Rational PROTAC design enabled by integrated in silico molecular modeling and in vitro biomimetic affinity assessment

通过集成的计算机分子建模和体外实验实现合理的PROTAC设计 仿生亲和性评价 蛋白水解靶向嵌合体，或PROTAC，近年来受到了相当大的关注， 与传统的抑制剂相比，这是一类新的药物。这些小分子靶向选择性降解 利用细胞的天然蛋白质降解机制，包括蛋白体， 溶酶体PROTAC的好处来自于一种完全不同的蛋白质靶向模式， 提供了一种独特的途径来靶向以前“不可药物治疗”的蛋白质，并允许较小的剂量， 副作用小。然而，其复杂的作用机制使得人们对 了解不同阶段的分子相互作用，特别是控制和稳定分子的因素。 导致泛素化和去除的三元复合物。另外，现有技术缺乏策略 模拟并确认PROTAC的连接区在影响弹头亲和力方面的作用 并有助于复合物的稳定性。目前还没有包括动态膜的报道 在分子识别建模中。一个新的技术平台，可以确定影响的关键参数 形成稳定的三元复合物，并具有筛选分子相互作用的能力， 非常需要关于结构性见解的信息。 为了满足未满足的需求，我们建议通过计算机模拟结合 建模和基于体外表面等离子体共振（SPR）的亲和力评估。我们的目标是找出 这些特性导致形成稳定的三元复合物，从而实现高效的PROTAC设计。建立和 为了证明技术上的可行性，我们将研究间变性淋巴瘤激酶（ALK），一种跨膜受体 酪氨酸激酶，其是多种癌症的重要药物靶标，以及E3连接酶CRBN，其具有 用于促进蛋白质降解。具体而言，我们提出三个目标：目标1。建立一个分子 PROTAC设计的建模平台。该平台整合了蛋白质动力学和来自 实验，并可以适应各种实验设置，如膜环境中使用的SPR。目的 2.构建和表征模型PROTAC和仿生膜。这包括生成 PROTAC相容的膜模拟物和膜结合的界面的结构表征 用于SPR分析的蛋白质。目标3. PROTAC候选化合物在仿生中的相互作用研究 膜。这包括在线体外实验和筛选PROTAC， 三元配合物稳定的PROTAC复合物的鉴定将用于进一步探索 理解系统的结构-功能关系。

Rational PROTAC design enabled by integrated in silico molecular modeling and in vitro biomimetic affinity assessment Proteolysis targeting chimeras, or PROTACs, have received considerable attention in recent years as a new class of drugs as compared to traditional inhibitors. These small molecules target selective degradation of proteins of interest utilizing cell’s native protein degradation machinery including proteosomes and lysosomes. The benefits of PROTACs stem from an entirely different paradigm of protein targeting, which provides a unique path to target previously “undruggable” proteins and allows for smaller doses and thus lower side-effects. However, the complex mechanism of action has left a large knowledge gap towards the understanding of molecular interactions in different stages, especially on factors that control and stabilize the ternary complex that leads to ubiquitination and removal. In addition, existing technology lacks of strategies to model and confirm the linker region of the PROTAC for their roles in affecting the affinities of the warheads and contributing to the stability of the complex. There has been no report that includes the dynamic membrane in molecular recognition modeling. A new technical platform that can identify key parameters that impact formation of stable ternary complex and has the capability of screening molecular interactions with detailed information on structural insights is highly desired. To fill the unmet need, we propose to develop a collaborative work plan via a combination of in silico modeling and in vitro surface plasmon resonance (SPR)-based affinity assessment. We aim to identify features that lead to formation of stable ternary complexes for efficient PROTAC design. To establish and prove the technical feasibility, we will study anaplastic lymphoma kinase (ALK), a transmembrane receptor tyrosine kinase that is an important drug target for a variety of cancers, and an E3 ligase CRBN which has been used to promote protein degradation. Specifically, we propose three aims: Aim 1. Establish a molecular modeling platform for rational PROTAC design. The platform incorporates protein dynamics and inputs from experiments and can adapt various experimental settings such as membrane environment used in SPR. Aim 2. Build and characterize modeled PROTACs and biomimetic membranes. This includes generating PROTAC-compatible membrane mimics and structural characterization of the interfaces for membrane-bound proteins for SPR analysis. Aim 3. Investigate interaction properties of PROTAC candidates’ in biomimetic membranes. This includes on-line in vitro experiments and screening for the PROTACs at stabilizing the ternary complexes. The identification of stable PROTAC complexes will be used into further exploration into the understanding of the structure-function relationship of the system.