Disrupting E3 ubiquitin ligase protein-protein interactions in cancer: chemical modalities, mechanisms, and therapeutic opportunities.

Ciz1 is a nuclear protein that associates with cyclin A – cyclin dependent kinase 2 (CDK2) and facilitates the initiation of DNA replication. Ciz1 overexpression has been linked to common cancer types, including breast, colon, prostate, lung, and liver cancers. This suggests that identification of mechanisms that regulate Ciz1 levels may represent potential drug targets in cancer. This work identifies that CDK2 and DDK activity are required to maintain Ciz1 levels. Chemical or genetic inhibition of CDK2 or DDK (Cdc7-Dbf4) activity in murine fibroblasts reduced Ciz1 levels. Further analysis demonstrated that CDK and DDK activity promotes Ciz1 accumulation in G1 phase by reducing ubiquitin proteasome system (UPS) mediated degradation. Furthermore, Ciz1 levels are actively controlled by the proteasome, as inhibition of protein translation rapidly reduced Ciz1 levels, and this is reversed by proteasomal inhibition. The data suggest a model where Ciz1 is regulated by opposing kinase and UPS activities, leading to Ciz1 accumulation in response to rising kinase activity in G1 phase, and its degradation later in the cell cycle. Significantly, human prostate adenocarcinoma (PC3) and oestrogen receptor positive breast cancer (MCF7) cell lines require Ciz1 for efficient proliferation. The data demonstrate that Ciz1 levels can be reduced with CDK2/ DDK inhibitors via proteasomally mediated degradation in human cancer cell lines similarly to normal fibroblasts. In PC3 and MCF7 cell lines, repurposing small molecule CDK2 inhibitors efficiently reduce Ciz1 levels, decrease E2F mediated transcription and proliferation. The targeted depletion of Ciz1 via CDK2/ DDK inhibition and UPS mediated degradation requires a functional E3 ligase to be effective. As a first step towards identifying the regulatory E3 ligase(s), a biochemical fractionation and mass spectrometry approach revealed three putative E3 ligases: UBR5, FBXO8 and UBE2O, which require further characterisation. Taken together, this work suggests that deregulation of CDK activity or inactivation of UPS signalling may promote Ciz1 overexpression in specific cancers. Importantly, Ciz1 is required for proliferation of some cancer cell lines, suggesting that approaches, which reduce Ciz1 levels may be of clinical benefit. Therefore, the identification of the regulatory mechanisms that control Ciz1 levels, represent potential targets in Ciz1 dependent cancers.

An E3 ligase guide to the galaxy of small-molecule-induced protein degradation

Advances and opportunities in targeted protein degradation

Targeted protein degradation (TPD) provides new therapeutic opportunities beyond traditional inhibitors. TPD relies on the ability to induce proximity between an E3 ligase and the target of interest, harnessing the ubiquitin proteasome system to ubiquitylate and degrade the target. This proximity can be induced by either monofunctional ligands (molecular glues) or bifunctional molecules that tether ligases and target ligands together. DNA encoded libraries (DELs) provide rapid access to diverse chemical space for ligand discovery and, by their design, facilitate the development of both molecular glues and bifunctional degraders.

Molecular glue (MG) degraders, small molecules with significant therapeutic potential for targeting undruggable proteins, are emerging as new modality in drug discovery. Profiling the E3 ligase interactome induced by MG degraders provides insights into their mechanism of action and identifies clinically relevant neosubstrates for degradation, thereby offering new therapeutic opportunities. However, established methods face significant challenges in comprehensive and accurate profiling of MG degrader-induced E3 ligase interactome. Herein, we introduce the concept of globally cross-linking profiling of the MG degrader-induced E3 ligase interactome in living cells, achieved by integrating genetic code expansion technology with mass spectrometry-based proteomics. Our approach presents an efficient and robust strategy for identifying neosubstrates recruited to cereblon E3 ligase by the known degraders CC-885 and DKY709, offering valuable insights for clinical evaluation and significantly expanding their target space. Moreover, we developed two novel MG degraders with potent antiproliferative effects on cancer cells, and application of our method identified neosubstrates, revealing a previously unrecognized target landscape and advancing our understanding of E3 ligase-neosubstrate interactions. Overall, our study provides a powerful tool for neosubstrate identification and expanding target space of E3 ligase, opening new opportunities for developing next-generation MG degraders to address the clinical challenge of undruggable targets.

