Carbohydrate Mimicry and Enzyme Inhibition

我将于2014年7月结束研究副总裁的任期，因为我已经累积了两年的行政休假，我将全职回到研究工作中。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（糖仿制品）或功能（糖）肽模拟物，并将其用于改进诊断试剂、疫苗或治疗药物的设计。从已发表的工作中可以看出，在上一次赠款期间，所有这些领域都取得了重大进展；这在common CV （CCV）中得到了总结。目前的建议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调查，最终的结果是体外酶抑制和/或体外细胞生长抑制。机制的见解源于我们对酶促反应中假定的过渡状态的了解，这是我们以前成功的结果。我们的设计策略基于一个公认的前提，即过渡态（TS）类似物将是酶抑制剂的最佳候选物，但在缺乏对TS的详细了解的情况下，一个近似且可行的策略是尽可能估计与TS相似的中间体。然而，我们建议进一步利用抑制剂和酶之间额外相互作用获得的能量，而不一定是在催化位点。此外，通过与一级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具有高亲和力，并且可能避免新抗性的发展，因为可能需要多个亚位点的多重突变来阻止化合物与靶酶的相互作用。我们在这里提出了两种酶系统，我们通过使用这种方法寻求有效的抑制剂。在高尔基甘露糖苷酶II （GMII）的情况下，除了催化位点外，锚定位点、保持位点和锌配位也被确定为关键成分。在甲型流感神经氨酸酶的情况下，除了催化位点，150和430亚位点可以用来提供额外的接触。在GMII的情况下，我们利用了我们早期的发现，即一类新的磺酸离子是肠道葡萄糖苷酶的纳摩尔抑制剂，并建议使用这种设计概念在催化亚位点结合。值得注意的是，这些分子不作为烷基化剂或不可逆抑制剂。在使用第三种酶，UDP-Galp突变酶（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欺骗酶。机理分析将与分子动力学计算相结合，并在可能的情况下辅以STD核磁共振或x射线结构数据，以设计下一代候选材料。本研究的结果对碳水化合物加工酶的基本认识以及碳水化合物拟态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.