Driving E3 Ligase Substrate Specificity for Targeted Protein Degradation: Lessons from Nature and the Laboratory.

Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.

A cofactor-induced repressive type of transcription factor condensation can be induced by synthetic peptides to suppress tumorigenesis.

Transcriptional factors (TFs) act as key determinants of cell death and survival by differentially modulating gene expression. Here, we identified many TFs, including TEAD4, that form condensates in stressed cells. In contrast to YAP-induced transcription-activating condensates of TEAD4, we found that co-factors such as VGLL4 and RFXANK alternatively induced repressive TEAD4 condensates to trigger cell death upon glucose starvation. Focusing on VGLL4, we demonstrated that heterotypic interactions between TEAD4 and VGLL4 favor the oligomerization and assembly of large TEAD4 condensates with a nonclassical inhibitory function, i.e., causing DNA/chromatin to be aggregated and entangled, which eventually impede gene expression. Based on these findings, we engineered a peptide derived from the TEAD4-binding motif of VGLL4 to selectively induce TEAD4 repressive condensation. This "glue" peptide displayed a strong antitumor effect in genetic and xenograft mouse models of gastric cancer via inhibition of TEAD4-related gene transcription. This new type of repressive TF phase separation exemplifies how cofactors can orchestrate opposite functions of a given TF, and offers potential new antitumor strategies via artificial induction of repressive condensation.

From Thalidomide to Rational Molecular Glue Design for Targeted Protein Degradation.

Thalidomide and its derivatives are powerful cancer therapeutics that are among the best-understood molecular glue degraders (MGDs). These drugs selectively reprogram the E3 ubiquitin ligase cereblon (CRBN) to commit target proteins for degradation by the ubiquitin-proteasome system. MGDs create novel recognition interfaces on the surface of the E3 ligase that engage in induced protein-protein interactions with neosubstrates. Molecular insight into their mechanism of action opens exciting opportunities to engage a plethora of targets through a specific recognition motif, the G-loop. Our analysis shows that current CRBN-based MGDs can in principle recognize over 2,500 proteins in the human proteome that contain a G-loop. We review recent advances in tuning the specificity between CRBN and its MGD-induced neosubstrates and deduce a set of simple rules that govern these interactions. We conclude that rational MGD design efforts will enable selective degradation of many more proteins, expanding this therapeutic modality to more disease areas.

Applications of protein ubiquitylation and deubiquitylation in drug discovery.

The ubiquitin (Ub)-proteasome system (UPS) is the major machinery mediating specific protein turnover in eukaryotic cells. By ubiquitylating unwanted, damaged, or harmful proteins and driving their degradation, UPS is involved in many important cellular processes. Several new UPS-based technologies, including molecular glue degraders and PROTACs (proteolysis-targeting chimeras) to promote protein degradation, and DUBTACs (deubiquitinase-targeting chimeras) to increase protein stability, have been developed. By specifically inducing the interactions between different Ub ligases and targeted proteins that are not otherwise related, molecular glue degraders and PROTACs degrade targeted proteins via the UPS; in contrast, by inducing the proximity of targeted proteins to deubiquitinases, DUBTACs are created to clear degradable poly-Ub chains to stabilize targeted proteins. In this review, we summarize the recent research progress in molecular glue degraders, PROTACs, and DUBTACs and their applications. We discuss immunomodulatory drugs, sulfonamides, cyclin-dependent kinase-targeting molecular glue degraders, and new development of PROTACs. We also introduce the principle of DUBTAC and its applications. Finally, we propose a few future directions of these three technologies related to targeted protein homeostasis.

Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.

Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila.

Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 µm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.

Development and therapeutic potential of GSPT1 molecular glue degraders: A medicinal chemistry perspective.

Unprecedented therapeutic targeting of previously undruggable proteins has now been achieved by molecular-glue-mediated proximity-induced degradation. As a small GTPase, G1 to S phase transition 1 (GSPT1) interacts with eRF1, the translation termination factor, to facilitate the process of translation termination. Studied demonstrated that GSPT1 plays a vital role in the acute myeloid leukemia (AML) and MYC-driven lung cancer. Thus, molecular glue (MG) degraders targeting GSPT1 is a novel and promising approach for treating AML and MYC-driven cancers. In this Perspective, we briefly summarize the structural and functional aspects of GSPT1, highlighting the latest advances and challenges in MG degraders, as well as some representative patents. The structure-activity relationships, mechanism of action and pharmacokinetic features of MG degraders are emphasized to provide a comprehensive compendium on the rational design of GSPT1 MG degraders. We hope to provide an updated overview, and design guide for strategies targeting GSPT1 for the treatment of cancer.

