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Determination of mode of action for novel synthetic antifungal agents using reversal assay method

机译:使用逆向测定法测定新型合成抗真菌剂的作用方式

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A classical method of identifying the mode of action is reversal assay, which has been used in the present study. The basis of this process is to reverse biological activity of a drug by adding metabolic intermediates or final product of a given pathway. Here, we have used two synthetic antifungal compounds, A and B which are not structurally similar to current antifungal drugs. Synthetic medium RPMI 1640 enriched by 2% glucose and Saccharomyces cerevisiae BY 4743 as the test microorganism was used for reversal assay.Fluorouracil, and benodanil were used as positive controls in assessing the reversal effect of nucleic acids and TCA cycle intermediates, respectively. Reversal assays was performed in 96-well flat-bottomed microplates. Reversing concentration of each nucleotide, amino acid or TCA cycle intermediates was determined through preparation of twofold serial dilutions across the concentration range (0-200 μg/ml) of each compound in the above medium containing the minimum inhibitory concetration of either compound A or B. In this study, reversal assay showed to be effective in identifying the inhibition sites and hence leading us to the mechanism of action. Our synthetic agents had effect on inhibition of nucleic acid pathway (compound A) and aliphatic amino acid synthesis and to a lesser extent on TCA cycle (compound B). Further study to identify the main target of each compound is underway.
机译:识别动作的模式的经典方法是逆转测定法,已在本研究中被使用。该方法的基础是通过将代谢中间体或给定途径的最终产物以扭转的药物的生物活性。在这里,我们已经使用了两种合成的抗真菌化合物,A和B在结构上不类似于目前抗真菌药物。合成培养基RPMI 1640富集了2%葡萄糖和酿酒酵母BY 4743作为测试微生物被用于反转assay.Fluorouracil,和麦锈灵用作评估的核酸和TCA循环中间体,分别的逆转作用的阳性对照。逆转测定法在96孔平底微量培养板进行。反转每个核苷酸的浓度,氨基酸或TCA循环中间体是通过在上述培养基含有化合物A或B的最小抑制concetration各化合物的制备两倍系列稀释液在整个浓度范围(0-200微克/毫升)来确定在本研究中,反转检测显示可有效地识别所述抑制站点,因此导致我们的作用机制。我们的合成剂对抑制核酸途径(化合物A)和脂肪族氨基酸的合成的,并就TCA循环(化合物B)在较小程度上的影响。进一步的研究,以确定每种化合物的主要对象是正在进行中。

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