RESUMEN
The lack of selective and safe in vivo IRE1α tool molecules has limited the evaluation of IRE1α as a viable target to treat multiple myeloma. Focus on improving the physicochemical properties of a literature compound by decreasing lipophilicity, molecular weight, and basicity allowed the discovery of a novel series with a favorable in vitro safety profile and good oral exposure. These efforts culminated in the identification of a potent and selective in vivo tool compound, G-5758, that was well tolerated following multiday oral administration of doses up to 500 mg/kg. G-5758 demonstrated comparable pharmacodynamic effects to induced IRE1 knockdown as measured by XBP1s levels in a multiple myeloma model (KMS-11).
Asunto(s)
Endorribonucleasas , Mieloma Múltiple , Proteínas Serina-Treonina Quinasas , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Humanos , Administración Oral , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Animales , Descubrimiento de Drogas , Ratones , Línea Celular Tumoral , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ratas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacocinética , Técnicas de Silenciamiento del Gen , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.
RESUMEN
The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.
Asunto(s)
Antineoplásicos , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Animales , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Línea Celular Tumoral , Mutación , Antineoplásicos/farmacología , Modelos Animales de EnfermedadRESUMEN
With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.
Asunto(s)
Descubrimiento de Drogas , Proteínas Proto-Oncogénicas p21(ras) , CinéticaRESUMEN
Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasas/metabolismo , Adenosina Trifosfato/química , Sitio Alostérico/efectos de los fármacos , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Endorribonucleasas/química , Técnicas de Inactivación de Genes , Humanos , Ligandos , Modelos Moleculares , Fosforilación , Conformación Proteica , Pliegue de Proteína , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Ribonucleasas/química , Respuesta de Proteína DesplegadaRESUMEN
Amino-quinazoline BRaf kinase inhibitor 2 was identified from a library screen as a modest inhibitor of the unfolded protein response (UPR) regulating potential anticancer target IRE1α. A combination of crystallographic and conformational considerations were used to guide structure-based attenuation of BRaf activity and optimization of IRE1α potency. Quinazoline 6-position modifications were found to provide up to 100-fold improvement in IRE1α cellular potency but were ineffective at reducing BRaf activity. A salt bridge contact with Glu651 in IRE1α was then targeted to build in selectivity over BRaf which instead possesses a histidine in this position (His539). Torsional angle analysis revealed that the quinazoline hinge binder core was ill-suited to accommodate the required conformation to effectively reach Glu651, prompting a change to the thienopyrimidine hinge binder. Resulting analogues such as 25 demonstrated good IRE1α cellular potency and imparted more than 1000-fold decrease in BRaf activity.
RESUMEN
Proprotein convertase subtilisin/kexin 9 (PCSK9) has become an important therapeutic target for lipid lowering, since it regulates low-density lipoprotein cholesterol (LDL-c) levels by binding to liver LDL receptors (LDLR) and effecting their intracellular degradation. However, the development of small molecule inhibitors is hampered by the lack of attractive PCSK9 target sites. We recently discovered helical peptides that are able to bind to a cryptic groove site on PCSK9, which is situated in proximity to the main LDLR binding site. Here, we designed potent bipartite PCSK9 inhibitors by appending organic moieties to a helical groove-binding peptide to reach a hydrophobic pocket in the proximal LDLR binding region. The ultimately designed 1-amino-4-phenylcyclohexane-1-carbonyl extension improved the peptide affinity by >100-fold, yielding organo-peptide antagonists that potently inhibited PCSK9 binding to LDLR and preserved cellular LDLR. These new bipartite antagonists have reduced mass and improved potency compared to the first-generation peptide antagonists, further validating the PCSK9 groove as a viable therapeutic target site.
Asunto(s)
Inhibidores de PCSK9 , Péptidos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Células Hep G2 , Humanos , Estructura Molecular , Péptidos/química , Péptidos/metabolismo , Proproteína Convertasa 9/química , Proproteína Convertasa 9/metabolismo , Unión Proteica , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
Asunto(s)
Endorribonucleasas/genética , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Anciano , Animales , Bortezomib/farmacología , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/antagonistas & inhibidores , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lenalidomida/farmacología , Masculino , Ratones , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-Box/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC50 of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.
Asunto(s)
Bencimidazoles/metabolismo , Diseño de Fármacos , Sondas Moleculares/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/química , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Quinolinas/química , Quinolinas/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Regulación Alostérica/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Escherichia coli/química , Hidrocarburos/química , Hidrocarburos/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Modelos Moleculares , Dominios Proteicos/efectos de los fármacosRESUMEN
Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.
Asunto(s)
Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida , Bloqueadores del Canal de Sodio Activado por Voltaje/análisis , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de Insectos , Potenciales de la Membrana , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Ratas , Veratridina , Venenos de AvispasRESUMEN
A novel, potent, and orally bioavailable inhibitor of the bromodomain of CBP, compound 35 (GNE-207), has been identified through SAR investigations focused on optimizing al bicyclic heteroarene to replace the aniline present in the published GNE-272 series. Compound 35â¯has excellent CBP potency (CBP IC50â¯=â¯1â¯nM, MYC EC50â¯=â¯18â¯nM), a selectively index ofâ¯>2500-fold against BRD4(1), and exhibits a good pharmacokinetic profile.
Asunto(s)
Compuestos de Bifenilo/química , Diseño de Fármacos , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Sitios de Unión , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/metabolismo , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Semivida , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Ratas , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the "hybridization" of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Asparagina/química , Asparagina/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Femenino , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones Endogámicos , Simulación del Acoplamiento Molecular , Proteínas Nucleares/antagonistas & inhibidores , Dominios Proteicos , Pirazoles/química , Piridinas/química , Quinolinas/química , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Indazoles/química , Indazoles/farmacología , Fenoles/química , Fenoles/farmacología , Piridinas/química , Piridinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/química , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/deficiencia , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.
Asunto(s)
Antineoplásicos/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteína de Unión a CREB/química , Perros , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , ARN/genética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P' helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Inhibidores de PCSK9 , Péptidos/metabolismo , Proproteína Convertasa 9/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Simulación del Acoplamiento Molecular , Biblioteca de Péptidos , Péptidos/aislamiento & purificaciónRESUMEN
Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.
Asunto(s)
Proteína p300 Asociada a E1A/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Proteína p300 Asociada a E1A/deficiencia , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones SCID , Terapia Molecular Dirigida , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Dominios Proteicos , Distribución Aleatoria , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 µL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by â¼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and nutrient consumption, while elevating titer, nuclear plasmid DNA (pDNA) copy numbers, and mRNA levels. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 3:1579-1588, 2017.
Asunto(s)
Formación de Anticuerpos/genética , Ensayos Analíticos de Alto Rendimiento , Inmunoglobulina G/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Células CHO , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cricetinae , Cricetulus , Células HEK293 , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Polietileneimina/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Bibliotecas de Moléculas Pequeñas/farmacología , TransfecciónRESUMEN
We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de Anillo en Puente/farmacología , Isoxazoles/farmacología , Oxazepinas/farmacología , Oxazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Perros , Células HEK293 , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de Anillo en Puente/síntesis química , Compuestos Heterocíclicos de Anillo en Puente/química , Humanos , Imidazoles/farmacología , Isoxazoles/síntesis química , Isoxazoles/química , Ratones , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Oxazepinas/síntesis química , Oxazepinas/química , Oxazoles/síntesis química , Oxazoles/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Quinasa de Factor Nuclear kappa BRESUMEN
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.