A New Co-Expressed P450 and UGT Platform for the Simultaneous Preparation of P450 Metabolites and Their Glucuronides

毒素、药物和生理上的吲哚化合物通过几种代谢途径被肝脏代谢。 酶的种类。尿苷二磷酸葡萄糖醛酸基转移酶（UGT）集中在 肝脏也存在于其他组织中。这些酶将UDP-葡糖醛酸（UDPGA）添加到化合物中， 从而增加其溶解度和在胆汁和尿中排泄的能力。 牛津生物医学研究所创造了一种专有的方法来处理巴斯德毕赤酵母菌株 优化表达细胞色素P450（P450）和氧化还原酶，导致不断增长的产品线 专为制药研究量身定制。这个过程将酵母转化为稳定的干粉（SDP） 格式，它表现出显着的有机溶剂和热耐受性沿着与延长催化时， 与传统的重组酶相比。拟议的多催化平台，能够同时 P450氧化，然后UGT葡萄糖醛酸化，将为制药研究人员提供一个有价值的工具， 制备葡萄糖醛酸苷，研究P450/UGT协同关系。 第一阶段的提议将涉及生产和优化三种催化系统：第一种毕赤酵母系统将 表达负责将UDP-葡萄糖转化为UDPGA的脱氢酶， 葡糖苷酸生成。第二个系统将在此平台上扩展，包括UGT 2B 7 酶以产生能够使4-甲基伞形酮葡萄糖醛酸化的催化剂。第三个也是最后一个系统 将CYP 3A 4和P450氧化还原酶（POR）添加到该表达系统中， 开发重点是优化1 '-羟基咪达唑仑葡糖苷酸生产。 实现这些目标将为制药研究提供一个强大的，易于使用的系统， 在一个系统中进行重要的代谢反应; P450氧化，然后进行UGT葡萄糖醛酸化。

Toxins, pharmaceuticals and physiologically indogenous compounds are metabolized by the liver via several classes of enzymes. The uridine diphosphate glucuronosyltransferases (UGTs) are concentrated in the liver but also present in other tissues. These enzymes add UDP-glucuronic acid (UDPGA) to a compound, thereby increasing its solubility and ability to be excreted in the bile and urine. Oxford Biomedical Research has created a proprietary method for processing a strain of Pichia pastoris optimized for expressing the cytochromes P450 (CYP) and oxidoreductase, resulting in a growing product line tailored to pharmaceutical research. This process transforms the yeast into a stabilized dry powder (SDP) format, which exhibits remarkable organic solvent and thermal tolerance along with extended catalysis when compared to traditional recombinant enzymes. The proposed multi-catalytic platform, capable of simultaneous P450 oxidation followed by UGT glucuronidation, would give the pharmaceutical researcher a valuable tool for preparing glucuronides as well as studying the P450/UGT synergistic relationship. This Phase I proposal will involve producing and optimizing three catalytic systems:the first Pichia system will express the dehydrogenase responsible for converting UDP-glucose to UDPGA, the cofactor required for glucuronide production. The second system will include expand on this platform and include the UGT2B7 enzyme to create a catalyst capable of glucuronidating 4-methylumbelliferone. The third and final system will add into this expression system the CYP3A4 and P450 oxidoreductase (POR) enzymes, with development focusing on the optimization of 1'-hydroxymidazolam glucuronide production. Achieving these goals will give pharmaceutical research a robust, easy-to-use system for performing two metabolically-important reactions in one system; P450 oxidation followed by UGT glucuronidation.