Design Of Proteolysis Targeting Chimeras Research Articles

Correlation between Membrane Permeability and the Intracellular Degradation Activity of Proteolysis-Targeting Chimeras.

Proteolysis-targeting chimeras (PROTACs) have attracted attention as an innovative drug modality that induces the selective degradation of target proteins. This technology shows higher activity than conventional inhibitors and holds great potential in the field of drug discovery. Optimization of the linker is essential for PROTACs to achieve sufficient activity, particularly with regard to cell membrane permeability. However, the correlation between membrane permeability and the activity of PROTACs has not been fully explored. To address this, we established a new molecular design approach to remove the linker and optimize PROTAC structure. These PROTAC compound groups were used to analyze the correlation between membrane permeability and activity using LC-tandem mass spectrometry (LC-MS/MS). Results revealed that the degradation activity of PROTACs fluctuates with increasing membrane permeability and changes in response to linker optimization, while sufficient proteolytic activity can be retained. These findings demonstrate the importance of considering the balance between membrane permeability and activity in PROTAC design and provide a new strategy for developing more effective PROTACs.

Integral Membrane Protein Degradation: A Novel Paradigm by PROTAC Technology

: Integral membrane proteins are attractive targets for therapeutic intervention due to their crucial roles in cellular functions and their involvement in various diseases. However, targeting these proteins for degradation has posed significant challenges due to their complex structures and diverse functions. The emergence of proteolysis-targeting chimeras (PROTAC) represents a groundbreaking paradigm shift in drug development, offering a novel approach to address the degradation of integral membrane proteins. This review explores the innovative application of PROTAC technology in inducing the degradation of integral membrane proteins. PROTACs are molecules that have two roles: they bring together specific target proteins and E3 ubiquitin ligases, which help with ubiquitination and the degradation of proteins by proteasomes. As PROTACs are made up of separate parts, they can be put together to make custom molecules that can target difficult targets, such as integral membrane proteins. This review covers recent developments and advancements in the design and optimisation of PROTACs tailored for integral membrane protein degradation. Furthermore, it also discusses the problems that come up when trying to target membrane proteins and how PROTACs solve these problems by using ligands that can pass cell membranes and specifically interact with target proteins. It delves into the potential therapeutic implications of degrading integral membrane proteins using PROTACs and elucidates the impact of this novel approach in treating diseases that involve aberrant membrane protein expression or function by highlighting specific examples and case studies.

Advancing Proteolysis Targeting Chimera (PROTAC) Nanotechnology in Protein Homeostasis Reprograming for Disease Treatment.

Proteolysis targeting chimeras (PROTACs) represent a transformative class of therapeutic agents that leverage the intrinsic protein degradation machinery to modulate the hemostasis of key disease-associated proteins selectively. Although several PROTACs have been approved for clinical application, suboptimal therapeutic efficacy and potential adverse side effects remain challenging. Benefiting from the enhanced targeted delivery, reduced systemic toxicity, and improved bioavailability, nanomedicines can be tailored with precision to integrate with PROTACs which hold significant potential to facilitate PROTAC nanomedicines (nano-PROTACs) for clinical translation with enhanced efficacy and reduced side effects. In this review, we provide an overview of the recent progress in the convergence of nanotechnology with PROTAC design, leveraging the inherent properties of nanomaterials, such as lipids, polymers, inorganic nanoparticles, nanohydrogels, proteins, and nucleic acids, for precise PROTAC delivery. Additionally, we discuss the various categories of PROTAC targets and provide insights into their clinical translational potential, alongside the challenges that need to be addressed.

Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design.

Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.

DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation.

Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors' spacing and orientation. The resulting constructs revealed distance- and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.

A Novel Rational PROTACs Design and Validation via AI-Driven Drug Design Approach.

The rational design of novel drug candidates presents a formidable challenge in modern drug discovery. Proteolysis-targeting chimeras (PROTACs) drug design is particularly demanding due to their limited crystal structure availability and design of a viable small molecule to bridge the protein of interest (POI) and ubiquitin-protein ligase (E3). An integrated approach that combines superimposition techniques and deep neural networks is demonstrated in this study to leverage the power of deep learning and structural biology to generate structurally diverse molecules with enhanced binding affinities. The superimposition technique ensures the congruence of initial and new protein-ligand pairs, which are evaluated via subsequent comprehensive screening using the root-mean-square deviation (RMSD), binding free energy (BFE), and buried solvent-accessible surface area (SASA). The final candidates are subjected to the incorporation of molecular dynamics (MD) and free energy perturbation (FEP) simulations to provide a quantitative evaluation of relative binding energies, reinforcing the efficacy and reliability of the generated molecules. The outcomes of the generated novel PROTACs molecules exhibit comparable structural attributes while demonstrating superior binding affinities within the binding pockets when contrasted with those of the established cocrystal ternary complexes. To enhance the generalizability of the workflow, we chose the ternary structure of the cellular inhibitor of apoptosis protein 1 (cIAP1) and Bruton's Tyrosine Kinase (BTK) for validating the chemical properties generated from the processes. The new linker molecules additionally showed superior affinity from the simulations. In summary, this methodology serves as an effective workflow to align computational predictions with current limitations, thereby introducing a novel paradigm in AI-driven drug design.