Targeted protein posttranslational modifications by chemically induced proximity for cancer therapy.

Post-translational modifications (PTMs) regulate all aspects of protein function. Therefore, upstream regulators of PTMs, such as kinases, acetyltransferases, or methyltransferases, are potential therapeutic targets for human diseases, including cancer. To date, multiple inhibitors and/or agonists of these PTM upstream regulators are in clinical use, while others are still in development. However, these upstream regulators control not only the PTMs of disease-related target proteins but also other disease-irrelevant substrate proteins. Thus, nontargeted perturbing activities may introduce unwanted off-target toxicity issues that limit the use of these drugs in successful clinical applications. Therefore, alternative drugs that solely regulate a specific PTM of the disease-relevant protein target may provide a more precise effect in treating disease with relatively low side effects. To this end, chemically induced proximity has recently emerged as a powerful research tool, and several chemical inducers of proximity (CIPs) have been used to target and regulate protein ubiquitination, phosphorylation, acetylation, and glycosylation. These CIPs have a high potential to be translated into clinical drugs and several examples such as PROTACs and MGDs are now in clinical trials. Hence, more CIPs need to be developed to cover all types of PTMs, such as methylation and palmitoylation, thus providing a full spectrum of tools to regulate protein PTM in basic research and also in clinical application for effective cancer treatment.

IKZF2 Degradation: It's Time to Take into Account it When Designing Cereblon-Based PROTACs.

Proteolysis-targeting chimera (PROTAC) has become a very important means of protein degradation and a new way of disease treatment. In particular, PROTACs constructed with ligands for E3 ligase cereblon account for more than 90 % of the PROTACs currently in clinical research. Notably, CRBN ligands themselves are a class of molecular glue compounds capable of degrading neo-substrate proteins. Compared to the target proteins degradation, the degradation of neo-substrates, especially IKZF2, has not received enough attention. Therefore, this review summarizes the currently published IKZF2 degraders derived from articles and patents, which are conducive to the design of PROTACs with desired IKZF2 degradation from the perspective of medicinal chemistry.

Discovery of 14-3-3 PPI Stabilizers by Extension of an Amidine-Substituted Thiophene Fragment.

Protein-protein interaction (PPI) modulation is a promising approach in drug discovery with the potential to expand the 'druggable' proteome and develop new therapeutic strategies. While there have been significant advancements in methodologies for developing PPI inhibitors, there is a relative scarcity of literature describing the 'bottom-up' development of PPI stabilizers (Molecular Glues). The hub protein 14-3-3 and its interactome provide an excellent platform for exploring conceptual approaches to PPI modulation, including evolution of chemical matter for Molecular Glues. In this study, we employed a fragment extension strategy to discover stabilizers for the complex of 14-3-3 protein and an Estrogen Receptor alpha-derived peptide (ERα). A focused library of analogues derived from an amidine-substituted thiophene fragment enhanced the affinity of the 14-3-3/ERα complex up to 6.2-fold. Structure-activity relationship (SAR) analysis underscored the importance of the newly added, aromatic side chain with a certain degree of rigidity. X-ray structural analysis revealed a unique intermolecular π-π stacking binding mode of the most active analogues, resulting in the simultaneous binding of two molecules to the PPI binding pocket. Notably, analogue 11 displayed selective stabilization of the 14-3-3/ERα complex.

Characterizing the stabilization effects of stabilizers in protein-protein systems with end-point binding free energy calculations.

