RESUMEN
Small molecule chemical probes are valuable tools for interrogating protein biological functions and relevance as a therapeutic target. Rigorous validation of chemical probe parameters such as cellular potency and selectivity is critical to unequivocally linking biological and phenotypic data resulting from treatment with a chemical probe to the function of a specific target protein. A variety of modern technologies are available to evaluate cellular potency and selectivity, target engagement, and functional response biomarkers of chemical probe compounds. Here, we review these technologies and the rationales behind using them for the characterization and validation of chemical probes. In addition, large-scale phenotypic characterization of chemical probes through chemical genetic screening is increasingly leading to a wealth of information on the cellular pharmacology and disease involvement of potential therapeutic targets. Extensive compound validation approaches and integration of phenotypic information will lay foundations for further use of chemical probes in biological discovery.
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HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.
Asunto(s)
Linfocitos B/fisiología , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/genética , ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Animales , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Ratones , Ratones Endogámicos C57BL , Translocación Genética/genéticaRESUMEN
Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.
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Histonas/química , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Acetilación , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Genoma Humano , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteoma/análisisRESUMEN
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.
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The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.
RESUMEN
5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.
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Proteínas de Neoplasias , Biosíntesis de Proteínas , Proteínas de Neoplasias/genética , Canadá , Metilación , ARN Mensajero/genética , ARN/metabolismoRESUMEN
Dysregulation of histone lysine methyltransferases and demethylases is one of the major mechanisms driving the epigenetic reprogramming of transcriptional networks in castration-resistant prostate cancer (CRPC). In addition to their canonical histone targets, some of these factors can modify critical transcription factors, further impacting oncogenic transcription programs. Our recent report demonstrated that LSD1 can demethylate the lysine 270 of FOXA1 in prostate cancer (PCa) cells, leading to the stabilization of FOXA1 chromatin binding. This process enhances the activities of the androgen receptor and other transcription factors that rely on FOXA1 as a pioneer factor. However, the identity of the methyltransferase responsible for FOXA1 methylation and negative regulation of the FOXA1-LSD1 oncogenic axis remains unknown. SETD7 was initially identified as a transcriptional activator through its methylation of histone 3 lysine 4, but its function as a methyltransferase on nonhistone substrates remains poorly understood, particularly in the context of PCa progression. In this study, we reveal that SETD7 primarily acts as a transcriptional repressor in CRPC cells by functioning as the major methyltransferase targeting FOXA1-K270. This methylation disrupts FOXA1-mediated transcription. Consistent with its molecular function, we found that SETD7 confers tumor suppressor activity in PCa cells. Moreover, loss of SETD7 expression is significantly associated with PCa progression and tumor aggressiveness. Overall, our study provides mechanistic insights into the tumor-suppressive and transcriptional repression activities of SETD7 in mediating PCa progression and therapy resistance.
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Histonas , Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Histonas/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Lisina/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Metiltransferasas/metabolismo , Histona Demetilasas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismoRESUMEN
The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.
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Reparación del ADN , Replicación del ADN , ADN/biosíntesis , Endonucleasas/metabolismo , Inestabilidad Genómica , Ciclo Celular , ADN/química , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/química , Endonucleasas/genética , Activación Enzimática , Humanos , Complejos Multienzimáticos , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Recombinasas/genética , Recombinasas/metabolismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.
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Proteína BRCA1 , Neoplasias de la Mama , Animales , Femenino , Humanos , Ratones , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/metabolismo , Inestabilidad Genómica , Recurrencia Local de Neoplasia , Estructuras R-Loop , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet, some protein folds are easier to design than others. Previous work identified the 43-residue ÉßßÉ fold as especially challenging: The best designs had only a 2% success rate, compared to 39 to 87% success for other simple folds [G. J. Rocklin et al., Science 357, 168-175 (2017)]. This suggested the ÉßßÉ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over 10,000 new ÉßßÉ proteins and found over 3,000 of them to fold into stable structures using a high-throughput protease-based assay. NMR, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed ÉßßÉ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the ÉßßÉ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.
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Pliegue de Proteína , Proteínas , Secuencia de Aminoácidos , Dicroismo Circular , Deuterio , Péptido Hidrolasas , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/genéticaRESUMEN
Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.
