Structure-Guided Discovery of Novel Dual-Site FEM1B Ligands and Assessment of Their Use in Targeted Protein Degradation.

A 2-in-1 Strategy for the Discovery of Compact HDAC8 Protac Degraders

Proteolysis-targeting chimera (PROTAC) technology has emerged as a powerful therapeutic strategy in drug discovery. Conventional PROACs are heterobifunctional molecules composed of two distinct ligands that independently bind to a protein of interest (POI) and an E3 ligase. However, their inherently large molecular size often leads to suboptimal drug-like properties. In this study, we report a “2-in-1” PROTAC design strategy for developing more compact HDAC8 degraders by integrating the CRBN-recruiting ligand into the capping group of an HDAC8 warhead, thereby enabling a single structural motif to engage both HDAC8 and CRBN. Compared to our first generation HDAC8 degrader YX862, the new degraders exhibit improved selectivity, reduced molecular weight, improved overall drug-like properties, and, importantly, potent HDAC8 degradation in vivo. These findings highlight the therapeutic potential of this new class of HDAC8 degraders, and demonstrate a conceptual framework for integrating E3 ligase recruitment and target engagement into a more efficient and compact PROTAC design.

Design, Synthesis, Biological Studies and Putative Binding Mode of Novel Benzotriazolothalidomide Derivatives as Cereblon Ligands

ABSTRACT Targeted protein degradation (TPD) represents a new paradigm in drug discovery, encompassing several innovative strategies like proteolysis targeting chimeras (PROTACs). PROTACs work by linking the target protein of interest (POI) to an E3 ubiquitin ligase, marking it for ubiquitination, and subsequent degradation by the proteasome. The ligand space of E3 ligase cereblon (CRBN) mostly includes phthalimide‐based thalidomide 1 and its analogs, commonly known as cereblon E3 ligase modulators (CELMoDs) which have been approved for the treatment of multiple subtypes of non‐Hodgkin's lymphoma (NHL). Recently, benzotriazolo thalidomide (4) was reported which displayed similar binding mode like thalidomide, having relatively stronger binding affinity. Towards our aim of identifying new generation CRBN binding ligands, which can have use either in PROTAC or CELMoD chemical space, systematic design and SAR studies around 4, synthesis, biological, and docking experiments to understand putative binding mode have been carried out. As a result, a few compounds were identified as potent CRBN ligands, including compound 13 having an IC50 value of 198 ± 7 nM. Cell viability assay of benzotriazolothalidomide 13 in human embryonic kidney cells (HEK293) suggest a high level of safety profile (SI = > 500). To showcase the drug likeness of this compound, it was tested in preliminary ADME assays, revealing its excellent aqueous solubility (161.87 ± 0.96 μM), and high in vitro metabolic stability in human liver microsome (CLint, app = 16.57 ± 0.19 μL/min/mg of protein; T1/2 = 104.57 ± 1.22 min) and thus offers the scope for further optimization of the compound.

High-throughput identification of carboxylate-free ligands for the E3 ligase Kelch domain-containing protein 2.

Expanding the repertoire of usable E3 ubiquitin ligases remains a critical challenge in the field of targeted protein degradation. For the emerging E3 ligase Kelch domain-containing protein 2 (KLHDC2), currently reported ligands have a carboxylate moiety in common that mimics the natural degron but compromises cellular permeability. Here we report the discovery of carboxylate-free ligands for KLHDC2 through high-throughput screening and cellular functional evaluation. A fluorescence polarization-based screening assay identified NL1 as a micromolar KLHDC2 binder, which was confirmed by orthogonal biophysical methods. Although the structure-activity study did not improve affinity, a degron reporter assay demonstrated measurable cellular target engagement. Encouraged by these results, an expanded screen further identified NL2 as a neutral ligand with improved cellular activity. Computational analysis suggests that NL2 adopts a distinct binding mode, in which the conformational flexibility of two loops of KLHDC2 enables accommodation of its aromatic scaffold, while hydrophobic and π-driven interactions compensate for the canonical ionic contacts of other carboxylate-containing ligands. This work demonstrates that KLHDC2 can accommodate non-carboxylate scaffolds and expands the chemical space for developing KLHDC2-based targeted protein degradation strategies.

Post-translational chemical modification of E3 ligase for efficient target protein degradation.

This work reports the first ligand-directed chemical strategy that converts transient PROTAC-mediated ternary complex formation into binary target recognition via post-translational chemical modification of an E3 ligase, and believes it could provide a platform for next-generation targeted protein degraders to overcome the current limitation of PROTAC approach.

Expanding the Ligandable Chemical Space of OTUB1 through Discovery of a Four-Membered-Ring Recruiter Chemotype

Deubiquitinase-targeting chimeras (DUBTACs) have emerged as a promising strategy for targeted protein stabilization, but their broader application remains limited by the scarcity of ligandable deubiquitinase recruiters. Here, we report a previously unexplored four-membered-ring OTUB1 recruiter chemotype. Through systematic structure–activity relationship studies, we identified compound 21 (MS2159) as a potent and selective covalent OTUB1 ligand. Biochemical and intact protein mass spectrometric analyses demonstrated that MS2159 selectively engages the non-catalytic C23 residue of OTUB1, shows minimal reactivity toward other tested proteins, and preserves OTUB1 deubiquitinase activity. Conjugation of MS2159 with the CFTR ligand lumacaftor yielded compound 25 (MS2134), which effectively stabilized ΔF508-CFTR. Collectively, these findings establish a new OTUB1 recruiter scaffold, expand the ligandable chemical space of OTUB1, and provide additional opportunities for developing next-generation DUBTACs.

A Proximity-Driven Functional Screening Platform for the Discovery of Noninhibitory USP7 Ligand Enabling Targeted Protein Stabilization