PROTAC-DB 3.0: an updated database of PROTACs with extended pharmacokinetic parameters.

Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic technology that leverages the ubiquitin-proteasome system to target protein degradation. Due to its event-driven mechanistic characteristics, PROTAC has the potential to regulate traditionally non-druggable targets. Recently, AI-aided drug design has accelerated the development of PROTAC drugs. However, the rational design of PROTACs remains a considerable challenge. Here, we present an updated online database, PROTAC-DB 3.0. In this third version, we have expanded the database to include 6111 PROTACs (87% increase compared to the 2.0 version). Additionally, the database now contains 569 warheads (small molecules targeting the protein), 2753 linkers, and 107 E3 ligands (small molecules recruiting E3 ligases). The number of target-PROTAC-E3 ternary complex structures has also increased to 959. Recognizing the importance of druggability in PROTAC design, we have incorporated pharmacokinetic data to PROTAC-DB 3.0. To enhance user experience, we have added features for sorting based on molecular similarity and literature publication date. PROTAC-DB 3.0 is accessible at http://cadd.zju.edu.cn/protacdb/.

Harnessing PROTACs to combat H5N1 influenza: A new frontier in viral destruction.

H5N1, a highly pathogenic avian influenza virus, poses an ongoing and significant threat to global public health, primarily due to its potential to cause severe respiratory illness and high mortality rates in humans. Despite extensive efforts in vaccination and antiviral therapy, H5N1 continues to exhibit high mutation rates, resulting in recurrent outbreaks and the emergence of drug-resistant strains. Traditional antiviral therapies, such as neuraminidase inhibitors and M2 ion channel blockers, have demonstrated limited efficacy, necessitating the exploration of innovative therapeutic strategies. Proteolysis-targeting chimeras (PROTACs) emerge as a novel and promising approach, leveraging the ubiquitin-proteasome system to selectively degrade pathogenic proteins. Unlike conventional inhibitors that only block protein function, PROTACs eliminate the target protein, providing a sustained therapeutic effect and potentially reducing the development of resistance. This paper offers a comprehensive examination of the current landscape of H5N1 infections, detailing the pathogenesis and challenges associated with existing treatments. It further explores the mechanism of action, design, and therapeutic potential of PROTACs in inhibiting H5N1. By targeting essential viral proteins, such as hemagglutinin and the RNA-dependent RNA polymerase complex, PROTACs hold the potential to revolutionize the treatment of H5N1 infections, offering a new frontier in antiviral therapy.

DiffPROTACs is a deep learning-based generator for proteolysis targeting chimeras.

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.

PROTAC-induced Protein Functional Dynamics in Targeted Protein Degradation.

PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain containing protein 4 (BRD4BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC dependent protein dynamics facilitate the arrangement of surface lysine residues of BRD4BD1 into the catalytic pocket of E2/ubiquitin for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a dynamic perspective on ligand induced protein degradation, providing insights to guide future PROTAC design endeavors.

PROTACable Is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning To Automate the De Novo Design of PROTACs.

Proteolysis-targeting chimeras (PROTACs) that engage two biological targets at once are a promising technology in degrading clinically relevant protein targets. Since factors that influence the biological activities of PROTACs are more complex than those of a small molecule drug, we explored a combination of computational chemistry and deep learning strategies to forecast PROTAC activity and enable automated design. A new method named PROTACable was developed for the de novo design of PROTACs, which includes a robust 3-D modeling workflow to model PROTAC ternary complexes using a library of E3 ligase and linker and an SE(3)-equivariant graph transformer network to predict the activity of newly designed PROTACs. PROTACable is available at https://github.com/giaguaro/PROTACable/.

Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.

PROTAC-Design-Evaluator (PRODE): An Advanced Method for In-Silico PROTAC Design.

PROTAC (proteolysis-targeting chimeras) is a rapidly evolving technology to target undruggable targets. The mechanism by which this happens is when a bifunctional molecule binds to a target protein and also brings an E3 ubiquitin ligase in proximity to trigger ubiquitination and degradation of the target protein. Yet, in-silico-driven approaches to design these heterobifunctional molecules that have the desired functional properties to induce proximity between the target protein and E3 ligase remain to be established. In this paper, we present a novel in-silico method for PROTAC design and to demonstrate the validity of our approach, we show that for a BRD4-VHL-PROTAC-mediated ternary complex known in the literature, we are able to reproduce the PROTAC binding mode, the structure of the ternary complex formed therein, and the free energy (ΔG) thermodynamics favoring ternary complexation through theoretical/computational methodologies. Further, we demonstrate the use of thermal titration molecule dynamics (TTMD) to differentiate the stability of PROTAC-mediated ternary complexes. We employ the proposed methodology to design a PROTAC for a new system of FGFR1-MDM2 to degrade the FGFR1 (fibroblast growth factor receptor 1) that is overexpressed in cancer. Our work presented here and named as PROTAC-Designer-Evaluator (PRODE) contributes to the growing literature of in-silico approaches to PROTAC design and evaluation by incorporating the latest in-silico methods and demonstrates advancement over previously published PROTAC in-silico literature.

