High Accuracy Prediction of PROTAC Complex Structures.

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders

Targeted protein degradation has emerged as a promising strategy for developing novel therapeutics, particularly for "undruggable" disease-related proteins. One approach is the use of PROteolysis TArgeting Chimeras (PROTACs) degraders, which induce the formation of ternary complexes between the target protein and E3 ligase, leading to ubiquitination and degradation of the target protein. Understanding the conformational behavior of PROTACs in solutions and how it relates to their pharmacokinetic properties and membrane permeability is crucial for optimizing PROTAC design and efficacy. Due to the large size and flexibility of PROTACs and their chameleonic character, it is essential to understand their conformational ensembles, and how they depend on the environment. Here, we introduce a novel methodology for exploring PROTAC conformational behavior using atomistic simulations. We employ the enhanced sampling method parallel bias metadynamics, where we bias generic local collective variables, specifically all rotatable dihedral angles, thereby avoiding the considerable challenge of identifying suitable global collective variables for biasing. The methodology allows for obtaining free energy surfaces of global CVs via reweighting. We apply the method to the prototypical case of the MZ1 PROTAC degrader, which targets bromodomain-containing protein-4 (BRD4) for degradation via the von Hippel-Lindau (VHL) E3 ligase, and elucidate its conformational behavior in different solvents, allowing us to gain insights into the chameleonic property of MZ1. Our results confirm that MZ1 adopts distinct conformations depending on the solvent, exhibiting collapsed conformations in water and chloroform, and extended conformations in DMSO. Collapsed conformations in chloroform have been correlated to increased cell permeability. Thus, our results show that MZ1 takes on conformations suitable for membrane permeation in apolar environments. Our methodological framework is generally applicable to large flexible molecules like PROTACs, and the results demonstrate its efficiency, laying the groundwork for similar investigations for other PROTACs and other "beyond-rule-of-5" drug candidates. This work provides valuable insights into the design and optimization of PROTACs, ultimately contributing to developing novel therapeutics for "undruggable" proteins.

PROTACs: A novel strategy for cancer therapy.

Proteolysis-targeting chimeras (PROTACs) are a class of bifunctional molecules that can induce the ubiquitin degradation of its target protein by hijacking the E3 ligase to form a target protein-PROTAC-E3 ligase ternary complex. Its underlying principle has inspired the development of a wide range of protein degraders that are similar to or beyond PROTACs in recent years. The formation of the ternary complexes is the key to the success of PROTAC-induced protein degradation. Nevertheless, the lack of effective ternary complex modeling techniques has limited the application of computer-aided drug discovery tools to this emerging and fast developing new land in drug industry. Thus, in this study, we explored the application of the more physically sound molecular dynamics simulation and the molecular mechanics combined with the generalized Born and surface area continuum solvation (MM/GBSA) method to solve the underlying three-body problem in PROTAC modeling. We first verified the accuracy of our approach using a series of known Brd4 BD2 degraders. The calculated binding energy showed a good correlation with the experimental Kd values. The modeling of a unique property, namely, the α value, for PROTACs was also first and accurately performed to our best knowledge. The results also demonstrated the importance of PROTAC-induced protein-protein interactions in its modeling, either qualitatively or quantitatively. Finally, by standing on the success of earlier docking-based approaches, our protocol was also applied as a rescoring function in pose prediction. The results showed a notable improvement in reranking the initial poses generated from a modified Rosetta method, which was reportedly one of the best among a handful of PROTAC modeling approaches available in this field. We hope this work could provide a practical protocol and more insights to study the binding and the design of PROTACs and other protein degraders.

Targeted protein degradation through PROteolysis TArgeting Chimeras (PROTACs) is a relatively new modality in cellular interventions. The minimum requirement for PROTACs to function is forming a tertiary complex of the protein of interest (POI), E3 ligase, and the molecular glue PROTAC. Here, we propose a new approach to modulate the nano-environment interactome of a non-protein target through a plausible quaternary complex of interactome-biomolecule of interest (BOI)-PROTAC and E3 ligase. We report nucleic acid-targeting PROTAC (NA-TAC) molecules by conjugating DNA-binding and E3 ligase ligands. We demonstrate that NA-TACs can target the G-quadruplex DNA and induce elevated DNA damage and cytotoxicity compared to the conventional G-quadruplex binding ligands. Our new class of NA-TACs lays the foundation for small molecule-based non-protein targeting PROTACs for interactome and nanoenvironment mapping and nucleic acid-targeted precision medicines.

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.

