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

通过从头设计结合小分子药物的蛋白质来探索分子识别的规则。

• 批准号: 10463468
• 负责人: Lee Schnaider
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463468
• 关键词: Achievement; Affinity; Agreement; Algorithms; Amines; Amino Acids; Anthracycline; Basic Science; Benchmarking; Binding; Binding Proteins; Binding Sites; Biocompatible Materials; Biological; Biological Assay; Biotechnology; Calorimetry; Carrier Proteins; Cell Survival; Cell model; Chemicals; Circular Dichroism; Cold Chains; Collaborations; Complex; Confocal Microscopy; Conjugated Carrier; Crystallization; Dependence; Development; Dimensions; Doxorubicin; Drug Carriers; Drug Controls; Drug Delivery Systems; Drug Design; Elements; Emerging Technologies; Environment; Evaluation; Fluorescence; Fluorescence Polarization; Future; Future Generations; Histidine; Hydroxyl Radical; Imidazole; Libraries; Ligands; Ligation; Logistics; Major Histocompatibility Complex; Methodology; Nature; Peptides; Pharmaceutical Preparations; Physiological; Positioning Attribute; Protein Engineering; Proteins; Proteolysis; Rapid screening; Research; Resolution; Resources; Side; Specificity; Structure; Testing; Therapeutic; Time; Titrations; Topoisomerase-II Inhibitor; Training; Vertebral column; X-Ray Crystallography; base; biophysical properties; chemical group; chemical synthesis; controlled release; delivery vehicle; design; experimental study; functional group; immunogenicity; improved; innovation; iterative design; member; molecular recognition; multidisciplinary; novel; novel drug class; small molecule; thermostability; training opportunity; tumor microenvironment; wound healing

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.

项目摘要/摘要： 从头开始设计配体结合蛋白是对分子识别原理的终极考验 蛋白质。最近在DeGrado实验室开发的新兴技术使我们第一次能够 只需一个计算步骤，就可以在完全合成的、设计好的蛋白质中设计封闭腔中的小分子。 在这里，我们建议通过以下方式加强我们对蛋白质-小分子相互作用的新理解 具有高热稳定性、结合亲和力和特异性的治疗结合蛋白的从头设计 作为受控释放能力。作为概念验证，我们将设计一种紧密和 与阿霉素特异性结合，阿霉素是拓扑异构酶II抑制剂中的一种典型成员。 这种固有的荧光和高度复杂的分子具有多个官能团来靶向，密切相关 结构衍生物，以及几种商业上可用的连接物和载体，使其成为 该项目的主要基本设计方面的概念，以及潜在的药物输送应用。 蛋白质载体的设计将通过测试和扩展我们新开发的Combs(合作模体)来完成 对于结合位点)算法，与允许精确设计的参数蛋白质设计相结合 高度稳定的螺旋束。我们将进一步提高我们的设计能力，利用组氨酸残基来 在生理pH下促进药物结合，并在酸性肿瘤微环境中实现受控释放， 由于咪唑侧链的pKA。最好的计算得分设计将是细菌学的 合成使其能够快速筛选其折叠和结合能力。X射线结晶学将是 进行以确定结构-功能关系，并确定结果结构与我们的 设计。这些结果将被用于载波的迭代设计。载体的生物活性 将通过细胞活性、增殖和伤口愈合分析以及共聚焦显微镜进行评估。这些 将告知载体的未来设计，并允许我们微调在 阿霉素结合。重要的是，载体将保持较小，以便以后的化学合成包括 所有D-氨基酸，以避免早期蛋白分解和相关的免疫原性(正如所有D配置的多肽 未由主要组织相容性复合体显示)。这种以函数为导向的方法将允许我们获得 对蛋白质-小分子相互作用的原子水平控制，虽然这一提议在本质上是基本的， 这些设计原则最终将有助于发展一类新的航空公司。可持续发展战略 这种药物载体的设计方法与我的科学背景高度一致，并代表了 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， 在德格拉多团队的合作精神支持下，结合我们与陈教授和 Kortemme教授以及加州大学旧金山分校的机构资源和独特的多学科环境。