TRL-PROTAC: A pre-trained generator of PROTACs targeting specific proteins optimized by reinforcement learning

Proteolysis-targeting chimeras (PROTACs) introduce a novel paradigm in drug development, incorporating three essential components: the warhead, the E3 ligand, and the linker. The complexity of the ternary structure, particularly the intricate design of the linker, presents a significant challenge in PROTACs drug design. Here an integrated protocol for design and evaluation of PROTACs targeting specific proteins, called TRL-PROTAC is proposed. TRL-PROTAC is focused on the de novo design of complete PROTACs by effectively joining the designed ligands targeting the proteins of interest (POI) with linkers. The ligands for POIs and E3 ligases are generated by a molecular generation model for targeting proteins, and the linker design is generated by a sequence-to-sequence model consisting of a transformer variant and the policy-based reinforcement learning method which is employed to optimize the reward values for generating PROTACs. The three components are then integrated and optimized based on their pharmacokinetic (PK) and degradation (DEG) properties. The experimental results have strongly confirmed that TRL-PROTAC is superior in optimizing properties. For existing PROTACs, TRL-PROTAC improves DEG scores by 0.45 and lowers PK scores by 1.20. Moreover, TRL-PROTAC enhances binding affinity by 2.15 in PROTACs generated from scratch.

Design and synthesis of nucleic acid nano-environment interactome-targeting small molecule PROTACs and their anticancer activity.

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.

Peptide-based PROTACs: Current Challenges and Future Perspectives.

Proteolysis-targeting chimeras (PROTACs) are an attractive means to target previously undruggable or drug-resistant mutant proteins. While small molecule-based PROTACs are stable and can cross cell membranes, there is limited availability of suitable small molecule warheads capable of recruiting proteins to an E3 ubiquitin ligase for degradation. With advances in structural biology and in silico protein structure prediction, it is now becoming easier to define highly selective peptides suitable for PROTAC design. As a result, peptide-based PROTACs are becoming a feasible proposition for targeting previously "undruggable" proteins not amenable to small molecule inhibition. In this review, we summarize recent progress in the design and application of peptide-based PROTACs as well as several practical approaches for obtaining candidate peptides for PROTACs. We also discuss the major hurdles preventing the translation of peptide-based PROTACs from bench to bedside, such as their delivery and bioavailability, with the aim of stimulating discussion about how best to accelerate the clinical development of peptide- based PROTACs in the near future.

In Silico Design, Synthesis, and Evaluation of PROTAC Against Hematopoietic Prostaglandin D Synthase.

Chemical protein knockdown technology using proteolysis-targeting chimeras (PROTACs) to hijack the endogenous ubiquitin-proteasome system is a powerful strategy to degrade disease-related proteins. This chapter describes in silico design of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, PROTAC(H-PGDS), using a docking simulation of the ternary complex of H-PGDS/PROTAC/E3 ligase as well as the synthesis of the designed PROTAC(H-PGDS)s and evaluation of their H-PGDS degradation activity.

Covalent PROTAC design method based on a sulfonyl pyridone probe.

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel-Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 μM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.

Proteolysis-targeting chimeras with reduced off-targets.

Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a high-throughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.

Rapid PROTAC Discovery Platform: Nanomole-Scale Array Synthesis and Direct Screening of Reaction Mixtures.

Precise length, shape, and linker attachment points are all integral components to designing efficacious proteolysis targeting chimeras (PROTACs). Due to the synthetic complexity of these heterobifunctional degraders and the difficulty of computational modeling to aid PROTAC design, the exploration of structure-activity relationships remains mostly empirical, which requires a significant investment of time and resources. To facilitate rapid hit finding, we developed capabilities for PROTAC parallel synthesis and purification by harnessing an array of preformed E3-ligand-linker intermediates. In the next iteration of this approach, we developed a rapid, nanomole-scale PROTAC synthesis methodology using amide coupling that enables direct screening of nonpurified reaction mixtures in cell-based degradation assays, as well as logD and EPSA measurements. This approach greatly expands and accelerates PROTAC SAR exploration (5 days instead of several weeks) as well as avoids laborious and solvent-demanding purification of the reaction mixtures, thus making it an economical and more sustainable methodology for PROTAC hit finding.