RESUMEN
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
Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.
Asunto(s)
Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismo , Neoplasias/enzimología , Transducción de Señal/fisiología , TYK2 Quinasa/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Dimerización , Activación Enzimática/fisiología , Humanos , Insectos/citología , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Janus Quinasa 3/genética , Mutación , Neoplasias/genética , Estructura Terciaria de Proteína , Relación Estructura-Actividad , TYK2 Quinasa/química , TYK2 Quinasa/genéticaRESUMEN
Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Indazoles/síntesis química , Indazoles/química , Indazoles/farmacología , Ratones , Modelos Moleculares , Proteínas Proto-Oncogénicas c-pim-1/química , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
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
Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirazoles/química , Pirimidinas/química , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Cinética , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
VPS34 is a class III phosphoinositide 3-kinase involved in endosomal trafficking and autophagosome formation. Inhibitors of VPS34 were believed to have value as anticancer agents, but genetic and pharmacological data suggest that sustained inhibition of VPS34 kinase activity may not be well tolerated. Here we disclose the identification of a novel series of dihydropyrazolopyrazinone compounds represented by compound 5 as potent, selective, and orally bioavailable VPS34 inhibitors through a structure-based design strategy. A water-interacting hydrogen bond acceptor within an appropriate distance to a hinge-binding element was found to afford significant VPS34 potency across chemical scaffolds. The selectivity of compound 5 over PIK family kinases arises from interactions between the hinge-binding element and the pseudo-gatekeeper residue Met682. As recent in vivo pharmacology data suggests that sustained inhibition of VPS34 kinase activity may not be tolerated, structure-activity relationships leading to VPS34 inhibition may be helpful for avoiding this target in other ATP-competitive kinase programs.
Asunto(s)
Antineoplásicos , Fosfatidilinositol 3-Quinasas Clase III , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Autofagia , Endosomas , Fosfatidilinositol 3-Quinasa/metabolismo , FosforilaciónRESUMEN
Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.
Asunto(s)
Neoplasias de la Mama , Fosfatidilinositol 3-Quinasas , Humanos , Femenino , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , MutaciónRESUMEN
Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.
Asunto(s)
Anticuerpos Biespecíficos , Mieloma Múltiple , Antígeno de Maduración de Linfocitos B , Humanos , Mieloma Múltiple/tratamiento farmacológico , Fagocitosis , Receptores de IgGRESUMEN
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
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
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animales , Antibacterianos/farmacología , Sitios de Unión , Mapeo Epitopo , Epítopos/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.
Asunto(s)
Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirazoles/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Perros , Femenino , Humanos , Macaca fascicularis , Células de Riñón Canino Madin Darby , Masculino , Ratones SCID , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Pirazoles/síntesis química , Pirazoles/metabolismo , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytokines and interferons initiate intracellular signaling via receptor dimerization and activation of Janus kinases (JAKs). How JAKs structurally respond to changes in receptor conformation induced by ligand binding is not known. Here, we present two crystal structures of the human JAK2 FERM and SH2 domains bound to Leptin receptor (LEPR) and Erythropoietin receptor (EPOR), which identify a novel dimeric conformation for JAK2. This 2:2 JAK2/receptor dimer, observed in both structures, identifies a previously uncharacterized receptor interaction essential to dimer formation that is mediated by a membrane-proximal peptide motif called the 'switch' region. Mutation of the receptor switch region disrupts STAT phosphorylation but does not affect JAK2 binding, indicating that receptor-mediated formation of the JAK2 FERM dimer is required for kinase activation. These data uncover the structural and molecular basis for how a cytokine-bound active receptor dimer brings together two JAK2 molecules to stimulate JAK2 kinase activity.
