Carbohydrate Mimicry and Enzyme Inhibition

我将于2014年7月结束我负责研究的副总裁的任期，由于我已经积累了两年的行政休假，我将全职回到研究领域。我们的实验室积极了解其他配体模拟碳水化合物的分子机制，如合成碳水化合物模拟物(GlycoMimtics)或功能(Glyco)肽模拟物，并将其开发用于设计改进的诊断试剂、疫苗或疗法。在上一次赠款期间，所有这些领域都取得了重大进展，这从出版的工作中可见一斑；这一点已在共同履历(CCV)中进行了总结。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，并提出了初步的合成和机理探讨，最终读出是体外酶抑制和/或体外基于细胞的生长抑制。机械论的见解来自我们对酶反应中假定的过渡态的知识，这得益于我们以前的成功。我们的设计策略是基于一个广为接受的前提，即过渡态(TS)类似物将是作为酶抑制剂的最佳候选者，但在缺乏TS的详细知识的情况下，一个近似和可行的策略是尽可能地估计一个类似TS的中间体。然而，我们建议进一步利用从抑制剂和酶之间的额外相互作用中获得的能量，而不一定是在催化位置。此外，通过与主要催化位点和其他亚位点的相互作用，我们认为这些化合物将以高亲和力发挥作用，并可能避免新的耐药性的产生，因为可能需要多个突变(在几个亚位点上)来阻止化合物与目标酶的相互作用。我们在这里介绍了两个酶系统，我们使用这一方法来寻找有效的抑制剂。在高尔基甘露糖苷酶II(GMII)中，除了催化中心外，锚定中心、保持中心和锌配位都被确定为关键成分。在甲型流感神经氨酸酶的情况下，除了催化部位外，150和430个亚位点可以用来提供额外的联系。在GMII的案例中，我们利用了我们早先的发现，即一类新的硫离子是肠道葡萄糖苷酶的纳米分子抑制剂，并建议使用这一设计概念来结合催化亚基。值得注意的是，这些分子既不起烷化剂作用，也不起不可逆转的抑制作用。在第三种酶，UDP-Galp变位酶(UGM)的单独策略中，我们建议通过提供导致非生产性事件的替代底物来复制该酶。机理上的洞察力将与分子动力学计算相结合，并在可能的情况下辅之以标准核磁共振或X射线结构数据，以设计下一代候选者。这项研究的结果对于基本理解碳水化合物加工酶和碳水化合物模拟的性质和起源具有重要意义。

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.