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最终考验 蛋白质。 DeGrado 实验室最近开发的新兴技术首次使我们能够 通过单个计算步骤，在完全合成、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式增强我们对蛋白质-小分子相互作用的新理解： 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 作为受控释放能力。作为概念验证，我们将设计一种紧密且紧密结合的蛋白质载体 特异性结合阿霉素，阿霉素是蒽环类拓扑异构酶 II 抑制剂的典型成员。 这种本质上荧光且高度复杂的分子具有多个可靶向的官能团，这些官能团密切相关 结构衍生物，以及几种市售的缀合物和载体，使其成为理想的证明 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。 蛋白质载体设计将通过测试和扩展我们新开发的 COMBS（合作主题）来完成 结合位点）算法，结合参数化蛋白质设计，可以精确设计 高度稳定的螺旋束。我们将进一步增强我们的设计能力，利用组氨酸残基来 促进药物在生理pH下结合，并在酸性肿瘤微环境中实现控释， 由于咪唑侧链的 pKa。最好的计算评分设计将是细菌设计 合成以能够快速筛选其折叠和结合能力。 X射线晶体学将 进行以确定结构-功能关系并确定所得结构与我们的一致 设计。这些结果将用于航母的迭代设计。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估。这些 将为载体的未来设计提供信息，并使我们能够微调中使用的组氨酸残基的量 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，以避免早期蛋白水解和相关的免疫原性（因为全 D 配置的肽是 主要组织相容性复合物不显示）。这种函数导向的方法将使我们能够获得 蛋白质-小分子相互作用的原子水平控制，虽然这个提议本质上是基本的， 这些设计原则最终可以为新型航母的开发做出贡献。利用 这种药物载体的设计方法与我的科学背景高度兼容，并且代表了 这是其众多应用中的第一个。拟议的研究提供了极好的培训机会，这将是 在 DeGrado 团队的合作精神的支持下，结合我们与陈教授的合作以及 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。