BOTULINUM NEUROTOXIN SEROTYPE B INHIBITOR DESIGN

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这七种肉毒杆菌神经毒素(BoNTs)起源于肉毒杆菌，无一例外地是人类已知的最有效的毒素--绕过破伤风、眼镜蛇和蓖麻毒素的致命性。人类可能遇到毒素的情况很多，包括生物恐怖袭击、美容和非美容医疗应用以及食物中毒。事实上，尽管到目前为止，B血清型(BONT/B)并不是科学界关注的重点，但已经证明，每年由这类毒素引起的食物中毒病例中，有很大一部分是由B型血清型引起的。鉴于BONT血清型和它们共同的催化机制在蛋白质结构上的相似之处，假设BONT/B也可能被用作大规模攻击的一部分并不是不合理的。 最终，我们的研究目标是合成有效的BONT/B抑制剂，尽可能地对毒素具有微妙的特异性，以避免与体内其他大分子的非特异性相互作用，从而避免意外的副作用。 为此，我们希望了解预先筛选出最佳活性的配体与全毒素催化亚单位BONT/B轻链(BLC)的活性部位之间的生物物理相互作用。将X射线衍射和分子模拟工具应用于截断形式的BLC(TBLC)将实现这一点；事实证明，全长结构很难结晶。虽然我们已经获得了第一个tBLC晶体，但需要改进结晶条件以提高它们的质量，我们认为在4月中旬之前是可行的。此外，我们理想地希望优化现有的候选支架(S)和/或从头开始开发它们。 为了实现这一目标，多重溶剂晶体结构(MSCS)经验算法将被用来通过检测具有不同化学功能的有机溶剂在化学交联晶体中的定位来识别蛋白质表面的外位。这将有助于通过探索催化部位外部的结合部位来提高抑制剂结合的特异性，这些结合部位可能是具有特定静电或空间性质的化学基团所特有的。这种方法取决于tBLC结晶提纯的进展情况，在RapiData期间也可能采用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The seven botulinum neurotoxins (BoNTs) originate from the Clostridium botulinum species of bacteria and are, without exception, the most potent toxins known to man- bypassing tetanus, cobra, and ricin toxins in their lethal nature. The contexts in which humans may encounter toxins are many and include bioterrorist attacks, cosmetic and non-cosmetic medical applications, and food poisoning. Indeed, the B serotype (BoNT/B), while not at the forefront of the scientific community's attention heretofore, has been shown to be responsible for a significant portion of annual food poisoning cases caused by this family of toxins. Given the similarities in protein structure between BoNT serotypes and their common catalytic mechanisms, it is not unreasonable to suppose that BoNT/B may also be used as part of a large-scale attack. Ultimately, the goal of our studies is to synthesize efficacious BoNT/B inhibitors that are as exquisitely specific to the toxin as possible so as to avoid nonspecific interactions with other macromolecules in the body and hence unanticipated side effects. To this end, we hope to gain knowledge of the biophysical interactions between ligands pre-screened for optimal activity and the active site of the BoNT/B light chain (BLC), the catalytic subunit of the holotoxin. Applying x-ray diffraction and molecular modeling tools to a truncated form of the BLC (tBLC) will attain this; the full-length construct has proven difficult to crystallize. While we have attained the first tBLC crystals, refinement of the crystallization conditions is required to improve their quality and is something we deem feasible by mid April.Additionally, we would ideally like to optimize existing candidate scaffold(s) and/or develop them de novo. To achieve this goal, the Multiple Solvent Crystal Structures (MSCS) empirical algorithm will be employed to identify exo-sites on the protein surface by examining the localization of organic solvents with distinct chemical functionalities in chemically cross linked crystals. This will help improve specificity of inhibitor binding by exploring binding sites external to the catalytic site that may be specific for chemical groups with defined electrostatic or steric properties. This approach, depending on how tBLC crystallization refinement progresses, might also be possible to pursue during RapiData.