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Hydrogen-deuterium eXchange mass spectrometry (HDX-MS) is increasingly used in drug development to locate binding sites and to identify allosteric effects in drug/target interactions. However, the potential of this technique to quantitatively analyze drug candidate libraries remains largely unexplored. Here, a collection of 13 WDR5-targeting small molecules with surface plasmon resonance (SPR) dissociation coefficients (KD) ranging from 20 nM to â¼116 µM were characterized using differential HDX-MS (ΔHDX-MS). Conventional qualitative analysis of the ΔHDX-MS data set revealed the binding interfaces for all compounds and allosteric effects where present. We then demonstrated that ΔHDX-MS signal-to-noise (S/N) not only can rank library-relative affinity but also can accurately predict KD from a calibration curve constructed from high-quality SPR data. Three methods for S/N calculation are explored, each suitable for libraries with different characteristics. Our results demonstrate the potential for ΔHDX-MS use in drug candidate library affinity validation and/or determination while simultaneously characterizing structure.
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Resonancia por Plasmón de Superficie/métodos , HumanosRESUMEN
Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus-host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.
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COVID-19 , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Humanos , Péptido Hidrolasas , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
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Proteína-Arginina N-Metiltransferasas , Neoplasias de la Mama Triple Negativas , Biomarcadores , Línea Celular Tumoral , Humanos , Interferones/uso terapéutico , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
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Nucléolo Celular/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Sondas Moleculares/química , Dominios Proteicos , Proteínas Represoras/metabolismo , Metilación , Mieloma Múltiple/metabolismo , Nucleosomas/metabolismoRESUMEN
We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.
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Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Simulación del Acoplamiento Molecular , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Sitios de Unión , Dominios Proteicos , Humanos , Ligandos , Unión Proteica , Repeticiones WD40 , AlgoritmosRESUMEN
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder resulting from a CAG expansion in the huntingtin (HTT) gene, which leads to the production and accumulation of mutant huntingtin (mHTT). While primarily considered a disorder of the central nervous system, multiple changes have been described to occur throughout the body, including activation of the immune system. In other neurodegenerative disorders, activation of the immune system has been shown to include the production of antibodies against disease-associated pathological proteins. However, the existence of mHTT-targeted antibodies has never been reported. In this study, we assessed the presence and titer of antibodies recognizing HTT/mHTT in patients with HD (n = 66) and age- and gender-matched healthy controls (n = 66) using a combination of Western blotting and ELISA. Together, these analyses revealed that antibodies capable of recognizing HTT/mHTT were detectable in the plasma samples of all participants, including healthy controls. When antibody levels were monitored at different disease stages, it was observed that antibodies against full-length mHTT were highest in patients with severe disease while antibodies against HTTExon1 were elevated in patients with mild disease. Combined, these results suggest that antibodies detecting different forms of mHTT peak at different disease stages.
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Enfermedad de Huntington , Enfermedades Neurodegenerativas , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , AnticuerposRESUMEN
Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.
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Cromatina , Proteínas Nucleares , Proteínas de Unión al ADN/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteína Fosfatasa 1/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.
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Carcinoma Ductal Pancreático/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Pancreáticas/enzimología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Células HEK293 , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
Nuclear receptor-binding SET domain-containing 2 (NSD2) plays important roles in gene regulation, largely through its ability to dimethylate lysine 36 of histone 3 (H3K36me2). Despite aberrant activity of NSD2 reported in numerous cancers, efforts to selectively inhibit the catalytic activity of this protein with small molecules have been unsuccessful to date. Here, we report the development of UNC8153, a novel NSD2-targeted degrader that potently and selectively reduces the cellular levels of both NSD2 protein and the H3K36me2 chromatin mark. UNC8153 contains a simple warhead that confers proteasome-dependent degradation of NSD2 through a novel mechanism. Importantly, UNC8153-mediated reduction of H3K36me2 through the degradation of NSD2 results in the downregulation of pathological phenotypes in multiple myeloma cells including mild antiproliferative effects in MM1.S cells containing an activating point mutation and antiadhesive effects in KMS11 cells harboring the t(4;14) translocation that upregulates NSD2 expression.