Drug design targeting protein-protein interactions (PPIs) associated with the development of diseases has been one of the most important therapeutic strategies. Besides interrupting the PPIs with PPI inhibitors/blockers, increasing evidence shows that stabilizing the interaction between two interacting proteins may also benefit the therapy, such as the development of various types of molecular glues/stabilizers that mostly work by stabilizing the two interacting proteins to regulate the downstream biological effects. However, characterizing the stabilization effect of a stabilizer is usually hard or too complicated for traditional experiments since it involves ternary interactions [protein-protein-stabilizer (PPS) interaction]. Thus, developing reliable computational strategies will facilitate the discovery/design of molecular glues or PPI stabilizers. Here, by fully analyzing the energetic features of the binary interactions in the PPS ternary complex, we systematically investigated the performance of molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) methods on characterizing the stabilization effects of stabilizers in 14-3-3 systems. The results show that both MM/PBSA and MM/GBSA are powerful tools in distinguishing the stabilizers from the decoys (with area under the curves of 0.90-0.93 for all tested cases) and are reasonable for ranking protein-peptide interactions in the presence or absence of stabilizers as well (with the average Pearson correlation coefficient of ~0.6 at a relatively high dielectric constant for both methods). Moreover, to give a detailed picture of the stabilization effects, the stabilization mechanism is also analyzed from the structural and energetic points of view for individual systems containing strong or weak stabilizers. This study demonstrates a potential strategy to accelerate the discovery of PPI stabilizers.

Lessons from natural molecular glue degraders.

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.

Prevention of Seroma Formation and Its Sequelae After Axillary Lymph Node Dissection: An Up-to-Date Systematic Review and Guideline for Surgeons.

INTRODUCTION: Seroma formation after axillary lymph node dissection (ALND) remains a troublesome complication with significant morbidity. Numerous studies have tried to identify techniques to prevent seroma formation. The aim of this systematic review and network meta-analysis is to use available literature to identify the best intervention for prevention of seroma after standalone ALND. METHODS: A literature search was performed for all comparative articles regarding seroma formation in patients undergoing a standalone ALND or ALND with breast-conserving surgery in the last 25 years. Data regarding seroma formation, clinically significant seroma (CSS), surgical site infections (SSI), and hematomas were collected. The network meta-analysis was performed using a random effects model and the level of inconsistency was evaluated using the Bucher method. RESULTS: A total of 19 articles with 1962 patients were included. Ten different techniques to prevent seroma formation were described. When combining direct and indirect comparisons, axillary drainage until output is less than 50 ml per 24 h for two consecutive days results in significantly less CSS. The use of energy sealing devices, padding, tissue glue, or patches did not significantly reduce the incidence of CSS. When comparing the different techniques with regard to SSIs, no statistically significant differences were seen. CONCLUSIONS: To prevent CSS after ALND, axillary drainage is the most valuable and scientifically proven measure. On the basis of the results of this systematic review with network meta-analysis, removing the drain when output is < 50 ml per 24 h for two consecutive days irrespective of duration seems best. Since drainage policies vary widely, an evidence-based guideline is needed.

DNA Molecular Glue Assisted Bacterial Conjugative Transfer.

Bacterial conjugation, a commonly used method to horizontally transfer functional genes from donor to recipient strains, plays an important role in the genetic manipulation of bacteria for basic research and industrial production. Successful conjugation depends on the donor-recipient cell recognition and a tight mating junction formation. However, the efficiency of conjugative transfer is usually very low. In this work, we developed a new technique that employed DNA molecule "glue" to increase the match frequency and the interaction stability between the donor and recipient cells. We used two E. coli strains, ETZ and BL21, as a model system, and modified them with the complementary ssDNA oligonucleotides by click chemistry. The conjugation efficiency of the modified bacteria was improved more than 4 times from 10 %-46 %. This technique is simple and generalizable as it only relies on the active amino groups on the bacterial surface. It is expected to have broad applications in constructing engineered bacteria.

Seroma formation after mastectomy: A systematic review and network meta-analysis of different flap fixation techniques.

Flap fixation is the most promising solution to prevent seroma formation after mastectomy. In this systematic review with network meta-analysis (NMA), three different techniques were compared. The NMA included 25 articles, comprising 3423 patients, and revealed that sutures are superior to tissue glue in preventing clinically significant seroma. In addition, running sutures seemed to be superior to interrupted sutures. An RCT comparing these suture techniques seems necessary, given the quality and nature of existing literature.

Crbn-based molecular Glues: Breakthroughs and perspectives.

