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Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

ABSTRACT: Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of an E3 ubiquitin ligase) promotes POI dissociation. Collision-induced dissociation (CID) causes elimination of the nonperipheral PROTAC, producing an intact VCB-POI complex not seen in solution but consistent with PROTAC-induced protein-protein interactions. In addition, we used ion mobility spectrometry (IMS) and collisional activation to identify the source of this unexpected dissociation. Together, the evidence shows that this integrated approach can be used to screen for ternary complex formation and PROTAC-protein contacts and may report on PROTAC-induced protein-protein interactions, a characteristic correlated with PROTAC selectivity and efficacy.

SUBMITTER: Song JH

PROVIDER: S-EPMC8592818 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

Song Jong Hee JH Wagner Nicole D ND Yan Jing J Li Jing J Huang Richard Y-C RY Balog Aaron J AJ Newitt John A JA Chen Guodong G Gross Michael L ML

Cell chemical biology 20210602 10

Proteolysis-targeting chimeras (PROTACs) represent a new direction in small-molecule therapeutics whereby a heterobifunctional linker to a protein of interest (POI) induces its ubiquitination-based proteolysis by recruiting an E3 ligase. Here, we show that charge reduction, native mass spectrometry, and gas-phase activation methods combine for an in-depth analysis of a PROTAC-linked ternary complex. Electron capture dissociation (ECD) of the intact POI-PROTAC-VCB complex (a trimeric subunit of a ...[more]

PMID: 34081921

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Project description:Proteolysis-targeting chimaeras (PROTACs), which induce proteolysis by recruiting an E3 ligase to dock into a target protein, are acquiring popularity as a novel pharmacological modality because of the unique features of PROTAC, including high potency, low dosage, and effective on undruggable targets. While PROTACs are promising prospects as chemical probes and therapeutic agents, their discovery usually necessitates the synthesis of numerous analogues to explore variations on the chemical linker structure exhaustively. Without extensive trial and error, it is unknown how to link the two protein-recruiting moieties to facilitate the formation of a productive ternary complex. Although molecular docking-based and optimization pipelines have been designed to predict ternary complexes, guiding rational PROTAC design, they have suffered from limited predictive performance in the quality of the ternary structure and their ranks. Here, MEGA PROTAC has been designed to enhance the performance in quality and ranking of ternary structures. MEGA PROTAC employs MEGADOCK to execute docking for protein-protein complexes (PPCs). The docking establishes an initial exploration area for PPCs. A sequential filtration strategy combined with rank aggregation is employed to choose a subset of PPCs for grid search. Once candidate PPCs are selected, a grid search method is used separately for translation and rotation. The remaining proteins have been grouped into clusters, and MEGA PROTAC further filters these clusters based on the energy score of the proteins within each cluster. MEGA PROTAC utilises rank aggregation to choose the best clusters and then employs MEGADOCK to dock PROTAC into the selected PPCs, forming a ternary structure. Finally, MEGA PROTAC was tested on 22 cases to compare with the state-of-the-art method, Bayesian optimisation for ternary complex prediction (BOTCP). MEGA PROTAC outperformed BOTCP on 16 test cases out of 22 cases, achieving a higher maximum DockQ score with an 18% higher mean and 35% higher median. Also, MEGA PROTAC exhibited 75% superior ranks and a reduced cluster number for maximum DockQ score compared to BOTCP. Also, MEGA PROTAC outperforms BOTCP by achieving a twofold improvement in locating the first acceptable DockQ scores, with a more significant proportion of near-native structures within the detected cluster.

Dataset Information

Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

Publications

Native mass spectrometry and gas-phase fragmentation provide rapid and in-depth topological characterization of a PROTAC ternary complex.

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