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.

Proteolysis-targeting chimera (PROTAC) represents an emerging and highly promising protein degradation technology with the potential to overcome critical bottlenecks in traditional small molecule drug development. However, the scarcity of publicly available compound structure data has significantly limited computational drug discovery and artificial intelligence-driven modeling in this field. Patent represents an important yet underutilized source of novel chemical structures in medicinal chemistry. In this study, we systematically collected PROTAC-related patents and associated chemical structures disclosed therein. Through rigorous manual screening and expert annotation, we obtained 63,136 unique PROTAC compounds from 590 patent families, along with 252 targets. Additionally, we employed the ADMETlab 3.0 platform to systematically predict 120 physicochemical properties for all compounds. The dataset has been publicly shared on Figshare, providing a solid foundation for computational drug discovery and structure-activity relationship analyses.

Proteolysis-targeting chimeras (PROTACs) show promise in tumor treatment. However, the E3 ligases VHL and CRBN, commonly used in PROTAC, are highly expressed in only a few tumors, thus limiting the application scope and efficacy of PROTAC drugs. Furthermore, the lack of tumor specificity in PROTAC drugs can result in toxic side effects. Therefore, there is an urgent need to develop tumor-selective PROTAC drugs that do not rely on endogenous E3 ligases. In this study, we introduce the ClickRNA-PROTAC system, which involves the expression of a fusion protein of the E3 ubiquitin ligase SIAH1 and SNAPTag through mRNA transfection and recruits the protein of interest (POI) using bio-orthogonal click chemistry. ClickRNA-PROTAC can effectively degrade various proteins such as BRD4, KRAS, and NFκB simply by replacing the warhead molecules. By employing a tumor-specific mRNA-responsive translation strategy, ClickRNA-PROTAC can selectively degrade POIs in tumor cells. Furthermore, ClickRNA-PROTAC demonstrated strong efficacy in targeted cancer therapy in a xenograft mouse model of adrenocortical carcinoma. In conclusion, this approach offers several advantages, including independence from endogenous E3 ubiquitin ligases, tumor specificity, and programmability, thereby paving the way for the development of PROTAC drugs.

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).

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.

Protein-Protein, Genetic, and Chemical Interactions for Lai AC (2016):Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

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 Bashore FM (2023):PROTAC Linkerology Leads to an Optimized Bivalent Chemical Degrader of Polycomb Repressive Complex 2 (PRC2) Components. curated by BioGRID (https://thebiogrid.org); ABSTRACT: 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 ?M).

Protein-Protein, Genetic, and Chemical Interactions for Pu C (2023):Development of PROTAC degrader probe of CDK4/6 based on DCAF16. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Treatment of breast cancer has greatly evolved during the last decades, but triple negative breast cancer (TNBC) with a higher degree of malignancy cannot be directly and effectively treated. Abnormal cell cycle is generally found in human breast cancer and other malignant tumors, and cyclin-dependent kinases (CDK) 4/6, a cell cycle-related regulatory nuclear protein, is deemed as an effective target for breast cancer treatment so far. Since DCAF16 E3 ligase is also mainly distributed in the nucleus, in this study, by combining Palbociclib and DCAF16 E3 ligase ligand KB02 with different linkers, a series of DCAF16 based CDK4/6 degraders were designed and synthesized. Among them, compound A4 showed potent inhibitory activity against CDK4/6, and decreased the level of CDK4/6 protein in MDA-MB-231 cells in a concentration- and time-dependent manner. Moreover, the toxicity of A4 in normal cells showed 7 times lower than that of Palbociclib, and A4 exhibits therapeutic potential in MDA-MB-231 xenograft models in vivo. These findings indicate that A4, as a novel CDK4/6 degrader based on DCAF16, is worthy of further investigating for the treatment of TNBC.

The development of miniaturized high-throughput in situ screening platforms capable of handling the entire process of drug synthesis to final screening is essential for advancing drug discovery in the future. In this study, an approach based on combinatorial solid-phase synthesis, enabling the efficient synthesis of libraries of proteolysis targeting chimeras (PROTACs) in an array format is presented. This on-chip platform allows direct biological screening without the need for transfer steps. UV-induced release of target molecules into individual droplets facilitates further on-chip experimentation. Utilizing a mitogen-activated protein kinase kinases (MEK1/2) degrader as a template, a series of 132 novel PROTAC-like molecules is synthesized using solid-phase Ugi reaction. These compounds are further characterized using various methods, including matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging, while consuming only a few milligrams of starting materials in total. Furthermore, the feasibility of culturing cancer cells on the modified spots and quantifying the effect of MEK suppression is demonstrated. The miniaturized synthesis platform lays a foundation for high-throughput in situ biological screening of potent PROTACs for potential anticancer activity and offers the potential for accelerating the drug discovery process by integrating miniaturized synthesis and biological steps on the same array.

