Probing the rules of molecular recognition through the de novo design of proteins that bind small-molecule drugs.

项目概要/摘要： 从头开始设计配体结合蛋白质是对分子识别原理的最终测试， proteins. DeGrado实验室最近开发的新兴技术，使我们能够第一次 在一个计算步骤中，在完全合成的、设计的蛋白质的封闭腔中设计小分子。 在这里，我们建议通过以下方法来增强我们对蛋白质-小分子相互作用的新认识： 从头设计具有高热稳定性、结合亲和力和特异性的治疗结合蛋白，以及 作为控制释放能力。作为概念验证，我们将设计一种蛋白质载体， 特异性结合多柔比星（拓扑异构酶II抑制剂的蒽环类的原型成员）。 这种固有荧光和高度复杂的分子具有多个官能团，与靶向分子密切相关。 结构衍生物，以及几种市售的缀合物和载体，使其成为 概念的主要基本设计方面的这个项目，和潜在的药物输送应用。 蛋白质载体设计将通过测试和扩展我们新开发的COMBS（合作模体）来完成 结合位点）算法，结合参数蛋白质设计，其允许精确设计 高度稳定的螺旋束。我们将进一步提高我们的设计能力，利用组氨酸残基， 促进药物在生理pH下的结合，并能够在酸性肿瘤微环境中控制释放， 由于咪唑侧链的pKa。最好的计算得分设计将是细菌 合成以使得能够快速筛选它们的折叠和结合能力。X射线晶体学将是 进行，以确定结构-功能关系，并确定所得结构与我们的 的设计.这些结果将用于载波的迭代设计。载体的生物活性 将通过细胞活力、增殖和伤口愈合测定以及共聚焦显微镜进行评价。这些 将告知未来的载体设计，并允许我们微调组氨酸残基的量， 阿霉素结合。重要的是，载体将保持较小，以允许稍后的化学合成，包括 所有D-氨基酸，以避免早期蛋白水解和相关的免疫原性（因为全D构型的肽是 不被主要组织相容性复合体显示）。这种功能导向的方法将允许我们获得 蛋白质-小分子相互作用的原子级控制，虽然这一提议在本质上是基本的， 这些设计原理最终可有助于开发新的一类载体。利用 这种设计药物载体的方法与我的科学背景高度一致， 这是它众多应用中的第一个。拟议的研究提供了极好的培训机会， 在DeGrado集团的合作精神的支持下，结合我们与陈教授的合作， 教授Kortemme，以及UCSF的机构资源和独特的多学科环境。

Project Summary/Abstract: Designing ligand-binding proteins from scratch is the ultimate test of the principles of molecular recognition by proteins. Emerging technologies recently developed in the DeGrado lab, have enabled us, for the first time, to design small molecules in enclosed cavities in fully synthetic, designed proteins, in a single computational step. Here, we propose to enhance our newfound understanding of protein-small-molecule interactions through the de novo design of a therapeutic-binding protein with high thermostability, binding affinity and specificity, as well as controlled release capabilities. As a proof-of-concept, we will design a protein carrier that tightly and specifically binds doxorubicin, a prototypical member of the anthracycline class of topoisomerase II inhibitors. This intrinsically fluorescent and highly complex molecule has multiple functional groups to target, closely related structural derivatives, and several commercially available conjugates and carriers, making it an ideal proof of concept for both the main fundamental design aspect of this project, and the potential drug delivery application. Protein carrier design will be done by testing and extending our newly developed COMBS (Cooperative Motifs for Binding Sites) algorithm, in conjunction with parametric protein design which allows for the precise design of highly stable helical bundles. We will further enhance our design capabilities by utilizing histidine residues to facilitate drug binding at physiological pH, and enable controlled release in the acidic tumor microenvironment, owing to the pKa of the imidazole side chain. The best computationally scored designs will be bacterially synthesized to enable rapid screening of their folding and binding capabilities. X-ray crystallography will be carried out to determine structure-function relations and ascertain agreement of the resulting structure with our designs. These results will be utilized for the iterative design of the carriers. The biological activity of the carriers will be evaluated via cell viability, proliferation, and wound healing assays as well as confocal microscopy. These will inform on future designs of the carriers, and allow us to fine-tune the amount of histidine residues utilized in doxorubicin binding. Importantly, the carriers will be kept small, to allow for later chemical synthesis to include all D-amino acids, to avoid early proteolysis and associated immunogenicity (as all-D configured peptides are not displayed by major histocompatibility complexes). This function-directed approach will allow us to obtain atomic-level control of protein-small-molecule interactions, and while this proposal is fundamental in nature, these design principals can ultimately contribute to the development of a new class of carriers. The utilization of this approach for the design of drug carriers is highly compatible with my scientific background, and represents the first of its many applications. The proposed research offers excellent training opportunities which will be supported by the cooperative ethos of the DeGrado group, combined with our collaboration with Prof. Chen and Prof. Kortemme, and the institutional resources and unique multi-disciplinary environment at UCSF.