets. Recently, a protocol based on Raman microscopy has been developed that achieves these goals. Drug candidates are soaked into live bacterial cells and subsequently the cells are frozen and freeze-dried. The samples yield exemplary (nonresonance) Raman data that provide a measure of the number of drug molecules within each cell, as well as details of drug-target interactions. Results are discussed for two classes of compounds inhibiting either β-lactamase or dihydrofolate reductase enzymes in a number of Gram-positive or Gram-negative cell lines. The advantages of the present protocol are that it does not use labels and it can measure the kinetics of cell-compound uptake on the time scale of minutes. Spectroscopic interpretation is supported by in vitro Raman experiments. Studying drug-target interactions in aqueous solution and in single crystals can provide molecular level insights into drug-target interactions, which, in turn, provide the underpinnings of our understanding of data from bacterial cells. Thus, the applicability of X-ray crystallographic-derived data to in-cell chemistry can be tested.
Department of Biochemistry.|Department of Chemistry, Case Western Reserve University, Cleveland, Ohio.
Paul R
PR
Carey
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio.
Hossein
H
Heidari-Torkabadi
D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D055808:Drug Discovery / Q000379:methods*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D015203:Reproducibility of Results; D012996:Solutions; D013059:Spectrum Analysis, Raman / Q000295:instrumentation / Q000379:methods*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D065093:beta-Lactamase Inhibitors / Q000737:chemistry / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D012996:Solutions; D065093:beta-Lactamase Inhibitors
Raman crystallography; Raman microscopy; acyl-enzyme; dihydrofolate reductase; in-cell inhibitor population; rapid mix-rapid freeze; β-lactamase