To evaluate the performance of the proposed DegradeMaster and baseline methods, we collected data from the PROTAC-DB 3.0 database. The latest release of PROTAC-DB 3.0 comprises 9,380 PROTAC entries, including 569 warheads, 107 E3 ligands, and 5,753 linkers. Each entry includes detailed information such as the PROTAC's SMILES representation, and UniProt ID of the POI and the E3 ligase.

We first removed entries that lack critical information, e.g., the UniProt ID of the POI or E3 ligase. For degradation labels, we utilized both explicit DC50/Dmax values and implicit values inferred from experimental descriptions to predict PROTAC degradation activity. A PROTAC is considered to have low degradation activity if DC50 is greater than or equal to 100 nM and Dmax is below 80%, otherwise, it is labeled with high degradation activity. Crystal structures of POIs and E3 ligases are sourced from the Protein Data Bank (PDB), while proteins without available crystal structures are supplemented with predicted structures generated by AlphaFold 2. We apply Smina to dock the warhead and E3 ligand to POI and E3 ligase, respectively.

Using these criteria, we constructed a supervised PROTAC dataset consisting of 620 high-activity entries and 1,011 low-activity entries. Additionally, we curated a semi-supervised PROTAC dataset containing 8,603 entries in total, incorporating the same labeled subset as the supervised dataset.

Bivalent chemical degraders, otherwise known as proteolysis-targeting chimeras (PROTACs), have proven to be an efficient strategy for targeting overexpressed or mutated proteins in cancer. PROTACs provide an alternative approach to small-molecule inhibitors, which are restricted by occupancy-driven pharmacology, often resulting in acquired inhibitor resistance via compensatory increases in protein expression. Despite the advantages of bivalent chemical degraders, they often have suboptimal physicochemical properties and optimization for efficient degradation remains highly unpredictable. Herein, we report the development of a potent EED-targeted PRC2 degrader, UNC7700. UNC7700 contains a unique cis-cyclobutane linker and potently degrades PRC2 components EED (DC50 = 111 nM; Dmax = 84%), EZH2WT/EZH2Y641N (DC50 = 275 nM; Dmax = 86%), and to a lesser extent SUZ12 (Dmax = 44%) after 24 h in a diffuse large B-cell lymphoma DB cell line. Characterization of UNC7700 and related compounds for ternary complex formation and cellular permeability to provide a rationale for the observed improvement in degradation efficiency remained challenging. Importantly, UNC7700 dramatically reduces H3K27me3 levels and is anti-proliferative in DB cells (EC50 = 0.79 ± 0.53 μM).

Dataset: High-Throughput Miniaturized Synthesis of PROTAC-Like Molecules

Targeted protein degradation refers to the use of small molecule protein-protein dimerizers that recruit a ubiquitin E3 ligase to a protein of interest for chemically induced degradation. Here we combine systematic exploration of 51 degraders designed to broadly target the HDAC family of proteins with chemo-proteomics, to map the degradability of zinc-dependent HDAC proteins. Our dataset provides chemical leads for targeting HDACs 1-8 and 10, and investigates important aspects of degrader design such as recruited ligase and linker length and position, to assist chemical design prioritization. We discover that targeting HDACs often results in collateral degradation of the multi-protein complexes that HDACs belong to, offering a new mechanism of controlling chromatin structure. This database is an open source resource for facilitating accelerated degrader design and development for this epigenetic class of enzymes.

Protein-Protein, Genetic, and Chemical Interactions for Bondeson DP (2018):Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Inhibiting protein function selectively is a major goal of modern drug discovery. Here, we report a previously understudied benefit of small molecule proteolysis-targeting chimeras (PROTACs) that recruit E3Â ubiquitin ligases to target proteins for their ubiquitination and subsequent proteasome-mediated degradation. Using promiscuous CRBN- and VHL-recruiting PROTACs that bind >50 kinases, we show that only a subset of bound targets is degraded. The basis of this selectivity relies on protein-protein interactions between the E3 ubiquitin ligase and the target protein, as illustrated by engaged proteins that are not degraded as a result of unstable ternary complexes with PROTAC-recruited E3 ligases. In contrast, weak PROTAC:target protein affinity can be stabilized by high-affinity target:PROTAC:ligase trimer interactions, leading to efficient degradation. This study highlights design guidelines for generating potent PROTACs as well as possibilities for degrading undruggable proteins immune to traditional small-molecule inhibitors.