CRBN is a substrate receptor for the Cullin Ring E3 ubiquitin ligase 4 (CRL4) complex. It has been observed that CRBN can be exploited by small molecules to facilitate the recruitment and ubiquitination of non-natural CRL4 substrates, resulting in the degradation of neosubstrate through the ubiquitin-proteasome system. This phenomenon, known as molecular glue-induced protein degradation, has emerged as an innovative therapeutic approach in contrast to traditional small-molecule drugs. One key advantage of molecular glues, in comparison to conventional small-molecule drugs adhering to Lipinski's Rule of Five, is their ability to operate without the necessity for specific binding pockets on target proteins. This unique characteristic empowers molecular glues to interact with conventionally intractable protein targets, such as transcription factors and scaffold proteins. The ability to induce the degradation of these previously elusive targets by hijacking the ubiquitin-proteasome system presents a promising avenue for the treatment of recalcitrant diseases. Nevertheless, the rational design of molecular glues remains a formidable challenge due to the limited understanding of their mechanisms and actions. This review offers an overview of recent advances and breakthroughs in the field of CRBN-based molecular glues, while also exploring the prospects for a systematic approach to designing these compounds.

SuFEx-based chemical diversification for the systematic discovery of CRBN molecular glues.

Molecular glues are small molecules that stabilize protein-protein interactions, enabling new molecular pharmacologies, such as targeted protein degradation. They offer advantages over proteolysis targeting chimeras (PROTACs), which present challenges associated with the size and properties of heterobifunctional constructions, but glues lack the rational design principles analogous to PROTACs. One notable exception is the ability to alter the structure of Cereblon (CRBN)-based molecular glues and redirect their activity toward new neo-substrate proteins. We took a focused approach toward modifying the CRBN ligand, 5'-amino lenalidomide, to alter its neo-substrate specificity using high-throughput chemical diversification by parallelized sulfur(VI)-fluoride exchange (SuFEx) transformations. We synthesized over 3,000 analogs of 5'-amino lenalidomide using this approach and screened the crude products using a phenotypic screen for cell viability, identifying dozens of analogs with differentiated activity. We characterized four compounds that degrade G-to-S phase transition 1 (GSPT1) protein, providing a proof-of-concept model for SuFEx-based discovery of CRBN molecular glues.

Expanding the ligand spaces for E3 ligases for the design of protein degraders.

Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.

High-Performance Dopamine-Based Supramolecular Bio-Adhesives.

The need for wound closure or surgical procedures has been commonly met by the application of sutures. Unfortunately, these are often invasive or subject to contamination. Alternative solutions are offered by surgical adhesives that can be applied and set without major disruption; a new class of supramolecular-based adhesives provides potential solutions to some of these challenges. In this study, a series of polymers utilizing dopamine as a self-assembling unit are synthesized. It is found that these motifs act as extremely effective adhesives, with control over the mechanical strength of the adhesion and materials' tensile properties enabled by changing monomer feed ratios and levels of cross-linking. These materials significantly outperform commercially available bio-adhesives, showing yield strengths after adhesion at least two times higher than that of BioGlue and Tisseel, as well as the ability to re-adhere with significant recovery of adhesion strength. Promisingly, the materials are shown to be non-cytotoxic, with cell viability > 90%, and able to perform in aqueous environments without significant loss in strength. Finally, the removal of the materials, is possible using benign organic solvents such as ethanol. These properties all demonstrate the effectiveness of the materials as potential bio-adhesives, with potential advantages for use in surgery.

Molecular glues targeting GSPT1 in cancers: A potent therapy.

G1 to S phase transition 1 (GSPT1) is a key translation termination factor that significantly overexpressed in various cancer tissues and cells. Molecular glue is a kind of small molecule, which can bind to an E3 ligase such as cereblon (CRBN) and subsequently recruit neosubstrate proteins for ubiquitination-proteasomal degradation. This emerging therapeutic approach shows great potential in treating cancers and other diseases. This review aims to introduce current understanding of antitumor mechanism of molecular glues targeting GSPT1, summarize pharmacology profiles of existing molecular glues, and outline development strategies of novel molecular glues. The insights provided in this review will be valuable for future studies.