Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins

Dunn, Elyse; Cook, Gregory M.; Heikal, Adam

doi:10.1016/j.mimet.2015.11.012

Cited by 5 publications

(4 citation statements)

References 16 publications

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“…Bacteria are routinely grown, and antimicrobial screening conducted, under conditions optimized for growth in the laboratory (oxygenation, temperature, pH, nutrient availability), even though conditions under which the target organisms cause disease may differ considerably from laboratory conditions and in fact, may be ‘sub-optimal’ for growth (Cooper, 2013). Variations in screening conditions have been shown to identify different inhibitors both in target-based and phenotypic screens (Miller et al, 2009; Dunn et al, 2015). Furthermore, it has recently been shown that apparently simple antimicrobial killing assays are very sensitive to variations in culture conditions and bacterial growth phase (Harms et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria

et al. 2019

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In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis , Staphylococcus aureus , and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, β-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 μM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.

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Section: Introductionmentioning

confidence: 99%

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria

et al. 2019

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“…Type II NADH dehydrogenase (NDH-2) was purified and reconstituted into proteoliposomes as described by Dunn et al [2] , [3] . Detergent-solubilised (DS) NDH-2 or lipid-reconstituted (LR) NDH-2 were screened by end point 96-well assay (340 nm, Varioskan® Flash Thermo Scientific, in the presence of 100 μM menadione and 20 μM test compound.…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

“…Screening was carried out in technical triplicate against each compound library. NADH: menadione oxidoreductase inhibition/stimulation was calculated as a percentage of control activity for either DS or LR protein [2] . Compounds showing inhibition (≤70% control) or activation (≥130% control) in at least 2 of the 3 independent screening replicates were considered hits.…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

“…The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2] .…”

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Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

2016

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The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2].

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Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target

Hartuti

Inaoka

Komatsuya

et al. 2018

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins

Cited by 5 publications

References 16 publications

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria

Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target

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