Protein-Protein, Genetic, and Chemical Interactions for Raina K (2016):PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.

Official repository of the data, scripts and structures employed in and produced during the work presented in "Design and Computational Evaluation of Tubulin-Targeting PROTACs for Brain Cancer Therapy".

The content of the repository is organized as follows:

• Starting ligands databases:
  • Database_colchicine.zip : employed colchicine analogs in mol, pdb, mol2, and moe database format.
  • Database_combretastatin.zip : employed combretastatin derivatives in mol, pdb, and moe database format.
  • Database_ExperimentalStructures.zip : experimental compounds in mol, pdb, mol2, and moe database format.

• Tubulin homology models:
  • Tubulin_Models_1SA0.zip : fasta sequences, homology models, bound ligands, molecular dynamics input and output files, and clustering results for each of the 8 tubulin dimers considered in the study.

• Docking:
  • Docking_vina_colchicine.zip : Vina config files, compounds pdbqt, and docking results for colchicine analogs dataset.
  • Docking_1SA0.zip : config files, compounds pdb and pdbqt, and docking results for all the ligands dataset used, for both AutoDock and MOE.

• ADMET predictions:
  • ADMET.zip : Simulations Plus' ADMET Predictor results for colchicine analogs, combretastatins, and experimental compounds datasets.

• MMPBSA:
  • MMPBSA.zip : input and output files for molecular dynamics, clustering, and rmsd of the tubulin dimers containing tubulin beta III and VI, and the UBR1 structure. MMPBSA calculations for tubulin beta III and VI in complex with selected ligands.

• PROTAC structures:
  • PROTACs.zip : structures of the PROTACs designed from each of the seven most promising tubulin-binding ligands. Data provided in ChemDraw, mol, mol2, and moe database format.

• PROTAC-mediated ternary structure prediction in MOE:
  • Ternary_Complex_Prediction.zip : moe output files and databases of the PROTAC-mediated ternary complexes, obtained via docking simulations in MOE.
  • Ternary_Complex_Prediction_Best.zip : moe databases for the best PROTACs and the most reliable ternary complexes, extracted from the docking data stored in Ternary_Complex_Prediction.zip.

The mammalian SWI/SNF helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of SMARCA2 selectivity is likely essential to afford an acceptable therapeutic index and this has been a considerable challenge due to the homology between paralogs. Herein we report the discovery of the first potent and selective SMARCA2 proteolysis-targeting chimera (PROTAC) molecule. Selective degradation was achieved in the absence of selective PROTAC binding and translated to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling revealed no unexpected off-target degradation. Our study thus highlights the ability to transform a non-selective SMARCA2-binding ligand into a selective and efficacious in vivo SMARCA2 PROTAC, providing a potential therapeutic opportunity for SMARCA4 mutant patients.

The catalytic properties of proteolysis targeting chimeras (PROTACs) may lead to uncontrolled off-tissue target degradation that causes potential toxicity, limiting their clinical applications. The precise control of this technology in a tissue-selective manner can minimize the potential toxicity. Hypoxia is a hallmark of most solid tumors, accompanied by elevated levels of nitroreductase (NTR). Based on this character, we presented a type of NTR-responsive PROTACs to selectively degrade proteins of interest (POI) in tumor tissues. Compound 17-1 was the first NTR-responsive PROTAC synthesized by incorporating the caging group on the Von Hippel–Lindau (VHL) E3 ubiquitin ligase ligand. It could be activated by NTR to release the active PROTAC 17 to efficiently degrade the EGFR protein and subsequently exert antitumor efficacy. Thus, a general strategy for the precise control of PROTAC to induce POI degradation in tumor tissues by NTR was established, which provided a generalizable platform for the development of NTR-controlled PROTACs to achieve selective degradation.