Advances in molecular glue degraders for targeted protein degradation: Focus on NEK7, WEE1, CDK2, GSPT1 and VAV1

The role of TRIM proteins in chronic inflammation-associated musculoskeletal diseases.

Simple SummaryCyclin-dependent kinases (CDKs) are rich and viable therapeutic targets for various cancers. The emergence of event-driven pharmacology as an alternative to occupancy-driven pharmacology has begun to address the challenges associated with selectively targeting CDKs. In this review article, we summarize the CDK inhibitors that are currently in clinical trials. In addition, we provide an overview of PROTAC- and molecular glue-based strategies to modulate CDK function.The cyclin-dependent kinase (CDK) family of proteins play prominent roles in transcription, mRNA processing, and cell cycle regulation, making them attractive cancer targets. Palbociclib was the first FDA-approved CDK inhibitor that non-selectively targets the ATP binding sites of CDK4 and CDK6. In this review, we will briefly inventory CDK inhibitors that are either part of over 30 active clinical trials or recruiting patients. The lack of selectivity among CDKs and dose-limiting toxicities are major challenges associated with the development of CDK inhibitors. Proteolysis Targeting Chimeras (PROTACs) and Molecular Glues have emerged as alternative therapeutic modalities to target proteins. PROTACs and Molecular glues utilize the cellular protein degradation machinery to destroy the target protein. PROTACs are heterobifunctional molecules that form a ternary complex with the target protein and E3-ligase by making two distinct small molecule–protein interactions. On the other hand, Molecular glues function by converting the target protein into a “neo-substrate” for an E3 ligase. Unlike small molecule inhibitors, preclinical studies with CDK targeted PROTACs have exhibited improved CDK selectivity. Moreover, the efficacy of PROTACs and molecular glues are not tied to the dose of these molecular entities but to the formation of the ternary complex. Here, we provide an overview of PROTACs and molecular glues that modulate CDK function as emerging therapeutic modalities.

Molecular glue (MG) degraders include plant hormones and therapeutic drugs and have become a hot topic in drug discovery. Unlike bivalent proteolysis targeting chimeras (PROTACs), monovalent MGs can trigger the degradation of non-ligandable proteins by enhancing their interaction with E3 ubiquitin ligases. Here, I analyze the characteristics of natural MG degraders, contrast them with synthetic ones, and provide a rationale for optimizing MGs. In natural MG-based degradation systems, a stable complex is only formed when all three partners (MG, E3 ligase, and substrate) are present, while the affinities between any two components are either weak or undetectable. After the substrate is degraded, the MG will dissociate from its receptor (E3 ligase) due to their low micromolar affinity. In contrast, synthetic MGs, such as immunomodulatory drugs (IMiDs) and CR8, are potent inhibitors of their receptors by blocking the CRBN-native substrate interaction or by occupying the active site of CDK12. Inspired by nature, the affinities of IMiDs to CRBN can be reduced to make those compounds degraders without the E3-inhibitory activity, therefore, minimizing the interference with the physiological substrates of CRBN. Similarly, the CR8-CDK interaction can be weakened to uncouple the degrader function from the kinase inhibition. To mimic natural examples and reduce side effects, future development of MG degraders that lack the inhibitory activity should be considered.

Regulatory functions of the ubiquitin-proteasome system (UPS) are exercised mainly by the ubiquitin ligases and deubiquitinating enzymes. Degradation of apoptotic proteins by UPS is central to the maintenance of cell health, and deregulation of this process is associated with several diseases including tumors, neurodegenerative disorders, diabetes, and inflammation. Therefore, it is the view that interrogating protein turnover in cells can offer a strategy for delineating disease-causing mechanistic perturbations and facilitate identification of drug targets. In this review, we are summarizing an overview to elucidate the updated knowledge on the molecular interplay between the apoptosis and UPS pathways. We have condensed around 100 enzymes of UPS machinery from the literature that ubiquitinates or deubiquitinates the apoptotic proteins and regulates the cell fate. We have also provided a detailed insight into how the UPS proteins are able to fine-tune the intrinsic, extrinsic, and p53-mediated apoptotic pathways to regulate cell survival or cell death. This review provides a comprehensive overview of the potential of UPS players as a drug target for cancer and other human disorders.

