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1.
Antimicrob Agents Chemother ; 66(12): e0092122, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36448795

ABSTRACT

CUO246, a novel DNA gyrase/topoisomerase IV inhibitor, is active in vitro against a broad range of Gram-positive, fastidious Gram-negative, and atypical bacterial pathogens and retains activity against quinolone-resistant strains in circulation. The frequency of selection for single step mutants of wild-type S. aureus with reduced susceptibility to CUO246 was <4.64 × 10-9 at 4× and 8× MIC and remained low when using an isogenic QRDR mutant (<5.24 × 10-9 at 4× and 8× MIC). Biochemical assays indicated that CUO246 had potent inhibitory activity against both DNA gyrase (GyrAB) and topoisomerase IV (ParCE). Furthermore, CUO246 showed rapid bactericidal activity in time-kill assays and potent in vivo efficacy against S. aureus in a neutropenic murine thigh infection model. These results suggest that CUO246 may be useful in treating infections by various causative agents of acute skin and skin structure infections, respiratory tract infections, and sexually transmitted infections.


Subject(s)
DNA Gyrase , DNA Topoisomerase IV , Animals , Mice , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , Topoisomerase II Inhibitors/pharmacology , DNA, Bacterial , Staphylococcus aureus , Microbial Sensitivity Tests , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use
2.
J Am Chem Soc ; 142(9): 4445-4455, 2020 03 04.
Article in English | MEDLINE | ID: mdl-32064871

ABSTRACT

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.


Subject(s)
Acyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Pyrazoles/pharmacology , Acyltransferases/metabolism , Anti-Bacterial Agents/metabolism , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Imidazoles/metabolism , Microbial Sensitivity Tests , Protein Binding , Pyrazoles/metabolism
3.
Bioorg Med Chem Lett ; 25(17): 3468-75, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26189081

ABSTRACT

We describe the synthesis and evaluation of a library of variably-linked ciprofloxacin dimers. These structures unify and expand on the use of fluoroquinolones as probes throughout the antibiotic literature. A dimeric analog (19) showed enhanced inhibition of its intracellular target (DNA gyrase), and translation to antibacterial activity in whole cells was demonstrated. Overall, cell permeation was governed by physicochemical properties and bacterial type. A principal component analysis demonstrated that the dimers occupy a unique and privileged region of chemical space most similar to the macrolide class of antibiotics.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Infective Agents/chemical synthesis , Ciprofloxacin/chemical synthesis , DNA, Bacterial/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Ciprofloxacin/chemistry , Ciprofloxacin/pharmacology , Permeability
4.
Proc Natl Acad Sci U S A ; 110(21): 8696-701, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23650377

ABSTRACT

Organisms that use ammonium as the sole nitrogen source discriminate between [(15)N] and [(14)N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥ 0.89 µM), discrimination against the heavy isotope by the WT strain (-19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4(+) to NH3 + H(+). NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤ 0.18 µM), discrimination reflects an isotope effect for the NH4(+) channel AmtB (-14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (-10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.


Subject(s)
Cation Transport Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli K12/metabolism , Escherichia coli Proteins/metabolism , Nitrogen/metabolism , Quaternary Ammonium Compounds/metabolism , Ion Transport/physiology , Nitrogen Isotopes/metabolism
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