Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here, it is reported that the development of small molecule degraders of the envelope (E) protein of dengue virus. Two classes of bivalent E-degraders are developed by linking two previously reported E-binding small molecules, GNF-2, and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E-degrader with ABL inhibitory activity while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof of concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class of direct-acting antiviral drugs.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Polymerization of ZBTB transcription factors regulates chromatin occupancy.

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.

Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism.

Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

Preclinical efficacy of potent and selective menin-KMT2A inhibitor JNJ-75276617 in KMT2A- and NPM1-altered leukemias.

The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).

Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

Discovery of CRBN-dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.

Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While Cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-01 derivatives that target casein kinase 1 alpha (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and a rationale for the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.

Targeted protein degradation: from mechanisms to clinic.

Targeted protein degradation refers to the use of small molecules to induce the selective degradation of proteins. In its most common form, this degradation is achieved through ligand-mediated neo-interactions between ubiquitin E3 ligases - the principal waste disposal machines of a cell - and the protein targets of interest, resulting in ubiquitylation and subsequent proteasomal degradation. Notable advances have been made in biological and mechanistic understanding of serendipitously discovered degraders. This improved understanding and novel chemistry has not only provided clinical proof of concept for targeted protein degradation but has also led to rapid growth of the field, with dozens of investigational drugs in active clinical trials. Two distinct classes of protein degradation therapeutics are being widely explored: bifunctional PROTACs and molecular glue degraders, both of which have their unique advantages and challenges. Here, we review the current landscape of targeted protein degradation approaches and how they have parallels in biological processes. We also outline the ongoing clinical exploration of novel degraders and provide some perspectives on the directions the field might take.

Structural characterization of methylation-independent PP2A assembly guides alphafold2Multimer prediction of family-wide PP2A complexes.

Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissecting PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography, and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes.

Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.

Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

Continuous evolution of compact protein degradation tags regulated by selective molecular glues.

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

Development of sulfonyl fluoride chemical probes to advance the discovery of cereblon modulators.

Sulfonyl fluoride EM12-SF was developed previously to covalently engage a histidine residue in the sensor loop of cereblon (CRBN) in the E3 ubiquitin ligase complex CRL4CRBN. Here, we further develop the structure-activity relationships of additional sulfonyl fluoride containing ligands that possess a range of cereblon binding potencies in cells. Isoindoline EM364-SF, which lacks a key hydrogen bond acceptor present in CRBN molecular glues, was identified as a potent binder of CRBN. This led to the development of the reversible molecular glue CPD-2743, that retained cell-based binding affinity for CRBN and degraded the neosubstrate IKZF1 to the same extent as EM12, but unlike isoindolinones, lacked SALL4 degradation activity (a target linked to teratogenicity). CPD-2743 had high permeability and lacked efflux in Caco-2 cells, in contrast to the isoindolinone iberdomide. Our methodology expands the repertoire of sulfonyl exchange chemical biology via the advancement of medicinal chemistry design strategies.

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

HDAC6/aggresome processing pathway importance for inflammasome formation is context-dependent.

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1ß and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocyte cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context-dependent.

Design principles for cyclin K molecular glue degraders.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML.

ABSTRACT: Small molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)-rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

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.

Development of a covalent cereblon-based PROTAC employing a fluorosulfate warhead.

Many cereblon (CRBN) ligands have been used to develop proteolysis targeting chimeras (PROTACs), but all are reversible binders of the E3 ubiquitin ligase. We recently described the use of sulfonyl exchange chemistry to design binders that covalently engage histidine 353 in CRBN for the first time. Here we show that covalent CRBN ligands can be used to develop efficient PROTAC degraders. We demonstrate that the fluorosulfate PROTAC FS-ARV-825 covalently labels CRBN in vitro, and in cells the BRD4 degrader is insensitive to wash-out and competition by potent reversible CRBN ligands, reflecting enhanced pharmacodynamics. We anticipate that covalent CRBN-based PROTACs will enhance degradation efficiencies, thus expanding the scope of addressable targets using the heterobifunctional degrader modality.