Proteolysis targeting chimeras (PROTACs) have emerged as a promising strategy for cancer therapy in the field of drug discovery. PROTACs are heterobifunctional small molecules that bind to both a target protein and an E3 ubiquitin ligase, thus facilitating degradation of the target protein of interest. Benefits of PROTACs as an alternative therapeutic strategy include the ability to target inaccessible proteins, the regulation of target protein signaling pathways, and overcoming drug resistance in certain types of cancer. There are more than 600 known E3 ligases in the human genome, but current PROTAC technology has been limited to the use of CRBN, VHL, XIAP, and MDM2. Therefore, the discovery of novel ligands for different E3 ligases could further improve current PROTAC technology. An application that our research group has focused on is exploring the possibility of tissue selective E3 ligases that are overexpressed in cancer cells compared to normal tissue to design tissue selective PROTACs for a target of interest. We have observed that protein levels of the E3 ligase KLHL12 are low in normal tissue and high in cancer cells. Using structure-activity relationship (SAR) by nuclear magnetic resonance (NMR), we identified hits that bound to the KLHL12 E3 ligase, which were then optimized to bind in the range of 10-100 nM Kd by SPR. We then generated KLHL12 based PROTACS that target BRD4 and Bcl-xL using thalidomide and A-115463 derivatives, respectively. PROTAC activity was optimized by measuring protein target degradation in HT29 cells by an in-cell western experiment where our most potent degrader had an average DC50 of 5.05 nM and achieved 89.65% degradation. Mechanism studies were carried out to verify that the PROTACs behaved in both a proteasome and target dependent manner. Future studies will support further characterization of KLHL12 by investigating its expression patterns across various cancer tissues, its functional essentiality in cells, and its involvement in protein-protein interactions. Citation Format: Zoe A. Petros, John Lee Sensintaffar, Anish Kizhakkekkara Vadukoot, Kwangho Kim, Manikandan Thangaraj, Alex G. Waterson, Stephen W. Fesik. Characterization and validation of PROTACs that recruit KLHL12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7014.

The Ubiquitin Proteasome System (UPS) is responsible for the degradation of misfolded or aggregated proteins via a multistep ATP-dependent proteolytic mechanism. This process involves a cascade of ubiquitin (Ub) transfer steps from E1 to E2 to E3 ligase. The E3 ligase transfers Ub to a targeted protein that is brought to the proteasome for degradation. The inability of the UPS to remove misfolded or aggregated proteins due to UPS dysfunction is commonly observed in neurodegenerative diseases, such as Alzheimer’s disease (AD). UPS dysfunction in AD drives disease pathology and is associated with the common hallmarks such as amyloid-β (Aβ) accumulation and tau hyperphosphorylation, among others. E3 ligases are key members of the UPS machinery and dysfunction or changes in their expression can propagate other aberrant processes that accelerate AD pathology. The upregulation or downregulation of expression or activity of E3 ligases responsible for these processes results in changes in protein levels of E3 ligase substrates, many of which represent key proteins that propagate AD. A powerful way to better characterize UPS dysfunction in AD and the role of individual E3 ligases is via the use of high-quality chemical tools that bind and modulate specific E3 ligases. Furthermore, through combining gene editing with recent advances in 3D cell culture, in vitro modeling of AD in a dish has become more relevant and possible. These cell-based models of AD allow for study of specific pathways and mechanisms as well as characterization of the role E3 ligases play in driving AD. In this review, we outline the key mechanisms of UPS dysregulation linked to E3 ligases in AD and highlight the currently available chemical modulators. We present several key approaches for E3 ligase ligand discovery being employed with respect to distinct classes of E3 ligases. Where possible, specific examples of the use of cultured neurons to delineate E3 ligase biology have been captured. Finally, utilizing the available ligands for E3 ligases in the design of proteolysis targeting chimeras (PROTACs) to degrade aberrant proteins is a novel strategy for AD, and we explore the prospects of PROTACs as AD therapeutics.

Proteolysis targeting chimeras (PROTACs) enable degradation of disease-related proteins via E3 ligase recruitment. PROTACs often do not easily crystallize, and they are usually formulated in amorphous forms. Determining the key interactions that stabilize the solid drug forms is of high interest. Here, we determine the complete atomic-level structure of an amorphous Von Hippel-Lindau (VHL)-based SMARCA2 PROTAC (PROTAC 2) using nuclear magnetic resonance (NMR) crystallography. We find that PROTAC 2 is more disordered as compared to previously studied amorphous formulations, and that the three functional units of the molecule have distinct structural types. In contrast to smaller drug molecules, where intermolecular hydrogen bonding interactions were found to be the main stabilization mechanism for the amorphous solid form, for PROTAC 2 we postulate that, in analogy to glassy polymers, the main stabilization mechanism is the entropic contribution introduced by the overall flexibility, especially in the linker region of the molecule. We also note that the most populated conformations found in the amorphous form differ from those of bound PROTAC 2 in the ternary protein complex as determined via X-ray crystallography. Our results provide insight into key structural features that stabilize amorphous formulations, specifically for molecules that can target proteins previously considered undruggable.

