Carbohydrate Mimicry and Enzyme Inhibition

我将在2014年7月结束我的副总裁任期，并将全职返回研究，因为我已经积累了两年的行政休假。我们的实验室积极了解其他配体的碳水化合物模拟的分子机制，如合成的碳水化合物模拟物（糖模拟物）或功能性（糖）肽模拟物，并利用它们设计改进的诊断剂，疫苗或治疗剂。在上一个资助期内，所有这些领域都取得了重大进展，这一点从已发表的工作中可以看出;共同简历（CCV）中对此进行了总结。本提案侧重于哺乳动物和微生物来源的碳水化合物加工酶，并提出了一个主要的合成和机制的调查，最终的读数是在体外酶抑制和/或在体外细胞为基础的生长抑制。机制的见解来自我们的知识，假定的过渡状态，在酶促反应，通知我们以前的成功。我们的设计策略是基于公认的前提下，过渡态（TS）类似物将是作为酶抑制剂的最佳候选人，但在没有详细的知识的TS，一个近似的和可行的策略是估计最好的一个可以类似的TS中间。然而，我们建议进一步利用抑制剂和酶之间的额外相互作用所获得的能量，而不一定是在催化位点。此外，通过与主要催化位点和其他亚位点的相互作用，我们认为化合物将以高亲和力起作用，并可能避免新抗性的发展，因为可能需要多个突变（在几个亚位点中）来防止化合物与靶酶的相互作用。我们在这里提出了两个酶系统，我们寻求有效的抑制剂，通过使用这种方法。在高尔基甘露糖苷酶II（GMII）的情况下，除了催化位点，锚定位点，持有网站，和锌协调已被确定为关键组件。在甲型流感神经氨酸酶的情况下，除了催化位点之外，150和430亚位点可用于提供额外的接触。在GMII的情况下，我们已经利用了我们早期的发现，一类新的锍离子是肠葡萄糖苷酶的纳摩尔抑制剂，并建议使用这种设计概念结合在催化亚位点。值得注意的是，这些分子不作为烷基化剂或不可逆抑制剂。在一个单独的战略与第三种酶，UDP-半乳糖苷酶（UGM），我们建议通过提供一个替代底物，导致非生产性事件的欺骗酶。机制的见解将与分子动力学计算相结合，并在可能的情况下辅以STD NMR或X射线结构数据，以设计下一代候选物。这项研究的结果对碳水化合物加工酶的基本理解以及碳水化合物模拟的性质和起源具有影响。

I will finish my term as Vice President, Research in July, 2014 and will return full time to research since I have accumulated two years of administrative leave. Our laboratory is active in understanding the molecular mechanisms of carbohydrate mimicry by other ligands, such as synthetic carbohydrate mimics (glycomimetics) or functional (glyco)peptide mimics, and in their exploitation for the design of improved diagnostic agents, vaccines, or therapeutics. Over the last grant period, significant progress has been made in all of these areas, as evident from the published work; this has been summarized in the common CV (CCV). The present proposal focuses on carbohydrate-processing enzymes of mammalian and microbial origin, and presents a primarily synthetic and mechanistic inquiry, with the ultimate readout being in vitro enzyme inhibition and/or in vitro cell-based growth inhibition. The mechanistic insights derive from our knowledge of putative transition states in enzymatic reactions, informed by our previous successes. Our design strategy is based on the well accepted premise that a transition-state (TS) analogue would be the optimal candidate as an enzyme inhibitor, but in the absence of detailed knowledge of the TS, an approximate and viable strategy is to estimate as best one can an intermediate resembling the TS. However, we propose further to harness the energy gained from additional interactions between inhibitor and enzyme, not necessarily in the catalytic site. Furthermore, through interaction with both the primary catalytic site and other subsites, we contend that the compounds will act with high affinity, and potentially evade the development of new resistance as multiple mutations (in several subsites) may be required to prevent the compound’s interaction with the target enzymes. We present here two enzyme systems for which we seek effective inhibitors by use of this approach. In the case of Golgi Mannosidase II (GMII), in addition to the catalytic site, an anchoring site , a holding site, and Zn coordination have been identified as critical components. In the case of influenza A neuraminidases, in addition to the catalytic site, the 150 and 430 subsites could be utilized to provide additional contacts. In the case of GMII, we have capitalized on our earlier discovery that a new class of sulfonium ions are nanomolar inhibitors of intestinal glucosidase enzymes, and propose to use this design concept for binding in the catalytic subsite. Remarkably, these molecules do not act as alkylating agents or irreversible inhibitors. In a separate strategy with a third enzyme, UDP-Galp mutase (UGM), we propose to dupe the enzyme by supplying a surrogate substrate that leads to a non-productive event. The mechanistic insights will be coupled with molecular dynamics calculations, complemented by STD NMR or X-ray structural data when possible, to design the next-generation candidates. The results of this research have implications for the fundamental understanding of carbohydrate processing enzymes and the nature and origin of carbohydrate mimicry.