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.

Protein-Protein, Genetic, and Chemical Interactions for Kumarasamy V (2023):PROTAC-mediated CDK degradation differentially impacts cancer cell cycles due to heterogeneity in kinase dependencies. curated by BioGRID (https://thebiogrid.org); ABSTRACT: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibition yields differential cellular responses in multiple tumor models due to redundancy in cell cycle. We investigate whether the differential requirements of CDKs in multiple cell lines function as determinant of response to pharmacological agents that target these kinases.We utilized proteolysis-targeted chimeras (PROTACs) that are conjugated with palbociclib (Palbo-PROTAC) to degrade both CDK4 and CDK6. FN-POM was synthesized by chemically conjugating pomalidomide moiety with a multi-kinase inhibitor, FN-1501. Patient derived PDAC organoids and PDX model were utilized to investigate the effect of FN-POM in combination with palbociclib.Palbo-PROTAC mediates differential impact on cell cycle in different tumor models, indicating that the dependencies to CDK4 and 6 kinases are heterogenous. Cyclin E overexpression uncouples cell cycle from CDK4/6 and drives resistance to palbo-PROTAC. Elevated expression of P16INK4A antagonizes PROTAC-mediated degradation of CDK4 and 6. FN-POM degrades cyclin E and CDK2 and inhibits cell cycle progression in P16INK4A-high tumor models. Combination of palbociclib and FN-POM cooperatively inhibit tumor cell proliferation via RB activation.Resistance to CDK4/6 inhibition could be overcome by pharmacologically limiting Cyclin E/CDK2 complex and proves to be a potential therapeutic approach.

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.

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.

MD-224 is a PROTAC that induces degradation of the human protein MDM2. However, we have found that it also induces degradation of a subset of nuclear receptors. DIA was performed to assess the overall specificity of MD-224 in 293T cells and the colorectal cancer cell line SNU-C4.

PKMYT1 has recently emerged as a compelling therapeutic target for precision cancer therapy due to its synthetic lethality with oncogenic alterations such as CCNE1 amplification and mutations in FBXW7 and PPP2R1A. Current small molecule PKMYT1 inhibitors face limitations, such as insufficient molecular diversity and poor selectivity. We used our generative AI platform to develop a bifunctional PKMYT1 degrader by linking an entirely novel PKMYT1 inhibitor to an optimized cereblon (CRBN) binder. The lead PROTAC D16-M1P2 demonstrated dual mechanisms of PKMYT1 degradation and antagonism, with strong antiproliferative potency facilitated by high selectivity. It also exhibited favorable oral bioavailability, stronger pharmacodynamic effects relative to the PKMYT1 inhibitor alone, and robust antitumor response as a monotherapy in xenograft models. This first-in-class PROTAC serves as a precise chemical probe to explore PKMYT1 biology and a promising lead for further cancer therapy exploration.

Herein, we report a potent HDAC6 PROTAC, TO-1187, which selectively degrades HDAC6 in cellulo and demonstrates in vivo efficacy. The design of TO-1187 was achieved by linking our previously reported HDAC6 inhibitor, TO-317, to the cereblon (CRBN) E3 ligase ligand, pomalidomide. TO-1187 achieved monoselective HDAC6 degradation in human multiple myeloma cells, MM.1S, with a Dmax of 94% and a DC50 of 5.81 nM after 6 h. Importantly, at concentrations up to 25 μM, TO-1187 exhibited no cellular degradation of other HDACs. Proteomic evaluation confirmed a highly selective proteome-wide degradation profile, with HDAC6 the only protein observed to be depleted. Notably, TO-1187 did not impact the abundance of well-known CRBN neosubstrates, like IKZF1, IKZF3, CK1α, SALL4, and GSPT1. In vivo evaluation confirmed that TO-1187 efficiently degraded HDAC6 in mouse tissues, measured 6 h after intravenous injection. In summary, TO-1187 represents a viable candidate for advanced preclinical evaluation of HDAC6 biology.

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.

Reactivation of fetal hemoglobin expression by down-regulation of BCL11A is a promising treatment of -hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of -globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR/Cas9 gene editing. We leveraged the dTAG PROTAC platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by PRO-seq, proteomics, chromatin accessibility, and histone profiling. Among ≤ 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in < 2h. Robust HBG1/2 reactivation upon acute BCL11A-depletion occurred without loss of promoter 5methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci

This repository contains 48 structures predicted with AlphaFold 3, reported in the publication "Enhancing PROTAC Ternary Complex Prediction with Ligand Information in AlphaFold 3" by Francisco Erazo, Nils Dunlop, Farzaneh Jalalypour, Rocío Mercado. The accompaning code is available at: https://github.com/NilsDunlop/PROTACFold/.

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