Sulfotransferase Specificity and the Development of Sulfation Resistant Compounds

描述(由申请人提供)：本提案的目的是对人胞浆磺基转移酶的分子行为有一个深入和基本的了解。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’-磷酸硫酸盐)转移到小分子受体的羟基或胺基上来调节数百个小分子的受体相互作用。了解硫磺及其底物和变构调节剂之间的分子相互作用将极大地加深我们对这些酶在生物学中的作用的理解，并提供一种在体内控制硫磺活性的手段。目的I.我们发现SULT1A1利用正协同效应将选定底物的催化效率提高103-104倍。这是SULT领域的第一个积极协同的例子。这些惊人的催化增强的分子基础将被确定，并将确定引起正协同作用的底物特征，目的是了解底物反应活性是如何被控制的。目的II集中在硫代谢的一个重要且几乎未被探索的领域--硫磺功能的变构调节。文献描述了少量重要的药物和营养素(阿司匹林、塞来宝、庞斯泰和表没食子儿茶素没食子酸酯)。 约占干茶叶质量的12%)，通过在与底物分离的位置结合来调节硫磺。结合紧密，同工酶特异，具有生理相关性。某些化合物抑制酶的活性，而另一些化合物则改变酶的专一性并激活酶的周转。我们将确定第一个异构体结合的晶体结构--完成这一任务所需的晶体已经准备就绪。看到这些配体结合的变构口袋在原子分辨率下将改变我们对sulth新陈代谢的看法，并为控制sult活性提供新的机会。目的III.成百上千种FDA批准的药物通过硫酸盐灭活。阻止这种修饰预计会增加这些化合物在体内的活性形式的浓度和半衰期。经典的抑制策略是有害的，因为它们阻碍了基本的结果功能。因此，文献中没有描述实现这一目的的方法。我们最近对底物选择性的分子基础的洞察为一种新的策略奠定了基础，该策略可以在不抑制硫酸盐或改变化合物的受体结合亲和力的情况下防止硫酸盐化。我们将开发这一策略，并展示其治疗潜力。将识别防止硫酸盐化的侧链，并将其插入FDA批准的两种被硫酸盐化有效抑制生物活性的药物中：用于治疗晚期帕金森病的阿朴吗啡，以及大多数口服避孕药中的活性雌激素乙炔雌二醇。这些新化合物的受体亲和力将在哺乳动物细胞中进行测试，它们的新陈代谢将使用原代人类肝细胞进行评估。

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 (r), Ponstel (r) 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.