RESUMO
Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for â¼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.
Assuntos
Proteínas Quinases/metabolismo , Proteólise , Proteoma/metabolismo , Adulto , Linhagem Celular , Bases de Dados de Proteínas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Cromatina , Microscopia Crioeletrônica , Humanos , Cromatina/metabolismo , Cromatina/genética , Multimerização Proteica , Sítios de Ligação , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Polimerização , Células HEK293 , Regulação da Expressão GênicaRESUMO
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
Assuntos
Cromatina , Fatores de Transcrição , Humanos , Ligantes , Cromatina/genética , Fatores de Transcrição/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinas , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Complexos Multiproteicos , Mutação , Neoplasias , Proteína SMARCB1 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteólise , Ubiquitina/metabolismoRESUMO
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.
Assuntos
Leucemia Mieloide Aguda , Morfolinas , Proteína de Leucina Linfoide-Mieloide , Ftalimidas , Piperidonas , Humanos , Lenalidomida/uso terapêutico , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/genética , MutaçãoRESUMO
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.
RESUMO
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.
Assuntos
Ciclinas , Ubiquitina-Proteína Ligases , Ciclinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.
Assuntos
Polimerização/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/química , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Microscopia Crioeletrônica , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/ultraestrutura , Solventes , Biologia Sintética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Assuntos
Ciclinas/deficiência , Ciclinas/metabolismo , Proteólise/efeitos dos fármacos , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Purinas/toxicidade , Piridinas/toxicidade , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinação/efeitos dos fármacosRESUMO
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.
Assuntos
Inflamassomos , Leucócitos Mononucleares , Animais , Humanos , Camundongos , Linhagem Celular , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transporte Proteico/fisiologiaRESUMO
Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2-mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição Ikaros , Fatores Imunológicos/farmacologia , Linfoma de Células T , Mieloma Múltiplo , Ubiquitina-Proteína Ligases , Humanos , Fator de Transcrição Ikaros/metabolismo , Lenalidomida/farmacologia , Linfoma de Células T/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Targeted protein degradation relies on small molecules that induce new protein-protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.
RESUMO
Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.
Assuntos
Neoplasias , Quimera de Direcionamento de Proteólise , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity.
Assuntos
Proteínas de Ligação a DNA/efeitos dos fármacos , Fator de Transcrição Ikaros/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição Ikaros/genética , Células Jurkat , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chemical probes are essential tools for understanding biological systems and for credentialing potential biomedical targets. Programmed cell death 2 (PDCD2) is a member of the B-cell lymphoma 2 (Bcl-2) family of proteins, which are critical regulators of apoptosis. Here we report the discovery and characterization of 10 e, a first-in-class small molecule degrader of PDCD2. We discovered this PDCD2 degrader by serendipity using a chemical proteomics approach, in contrast to the conventional approach for making bivalent degraders starting from a known binding ligand targeting the protein of interest. Using 10 e as a pharmacological probe, we demonstrate that PDCD2 functions as a critical regulator of cell growth by modulating the progression of the cell cycle in T lymphoblasts. Our work provides a useful pharmacological probe for investigating PDCD2 function and highlights the use of chemical proteomics to discover selective small molecule degraders of unanticipated targets.
Assuntos
Proteínas Reguladoras de Apoptose , Linfoma de Células B , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteômica , Apoptose , Proliferação de CélulasRESUMO
Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.
Assuntos
AutofagiaRESUMO
The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 Å, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.
Assuntos
Indóis/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteólise/efeitos dos fármacos , Proteínas com Motivo de Reconhecimento de RNA/química , Sulfonamidas/química , Motivos de Aminoácidos , Animais , Benzamidas/química , Benzamidas/farmacologia , Microscopia Crioeletrônica , Transferência Ressonante de Energia de Fluorescência , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA , Spodoptera , Sulfonamidas/farmacologia , Ubiquitina-Proteína Ligases/química , XenopusRESUMO
BACKGROUND: Individuals with mood disorders frequently experience cognitive impairment, which impacts on the long-term trajectory of the disorders, including being associated with persisting difficulties in occupational and psychosocial functioning, residual mood symptoms, and relapse. Current first-line treatments for mood disorders do little to improve cognitive function. Targeting cognition in clinical research is thus considered a priority. This protocol outlines a prospectively-registered randomised controlled trial (RCT) which examines the impact of adding group-based Cognitive Remediation (CR) to Interpersonal and Social Rhythm Therapy (IPSRT-CR) for individuals with mood disorders. METHODS: This is a pragmatic, two-arm, single-blinded RCT comparing IPSRT-CR with IPSRT alone for adults (n = 100) with mood disorders (Major Depressive Disorder or Bipolar Disorder) with subjective cognitive difficulties, on discharge from Specialist Mental Health Services in Christchurch, New Zealand. Both treatment arms will receive a 12-month course of individual IPSRT (full dose = 24 sessions). At 6 months, randomisation to receive, or not, an 8-week group-based CR programme (Action-based Cognitive Remediation - New Zealand) will occur. The primary outcome will be change in Global Cognition between 6 and 12 months (treatment-end) in IPSRT-CR versus IPSRT alone. Secondary outcomes will be change in cognitive, functional, and mood outcomes at 6, 12, 18, and 24 months from baseline and exploratory outcomes include change in quality of life, medication adherence, rumination, and inflammatory markers between treatment arms. Outcome analyses will use an intention-to-treat approach. Sub-group analyses will assess the impact of baseline features on CR treatment response. Participants' experiences of their mood disorder, including treatment, will be examined using qualitative analysis. DISCUSSION: This will be the first RCT to combine group-based CR with an evidence-based psychotherapy for adults with mood disorders. The trial may provide valuable information regarding how we can help promote long-term recovery from mood disorders. Many issues have been considered in developing this protocol, including: recruitment of the spectrum of mood disorders, screening for cognitive impairment, dose and timing of the CR intervention, choice of comparator treatment, and choice of outcome measures. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry, ACTRN12619001080112 . Registered on 6 August 2019.