Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

SRSF2 plays an unexpected role as reader of m5C on mRNA, linking epitranscriptomics to cancer.

A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.

The 17th EFMC Short Course on Medicinal Chemistry on Small Molecule Protein Degraders.

The 17th EFMC Short Course on Medicinal Chemistry took place April 23-26, 2023 in Oegstgeest, near Leiden in the Netherlands. It covered for the first time the exciting topic of Targeted Protein Degradation (full title: Small Molecule Protein Degraders: A New Opportunity for Drug Design and Development). The course was oversubscribed, with 35 attendees and 6 instructors mainly from Europe but also from the US and South Africa, and representing both industry and academia. This report summarizes the successful event, key lectures given and topics discussed.

Harnessing UBR5 for targeted protein degradation of key transcriptional regulators.

Targeted protein degradation (TPD) has opened the door for drugging transcriptional regulators, yet the number of proteins targeted and E3 ligases utilized remain limited. Here, we highlight UBR5 and propose multiple strategies by which this E3 ligase could be modulated to drive degradation of key transcriptional targets implicated in disease.

Hidden modes of DNA binding by human nuclear receptors.

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA-protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

Target 2035 - an update on private sector contributions.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins. Here, we present a brief progress update on Target 2035 and highlight some of industry's contributions.

The importance of cellular degradation kinetics for understanding mechanisms in targeted protein degradation.

Targeted protein degradation has exploded over the past several years due to preclinical and early clinical therapeutic success of numerous compounds, and the emergence of new degradation modalities, which has broadened the definition of what a degrader is. The most characterized and well-studied small molecule degraders are molecular glues and proteolysis targeting chimeras (PROTACs). These degraders induce a ternary complex between a target protein, degrader, and E3 ligase component, resulting in ubiquitination and subsequent degradation of the target protein via the ubiquitin proteasomal system (UPS). This event-driven process requires success at all steps through a complex cascade of events. As more systems, degraders, and targets are tested, it has become increasingly clear that achieving degradation is only the first critical milestone in a degrader development program. Rather highly efficacious degraders require a combination of multiple optimized parameters: rapid degradation, high potency, high maximal degradation (Dmax), and sustained loss of target without re-dosing. Success to meet these more rigorous goals depends upon the ability to characterize and understand the dynamic cellular degradation profiles and relate them to the underlying mechanism for any given target treated with a specific concentration of degrader. From this starting point, optimization and fine tuning of multiple kinetic parameters such as how fast degradation occurs (the rate), how much of the target is degraded (the extent), and how long the target remains degraded (the duration) can be performed. In this review we explore the diversity of cellular kinetic degradation profiles which can arise after molecular glue and PROTAC treatment and the potential implications of these varying responses. As the overall degradation kinetics are a sum of individual mechanistic steps, each with their own kinetic contributions, we discuss the ways in which changes at any one of these steps could potentially influence the resultant kinetic degradation profiles. Looking forward, we address the importance in characterizing the kinetics of target protein loss in the early stages of degrader design and how this will enable more rapid discovery of therapeutic agents to elicit desired phenotypic outcomes.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."

Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity.

Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

Kinetic Detection of E3:PROTAC:Target Ternary Complexes Using NanoBRET Technology in Live Cells.

Heterobifunctional small-molecule degraders known as Proteolysis Targeting Chimeras (PROTACs) serve as a chemical bridge bringing into direct association a target protein with an active E3 ligase complex, called the ternary complex, to facilitate targeted protein degradation. This ternary complex formation is the first key mechanistic step in a cascade of events that results in ubiquitination and subsequent degradation of the target protein via the ubiquitin-proteasome pathway. The ternary complex, however, is a nonnative cellular complex; therefore, PROTAC compound design has many challenges to overcome to ensure successful formation, including achieving structural and electrostatic favorability among target and ligase. Due to these challenges, finding successful PROTACs typically requires testing of extensive libraries of heterobifunctional compounds with varying linkers and E3 handles. As PROTAC ternary complex formation is also critically dependent on cellular context, live cell assays and technologies for rapid and robust screening are highly enabling for triaging of early stage compounds. Here, we present cellular assays utilizing NanoBRET technology for the study of ternary complexes, showing examples with two most popular PROTAC E3 ligase components, VHL (von Hippel-Lindau disease tumor suppressor) and CRBN (Cereblon). These assays can be run in either endpoint or real-time kinetic formats, are compatible with high-throughput workflows, and provide insight into how altering the PROTAC chemical composition affects the formation and stability of the ternary complex in live cells.

Discovery and resistance mechanism of a selective CDK12 degrader.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle-Dependent Degradation of CDK2.

Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.

To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds using HiBiT CRISPR Cell Lines.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.

Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

Monitoring and deciphering protein degradation pathways inside cells.

A new series of therapeutic modalities resulting in degradation of target proteins, termed proteolysis targeting chimeras (PROTACs), hold significant therapeutic potential with possible prolonged pharmacodynamics, improved potency, and ability to target proteins previously thought of as "undruggable". PROTACs are heterobifunctional small molecules consisting of a target binding handle bridged via a chemical linker to an E3 ligase handle which recruit the E3 ligase and ubiquitin machinery to target proteins, resulting in subsequent ubiquitination and degradation of the target. With the generation of small molecule PROTAC compound libraries for drug discovery, it becomes essential to have sensitive screening technologies to rapidly profile activity and have assays which can clearly inform on performance at the various cellular steps required for PROTAC-mediated degradation. For PROTAC compounds, this has been particularly challenging using either biochemical or cellular assay approaches. Biochemical assays are highly informative for the first part of the degradation process, including optimization of compound binding to targets and interrogation of target:PROTAC:E3 ligase ternary complex formation, but struggle with the remaining steps; recruitment of ternary complex into larger active E3 ligase complexes, ubiquitination, and proteasomal degradation. On the other hand, cellular assays are excellent at determining if the PROTAC successfully degrades the target in its relevant setting but struggle as early development PROTAC compounds are often poorly cell-permeable given their high molecular weight. Additionally, if degradation is not observed in a cellular assay, it is difficult to deconvolute the reason why or at which step there was failure. In this review we will highlight the current approaches along with recent advances to overcome the challenges faced for cellular PROTAC screening, which will enable and advance drug discovery of therapeutic degradation compounds.

Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7.

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast, and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations.