Sulfotransferase Specificity and the Development of Sulfation Resistant Compounds

描述（由申请人提供）：本提案的目的是获得对人细胞质硫转移酶的分子行为的深入和基本的理解。这个由13个成员组成的酶家族通过将硫酰基（- so3）从核苷酸供体（PAPS, 3'-磷酸腺苷5'-硫酸磷酸）转移到小分子受体的羟基或胺基部分来调节数百个小分子的受体相互作用。了解SULT及其底物和变构调节剂之间的分子相互作用将大大加深我们对这些酶在生物学中的作用的理解，并提供一种控制体内SULT活性的方法。目的1 .我们发现SULT1A1利用正协同作用将选定底物的催化效率提高了103-104倍。这是SULT领域积极协同作用的第一个例子。这些令人惊叹的催化增强的分子基础将被确定，引发积极协同作用的底物特征将被确定，以了解如何控制硫酸盐-底物的反应性。Aim II的重点是硫代谢的一个重要的和几乎未开发的领域-硫代谢功能的变构调节。文献描述了少数重要的药物和营养素（阿司匹林，西乐葆®，Ponstel®和表没食子儿茶素没食子酸盐-）

DESCRIPTION (provided by applicant): The objective of this proposal is to obtain a deep and fundamental understanding of the molecular behavior of the human cytosolic sulfotransferases. This 13-member enzyme family regulates the receptor interactions of hundreds of small molecules by transferring the sulfuryl-group (-SO3) from a nucleotide donor (PAPS, 3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyl- or amine-moieties of small-molecule acceptors. Understanding the molecular interactions between SULTs and their substrates and allosteric modulators will substantially deepen our understanding of the roles of these enzymes in biology and provide a means of controlling SULT activity in-vivo. Aim I. We have discovered that SULT1A1 uses positive synergy to enhance the catalytic efficiencies of select substrates 103-104-fold. This is the first example of positive synergy in the SULT field. The molecular basis of these stunning catalytic enhancements will be determined, and the substrate features that elicit positive synergy will be identified with the goal of understanding how SULT-substrate reactivity is controlled. Aim II focuses on an important and virtually unexplored area in sulfur metabolism - the allosteric regulation of SULT function. The literature describes a small number of important drugs and nutrients (aspirin, Celebrex ®, Ponstel ® and epigallocatechin gallate - which comprises ~ 12% of the mass of dry tea leaves) that regulate SULTs by binding at sites separate from those of substrates. Binding is tight, isozyme specific and physiologically relevant. Certain compounds inhibit while others change the specificity and activate turnover of the enzyme. We will determine the first allostere-bound SULT structures - the crystals needed to do this are in-hand. Seeing these ligand-bound allosteric pockets at atomic resolution will change our perceptions of SULT metabolism and provide novel opportunities to control SULT activity. Aim III. Hundreds of FDA-approved drugs are inactivated by sulfation. Preventing this modification is expected to increase the concentration and half-lives of the active forms of these compounds in-vivo. Classical inhibition strategies are detrimental because they prevent essential SULT functions. Consequently, no means of achieving this end is described in the literature. Our recent insights into the molecular basis of SULT-substrate selectivity lay the foundations for a novel strategy to prevent sulfation without inhibiting SULTs or altering a compound's receptor-binding affinity. We will develop this strategy and demonstrate its therapeutic potential. Sidechains that prevent sulfation will be identified and inserted into two FDA-approved drugs whose bioactivities are potently suppressed by sulfation: apomorphine, used to treat late-stage Parkinson Disease, and ethinyl estradiol, the active estrogen in most oral contraceptives. The receptor affinities of these new compounds will be tested in mammalian cells and their metabolism will be evaluated using primary human hepatocytes.