Accurately predicting the degradation capabilities of proteolysis-targeting chimeras (PROTACs) for given target proteins and E3 ligases is important for PROTAC design. The distinctive ternary structure of PROTACs presents a challenge to traditional drug-target interaction prediction methods, necessitating more innovative approaches. While current state-of-the-art (SOTA) methods using graph neural networks (GNNs) can discern the molecular structure of PROTACs and proteins, thus enabling the efficient prediction of PROTACs' degradation capabilities, they rely heavily on limited crystal structure data of the POI-PROTAC-E3 ternary complex. This reliance underutilizes rich PROTAC experimental data and neglects intricate interaction relationships within ternary complexes. In this study, we propose a model based on cross-modal strategy and ternary attention technology, ET-PROTACs, to predict the targeted degradation capabilities of PROTACs. Our model capitalizes on the strengths of cross-modal methods by using equivariant GNN graph neural networks to process the graph structure and spatial coordinates of PROTAC molecules concurrently while utilizing sequence-based methods to learn the protein sequence information. This integration of cross-modal information is cohesively harnessed and channeled into a ternary attention mechanism, specially tailored for the unique structure of PROTACs, enabling the congruent modeling of both PROTAC and protein modalities. Experimental results demonstrate that the ET-PROTACs model outperforms existing SOTA methods. Moreover, visualizing attention scores illuminates crucial residues and atoms pivotal in specific POI-PROTAC-E3 interactions, thus offering invaluable insights and guidance for future pharmaceutical research. The codes of our model are available at https://github.com/GuanyuYue/ET-PROTACs.

Overexpression of anti-apoptotic proteins such as BCL-xL is a hallmark of various cancers and a major driver of resistance to conventional chemotherapies. While small-molecule BCL-xL inhibitors have shown promising outcomes, their clinical use is hindered by dose-limiting toxicities, especially thrombocytopenia. Proteolysis-targeting chimeras (PROTACs) offer a promising alternative by promoting selective degradation of target proteins via the ubiquitin-proteasome system, thereby reducing off-target effects associated with small molecule inhibitors. However, rational design and optimization of PROTACs remain challenging due to the need to balance simultaneous interactions with both an E3 ubiquitin ligase and the target protein. Here we employ native mass spectrometry (MS) as a rapid, label-free platform to screen and characterize the formation and stability of ternary complexes between BCL-xL, VHL E3 ligase complex (VCB), and various targeting PROTACs. Native MS enables direct detection of binary BCL-xL●PROTAC and ternary BCL-xL●PROTAC●VCB complexes and provides semi-quantitative insights into PROTAC affinity and cooperativity with both binding partners. Furthermore, we explore the dissociation pathways of these complexes in the gas phase using collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD), revealing distinct fragmentation and subunit release patterns that reflect the structural organization and gas-phase stability of the complexes. Variable-temperature ESI-MS (vT-ESI) further allows assessment of thermal stabilities of the complexes in solution. Together, our study demonstrates the power of native MS to both screen and mechanistically characterize PROTAC-induced ternary complex formation.

Proteolysis-targeting chimeras (PROTACs) are a valuable therapeutic method for degrading target proteins of interest. Their success depends on forming a stable ternary complex composed of PROTAC, the target protein, and the E3 ligase-a relationship that is inherently difficult to predict. Existing deep learning methods often fail to accurately model the 3D arrangement of this ternary structure or overlook the critical background information provided by sequences, which is essential for accurate degradation predictions. Herein, we introduce SE(3)-PROTACs, a PROTAC degradation prediction model designed specifically using a geometric deep learning architecture. An SE(3)-equivariant transformer encodes PROTAC substructures-the warhead, linker, and E3 ligand-as molecular graphs invariant to translation and rotation while retaining geometrical features relevant to chemistry. Pretrained evolutionary scale modeling (ESM) embeddings provide functional and structural context to target proteins and E3 ligases directly from sequences, thus bypassing the limitations inherent in incomplete structural data. A Pairwise Interaction Mechanism computes all-pairs compatibility scores between target and E3 ligase residue positions conditioned on the PROTAC scaffold, using mean-pooled sigmoid weighting to reweight each protein representation before classification. A benchmark dataset of 1979 curated PROTAC samples sourced from PROTAC-DB was used for model development and evaluation. SE(3)-PROTACs achieved a test accuracy of 80.81% on a held-out random-split test set, with consistent performance across the cluster-split (65.62%) and temporal-split (64.08%) evaluations. SE(3)-PROTACs outperform baseline models across random, cluster-based, and temporal evaluations, demonstrating strong generalization to new targets and compounds, and serving as a reliable computational pre-filter for prioritizing candidate degraders before experimental validation.

