Abstract 2024: High resolution limited proteolysis (HR-LiP), a novel structural proteomics approach for the prediction of small molecule-protein binding events

Abstract

Abstract High resolution profiling of drug-protein interactions and binding mechanisms remains a major hurdle during lead selection and optimization. A key milestone in structure-based drug design is compound binding site identification and characterization. Structure-activity relationship (SAR) studies traditionally utilize time and cost intensive techniques such as nuclear magnetic resonance (NMR), x-ray crystallography (X-ray) and cryo-electron microscopy (cryo-EM). To date, hydrogen-deuterium exchange (HDX) is the only mass spectrometry-based approach that has been extensively utilized. Further, SAR studies are often complicated by protein size (large proteins and/or oligomers) and location (membrane proteins), which can lead to protocol adaptations that can introduce artifacts. High resolution limited proteolysis (HR-LiP) is a high-throughput, high-resolution approach based on LiP-Quant that utilizes peptide-level resolution to characterize drug-protein interactions. Two well-characterized drug target proteins, bromodomain-containing protein 4 (BRD4) and transitional endoplasmic reticulum ATPase (VCP), were selected for analysis as they represent both difficult targets for structural biology (size and oligomerization respectively). BRD4 was targeted with a well characterized inhibitor (JQ1), while VCP was investigated for binding with a known autophagy activator. Using HR-LiP we identify the binding site of the BRD4 inhibitor JQ1 in the full-length protein, which is typically too large to be used directly in with conventional methods. Further, we confirm binding of the autophagy activator to VCP at a site known to increase VCP activity. For both compounds tested, our data are in good accordance with orthogonal data obtained by other structural protein approaches including HDX-MS and “click” chemistry-based chemoproteomics. We demonstrate that HR-LiP can be used to dissect small molecule-protein binding events, including compound binding site prediction for protein targets classically considered to be difficult and time intensive. Further, we show that this data can be used to shed light on the biological mechanisms driving a phenotypic response. Given its biological power, broad applicability and ease of implementation, we envision the use of HR-LiP as a routine approach for target validation and lead optimization in small molecule drug discovery pipelines. Citation Format: Nigel Beaton, Roland Bruderer, Yuehan Feng, Jagat Adhikari, Ivan Cornella-Taracido, Lukas Reiter. High resolution limited proteolysis (HR-LiP), a novel structural proteomics approach for the prediction of small molecule-protein binding events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2024.