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青教授正在开发新的方法来设计被称为单体的细胞渗透性抗体样小蛋白。虽然单抗已经为解剖细胞内信号通路提供了一种通用的研究工具，但设计用于治疗应用的穿透细胞的单体仍然是一个挑战。所提出的方法将选择性的化学交联引入到单体中以增强其结构，并与表面增压一起赋予工程单体的细胞渗透性。该项目将通过与纽约州立大学布法罗分校的几个以少数族裔为重点的项目合作，为未被充分代表的本科生提供多学科的研究机会。该项目的成果还将被整合到本科生的体验式学习网络中，以加强他们的课堂学习。本研究项目旨在优化正交交联单体支架，以设计以细胞内蛋白质为靶点的生物活性穿透细胞单体。通过将一种名为β-内酰胺-赖氨酸的基因编码的非规范氨基酸引入单体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；以及基于细菌表面显示的实验方法。优化的单体支架的实用性将通过设计针对致癌KRAS突变体的有效和选择性的细胞渗透性单体并检测疗效来评估。这些研究有望深入了解蛋白质拓扑和胞质转运之间的关系，这对于使用基于蛋白质的配体来研究生命过程至关重要。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.