Proteolysis targeting chimeras (PROTACs) represent a promising class of hetero-bivalent molecules that facilitate ubiquitination of a target protein by simultaneously binding and bringing together both the E3 enzyme and the target. These compounds consist of three structural components: two ligands one of which binds the protein of interest (POI) while the other binds an E3 ubiquitin ligase to promote POI ubiquitination, and a linker connecting both moieties. Recent developments in the field highlight the fact that linker composition and length play a crucial role in achieving optimal PROTAC properties, modulate binding kinetics and substantially impacts the potency and selectivity. In this review, the authors briefly discuss the recent findings in PROTAC design approaches with focus on the linker. For each PROTAC such linker parameters as chemical nature, length, hydrophilicity and rigidity have to be optimized to achieve improved stability, bioavailability cell membrane permeability and suitable spatial orientation between the target POI and the E3 ubiquitin ligase. Thus rational linker design with respect to composition, length and attachment sites is essential for the development of potent PROTAC compounds. Computer-aided design and novel innovative linker strategies, such as PROTAC shortening, photo-switchable PROTACs, in-cell click-formed CLIPTACs, “click chemistry” approaches are also discussed in the review.

The development of proteolysis targeting chimeras (PROTACs), which induce the degradation of target proteins by bringing them into proximity with cellular E3 ubiquitin ligases, has revolutionized drug development. While the human genome encodes more than 600 different E3 ligases, current PROTACs use only a handful of them, drastically limiting their full potential. Furthermore, many PROTAC development campaigns fail because the selected E3 ligase candidates are unable to induce degradation of the particular target of interest. As more and more ligands for novel E3 ligases are discovered, the chemical effort to identify the best E3 ligase for a given target is exploding. Therefore, a genetic system to identify degradation-causing E3 ligases and suitable target/E3 ligase pairs is urgently needed. Here, we used the well-established dimerization of the FKBP12 protein and FRB domain by rapamycin to bring the target protein WDR5 into proximity with candidate E3 ligases. Strikingly, this rapamycin-induced proximity assay (RiPA) revealed that VHL, but not Cereblon, is able to induce WDR5 degradation - a finding previously made by PROTACs, demonstrating its predictive power. By optimizing the steric arrangement of all components and fusing the target protein with a minimal luciferase, RiPA can identify the ideal E3 for any target protein of interest in living cells, significantly reducing and focusing the chemical effort in the early stages of PROTAC development.

PROteolysis TArgeting Chimeras (PROTACs) has recently emerged as a promising technology. However, the design of rational PROTACs, especially the linker component, remains challenging due to the absence of structure-activity relationships and experimental data. Leveraging the structural characteristics of PROTACs, fragment-based drug design (FBDD) provides a feasible approach for PROTAC research. Concurrently, artificial intelligence-generated content has attracted considerable attention, with diffusion models and Transformers emerging as indispensable tools in this field. In response, we present a new diffusion model, DiffPROTACs, harnessing the power of Transformers to learn and generate new PROTAC linkers based on given ligands. To introduce the essential inductive biases required for molecular generation, we propose the O(3) equivariant graph Transformer module, which augments Transformers with graph neural networks (GNNs), using Transformers to update nodes and GNNs to update the coordinates of PROTAC atoms. DiffPROTACs effectively competes with existing models and achieves comparable performance on two traditional FBDD datasets, ZINC and GEOM. To differentiate the molecular characteristics between PROTACs and traditional small molecules, we fine-tuned the model on our self-built PROTACs dataset, achieving a 93.86% validity rate for generated PROTACs. Additionally, we provide a generated PROTAC database for further research, which can be accessed at https://bailab.siais.shanghaitech.edu.cn/service/DiffPROTACs-generated.tgz. The corresponding code is available at https://github.com/Fenglei104/DiffPROTACs and the server is at https://bailab.siais.shanghaitech.edu.cn/services/diffprotacs.