Development of Cell-Penetrating Monobodies

在化学系生命过程化学（CLP）项目的支持下，纽约州立大学布法罗分校的林庆教授正在开发新的方法来设计细胞可渗透的抗体样小蛋白，称为单体。虽然单抗体提供了一个多功能的研究工具，解剖细胞内信号转导途径，它仍然是一个挑战，设计细胞穿透单抗体的治疗应用。所提出的方法将选择性化学交联引入单体中以增强其结构，并且与表面增压一起赋予工程化单体细胞渗透性。该项目将通过与纽约州立大学布法罗分校的几个以少数民族为重点的项目合作，为代表性不足的本科生提供多学科研究的机会。本研究旨在优化正交交联单体支架，以设计靶向细胞内蛋白的生物活性穿细胞单体。通过将一种称为β-内酰胺-赖氨酸的遗传编码的非规范氨基酸整合到单体中进行选择性化学交联，将探索两种互补的方法来增强单体支架的能力：基于深度学习的计算方法;以及基于细菌表面展示的实验方法。优化的单体支架的效用将通过设计靶向致癌KRAS突变体的有效和选择性的细胞可渗透单体并检查功效来评估。这些研究有望深入了解蛋白质拓扑结构和胞质转运之间的关系，这对于使用蛋白质配体研究生命过程至关重要。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Qing Lin of SUNY at Buffalo is developing new approaches for designing cell-permeable antibody-like small proteins called monobodies. While monobodies have provided a versatile research tool for dissecting the intracellular signaling pathways, it remains a challenge to design cell-penetrating monobodies for therapeutic applications. The proposed approaches introduce selective chemical crosslinking into monobodies to reinforce their structure, and together with surface supercharging endow cell permeability to the engineered monobodies. This project will provide multidisciplinary research opportunities to underrepresented undergraduate students through collaborations with several minority-focused programs at SUNY at Buffalo. The results of this project will also be integrated into the Experiential Learning Network for undergraduates to enhance their classroom learning.This research project seeks to optimize orthogonally crosslinked monobody scaffolds for the design of bioactive cell-penetrating monobodies targeting the intracellular proteins. By incorporating a genetically encoded noncanonical amino acid called beta-lactam-lysine into a monobody for selective chemical crosslinking, two complementary approaches will be explored to enhance the capability of the monobody scaffold: a computational approach based on deep learning; and an experimental approach based on bacterial surface display. The utility of the optimized monobody scaffolds will be evaluated by designing potent and selective cell-permeable monobodies targeting oncogenic KRAS mutants and examining efficacy. These studies are expected to provide insight into the relationship between protein topology and cytosolic transport, which is crucial for the use of protein-based ligands to study life processes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.