Molecular glues (MGs) are monovalent small molecules that induce an interaction between proteins (native or non-native partners) by altering the protein-protein interaction (PPI) interface toward a higher-affinity state. Enhancing the PPI between a protein and E3 ubiquitin ligase can lead to degradation of the partnering protein. Over the past decade, retrospective studies of clinical drugs identified that immunomodulatory drugs (e.g., thalidomide and analogues) and indisulam exhibit a molecular glue effect by driving the interaction between non-native substrates to CRBN and DCAF15 ligases, respectively. Ensuing reports of phenotypic screens focused on MG discovery have suggested that these molecules may be more common than initially anticipated. However, prospective discovery of MGs remains challenging. Thus, expanding the repertoire of MGs will enhance our understanding of principles for prospective design. Herein, we report the results of a CRISPR/Cas9 knockout screen of over 1000 ligases and ubiquitin proteasome system components in a BRD4 degradation assay with a JQ1-based monovalent degrader, compound 1a. We identified DCAF16, a substrate recognition component of the Cul4 ligase complex, as essential for compound activity, and we demonstrate that compound 1a drives the interaction between DCAF16 and BRD2/4 to promote target degradation. Taken together, our data suggest that compound 1a functions as an MG degrader between BRD2/4 and DCAF16 and provides a foundation for further mechanistic dissection to advance prospective MG discovery.

In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN : DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15 : DDA1 : DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15 : DDA1 : DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1 : 1 : 1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and its self-association into dimers and trimers at reduced salt concentration during structural analysis.

To date, most successful molecular glues have leveraged ubiquitously expressed E3 ligases, most notably Cereblon in the case of the thalidomide derivatives. However, a number of specific E3 ligases are highly over-expressed in certain tumors. Compounds that engage such tumor-specific E3 ligases might provide a unique opportunity to develop molecular glues with higher therapeutic indexes. One such tumor-specific E3 ligase is FBXO5, a member of the F-box family of E3 ligases. Expression analysis suggests this E3 ligase is highly expressed in a wide array of tumor tissues when compared to the corresponding normal tissue, and that within a given tumor type, higher FBXO5 expression correlates with advanced tumor stage and worse disease-free survival. Here, we describe a small molecule (MW~400 Daltons) that uses FBXO5 to function as a molecular glue degrader. This compound, CL-200, exerts cytotoxicity in the nanomolar range across a number of diverse tumor types. The cytotoxicity of CL-200 is abrogated when FBXO5 is knockdown using siRNA. We have further determined that CL-200 induces the FBXO5-dependent proteasomal degradation of at least two critical neosubstrates. One identified neosubstrate is the p150 isoform of ADAR1, an enzyme that catalyzes the posttranscriptional conversion of adenosine to inosine in double-stranded RNA (dsRNA). Evidence suggests that ADAR1p150 is the isoform that confers oncogenic and immune modulating effects in many tumors. Another neosubstrate targeted by CL-200 is c-myc, a critical transcription factor linked to a wide array of tumors. Of note, both p150 ADAR1 or c-myc have been viewed as difficult-to-drug if not undruggable targets. As such, these data suggests that CL-200 functions as a novel molecular glue degrader that engages the cancer-specific E3 ligase FBXO5 to degrade both p150 ADAR1 and c-myc. Structural alteration of CL-200 suggest that chemical modifications can be made that retain the engagement with FBXO5 while altering the neosubstrate profile. Together, these results demonstrate the possibility of leveraging cancer-specific E3 ligases to develop novel molecular glues. Citation Format: Yuan Liu, Mads Larsen, Bo Lin, Irene A. Cardenal, Jason R. Kennerdell, Toren Finkel, Bill B. Chen. Identification of a molecular glue degrader that engages a cancer-specific E3 ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6070.

Auxin is one of the main agents that regulate plant growth and development. Intricate crystallographic studies reveal how this hormone acts as a 'molecular glue' in mediating substrate–receptor interactions. The mechanism by which the plant hormone auxin regulates plant growth has puzzled scientists since Darwin's time. Auxin is known to regulate gene expression by binding to its receptor TIR1 and promoting ubiquitin-dependent degradation of Aux/IAA repressor proteins. Now the determination of the crystal structures of TIR1 in complexes with three different auxins and an Aux/IAA peptide shows auxin to act as a 'molecular glue' promoting interactions between the receptor and proteins targeted for degradation. As well as revealing auxin's mechanism, this work establishes the first structural model of a plant hormone receptor. Also, the discovery that a small molecule like auxin can regulate ubiquitin ligases suggests a novel strategy for developing therapeutics for human disorders associated with ubiquitin ligase defects. On the cover, auxin (shown as a spacefilling model) is seen in the cavity between TIR1 (blue) and IAA7 peptide (orange).

Reversing Cachexia