Protein degradation mediated by the proteolysis-targeting chimera (PROTAC) has emerged as an efficient strategy to accurately control intracellular protein levels. However, the development of PROTACs is limited by their systemic toxicity. Herein, we report a bioorthogonally activatable prodrug (BT-PROTAC) strategy to accurately control the activity of PROTACs. As a proof of concept, we introduced the highly reactive trans-cyclooctene into PROTAC molecule MZ1, the structure–acitivity relationships of which were well characterized previously, to construct the bioorthogonally activatable prodrug BT-PROTAC. Compared with MZ1, BT-PROTAC is incapable of degradation of BRD4 protein. However, BT-PROTAC can be activated by highly active tetrazine compound BODIPY-TZ in vitro. Furthermore, we could selectively degrade BRD4 protein in tumor tissue enabled by tumor-targeted tetrazine compound IR808-TZ. This strategy may represent an alternative to existing strategies and may be widely applied in the design of BT-PROTAC targeting other proteins.

Targeted proTargeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but it has not been systematically explored to what extent it influences target protein degradation. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a PROTAC which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required before concluding that a novel agent works as a bone-fide targeted protein degrader. tein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but it has not been systematically explored to what extent it influences target protein degradation. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a PROTAC which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required before concluding that a novel agent works as a bone-fide targeted protein degrader.

Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure–activity relationships of our bump-and-hole–PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo.

Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.

KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, one particular form of mutant KRAS, namely KRASG12C, can be targeted by small molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Although our lead degrader successfully engaged CRBN in cells, bound KRASG12C in vitro, induced CRBN/ KRASG12C dimerization, and degraded GFP-KRASG12C in GFP reporter cells in a CRBN-dependent manner, it failed to degrade endogenous KRASG12C in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively polyubiquitinate endogenous KRASG12C underlies the lack of activity. We discuss challenges for achieving targeted KRASG12C degradation and propose several possible solutions which may lead to efficient degradation of endogenous KRASG12C.

Our studies establish the unique properties of the cyclimids as versatile warheads in TPD and a systematic biochemical approach for quantifying ternary complex formation to predict their cellular degradation activity, which together will accelerate the development of novel CRBN-targeting bifunctional degraders.

Protein-Protein, Genetic, and Chemical Interactions for Smalley JP (2020):PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes. curated by BioGRID (https://thebiogrid.org); ABSTRACT: We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

input files for MD simulations, the restart structures in the PaCS-MD and OFLOOD, and analysis scripts

Protein-Protein, Genetic, and Chemical Interactions for Cyrus K (2010):Two-headed PROTAC: an effective new tool for targeted protein degradation. curated by BioGRID (https://thebiogrid.org); ABSTRACT:

Input files, topologies, cordinates, trajectory files, parameter setting files, and global/local minimum states about conformational search of PROTAC systems. Details of these files are given by README.

Protein-Protein, Genetic, and Chemical Interactions for Tinworth CP (2019):PROTAC-Mediated Degradation of Bruton's Tyrosine Kinase Is Inhibited by Covalent Binding. curated by BioGRID (https://thebiogrid.org); ABSTRACT: The impact of covalent binding on PROTAC-mediated degradation of BTK was investigated through the preparation of both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. It was determined that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC. These observations were consistently found when PROTACs that were able to recruit either IAP or cereblon E3 ligases were employed. Proteomics analysis determined that the use of a covalently bound PROTAC did not result in the degradation of covalently bound targets, while degradation was observed for some reversibly bound targets. This observation highlights the importance of catalysis for successful PROTAC-mediated degradation and highlights a potential caveat for the use of covalent target binders in PROTAC design.