Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex

The study focuses on the development and investigation of potent heterobifunctional degraders (Cmpd-1 and Cmpd-2) targeting PTPN2/N1, which are attractive immuno-oncology targets with potential applications in cancer immunotherapy. The degraders were designed to facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN and mediate potent PTPN2/N1 degradation in cells and mice. The research also aimed to provide mechanistic insights into the cooperative complex formation introduced by the degraders through a combination of structural approaches, including crystal structure determination, single-particle cryo-electron microscopy (cryo-EM), and molecular dynamic simulations.

The study employed a comprehensive structural workflow to characterize the interactions between the degraders, the target protein PTPN2/N1, and the E3 ubiquitin ligase CRBN-DDB1. Crystal structure analysis revealed the recognition of PTPN2 by the degrader, and high-resolution cryo-EM structure determination demonstrated large conformational heterogeneity of the ternary complex induced by the degraders. Molecular dynamic simulations further revealed a large rigid body movement of PTPN2 and illustrated dynamic interactions between PTPN2 and CRBN, highlighting the plasticity and dynamic nature of degrader-induced protein-protein interactions.

In addition, the study investigated the selectivity of the degraders across various phosphatases, demonstrating potent and selective degradation specifically for PTPN1/N2 in cells. The degraders facilitated cooperative complex assembly between PTPN1/N2 and CRBN-DDB1, leading to efficient PTPN1/N2 degradation in vitro and also exhibited in vivo potency in mouse spleens.

The findings from the molecular dynamic simulations and cryo-EM structure-based analyses revealed the dynamic nature of the degrader-induced cooperative ternary complexes, providing a deeper understanding of the structural mechanisms and dynamic interactions involved in the formation of the PTPN2/N1-CDRBN-DDB1 complex. The study's detailed characterization of the degraders and their effects on protein degradation has significant implications for cancer immunotherapy, offering insights into the potential development of new therapeutic strategies targeting challenging proteins such as PTPN2/N1. This work contributes to the understanding of degrader-induced protein-protein interactions and offers valuable information for guiding the rational design of improved degraders with potential applications in cancer immunotherapy.

The study aimed to develop potent PTPN2/N1 dual heterobifunctional degraders and investigate their ability to facilitate efficient complex assembly with E3 ubiquitin ligase CRL4 CRBN, mediate potent PTPN2/N1 degradation in cells and mice, and improve the response to immunotherapy in disease models. The degraders were designed and characterized using various biochemical assays and structural methods to understand the cooperative complex formation and potential applications in cancer immunotherapy.

The researchers used surface plasmon resonance (SPR) to assess the binding of the degraders to PTPN2/N1 and CRBN. They observed strong and specific interactions, and the affinities of the binding were quantified using steady-state kinetics. High Precision Streptavidin (SAX) biosensors were employed to study the binding to CRBN, and the affinities were calculated by globally fitting the obtained curves to a 1:1 binding model. Analytical size exclusion chromatography was used to analyze the complex formation between PTPN1/2, CRBN-DDB1, and the degraders, confirming the efficient assembly of the ternary complex.

Additionally, interaction between CRBN-DDB1 and Cmpd-1 was measured using MicroCal PEAQ-ITC and a thermal shift assay was performed to determine the melting temperature (Tm) of the PTPN2 constructs in the presence of the degraders. X-ray crystallography and cryo-electron microscopy (Cryo-EM) were employed to determine the structural details of the ternary complex formation. The study described the crystallization of PTPN2 with Cmpd-2 and the collection of diffraction data with subsequent structure determination and refinement steps. Cryo-EM specimen preparation and data acquisition were performed to visualize the complex, followed by data processing, model building, and refinement to obtain a high-resolution 3D reconstruction model. Molecular dynamics (MD) simulations were conducted to understand the dynamics of the PTPN2-Cmpd-1/CRBN-DDB1 complex. The study detailed the refinement, preparation steps, and simulations to optimize the system and establish a reference for monitoring the displacement of PTPN2 on the surface of CRBN. Synthesis of the molecules and hydrogen-deuterium exchange mass spectrometry (HDX-MS) were also described in the study to assess the level of deuterium incorporation into peptides and provide insights into the structural changes induced by the degraders at the protein level. The research provides a comprehensive structural and functional characterization of PTPN2/N1 dual heterobifunctional degraders and their interactions with the E3 ubiquitin ligase CRBN. The findings offer valuable insights into the cooperative complex formation introduced by the degraders, highlighting the potential applications of these degraders in cancer immunotherapy. The study presents a detailed structural workflow for understanding degrader-induced cooperative ternary complexes, shedding light on their mechanism of action and potential therapeutic implications.