Asunto(s)
Janus Quinasa 2/química , Fragmentos de Péptidos/química , Conformación Proteica , Receptores de Eritropoyetina/química , Receptores de Leptina/química , Cristalografía por Rayos X , Dimerización , Dominios FERM/genética , Humanos , Janus Quinasa 2/genética , Mutación , Fragmentos de Péptidos/genética , Fosforilación/genética , Unión Proteica/genética , Receptores de Eritropoyetina/genética , Receptores de Leptina/genética , Factores de Transcripción STAT/química , Factores de Transcripción STAT/genética , Transducción de Señal/genética , Dominios Homologos src/genéticaRESUMEN
An increasing number of assay detection technologies are routinely used in small molecule drug discovery and lead optimization. These assays range from solid-phase heterogeneous assays such as enzyme-linked immunosorbent assay and dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Boston, MA) to solution phase, bead-based assays such as electrochemiluminescence assay (BioVeris [Gaithersburg, MD] technology) and amplified luminescent proximity homogeneous assay (AlphaScreen, PerkinElmer Life and Analytical Sciences) to completely solution-based homogeneous assays such as time-resolved fluorescence resonance energy transfer and fluorescence polarization. The aim of this study is to compare these assay technologies and assess the advantages and disadvantages of each in the context of our efforts to develop small molecule antagonists to the melanoma inhibitor of apoptosis protein. In this study, seven peptides have been evaluated for their potencies in each assay format. Our results indicate that these assay technologies produce similar relative potencies; however, some methods may be more susceptible to interference than others. Consequently, the choice of the method used frequently depends on a number of factors in addition to assay reproducibility and performance, such as throughput of the assay, cost, compound interference, and ease of use.
Asunto(s)
Bioensayo/métodos , Evaluación Preclínica de Medicamentos/métodos , Péptidos/antagonistas & inhibidores , Péptidos/química , Proteínas/antagonistas & inhibidores , Proteínas/química , Biotina/química , Interpretación Estadística de Datos , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoresceínas/química , Polarización de Fluorescencia , Péptidos/síntesis químicaRESUMEN
Programmed cell death-1 (PD-1) is a coinhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). By titrating PD-1 signaling in a biochemical reconstitution system, we demonstrate that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1-recruited Shp2 phosphatase. We also show that CD28, but not the TCR, is preferentially dephosphorylated in response to PD-1 activation by PD-L1 in an intact cell system. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways play key roles in regulating effector T cell function and responses to anti-PD-L1/PD-1 therapy.
Asunto(s)
Antígeno B7-H1/metabolismo , Antígenos CD28/metabolismo , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Antígenos CD28/genética , Humanos , Inmunoterapia , Células Jurkat , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , FosforilaciónRESUMEN
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Estimulación Eléctrica , Fármacos actuantes sobre Aminoácidos Excitadores/química , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Glicina/metabolismo , Células HEK293 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Técnicas de Placa-Clamp , Dominios Proteicos/efectos de los fármacos , Dominios Proteicos/genética , Pirimidinonas/química , Pirimidinonas/farmacología , Receptores de N-Metil-D-Aspartato/genética , Sulfonamidas/química , Sulfonamidas/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , TransfecciónRESUMEN
Pim kinases have been identified as promising therapeutic targets for hematologic-oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as 20 (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge.
Asunto(s)
Indazoles/química , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Humanos , Indazoles/metabolismo , Indazoles/farmacocinética , Mucosa Intestinal/metabolismo , Simulación del Acoplamiento Molecular , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , RatasRESUMEN
The N-methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca2+ and Na+. NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimer's disease. Recently, we have disclosed GNE-0723 (1), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure-activity relationships, despite the structural similarity to GNE-0723. GNE-5729 (13), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.
RESUMEN
ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Ingeniería de Proteínas , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/genética , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular , Cinética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
EDA is a tumor necrosis factor family member involved in ectodermal development. Splice variants EDA-A1 and EDA-A2 differ only by the presence of Glu 308 and Val 309 in the expected receptor binding region of EDA-A1 but not EDA-A2. This two amino acid difference functions as a switch controlling receptor specificity. EDA-A1 binds only to EDAR, while EDA-A2 is specific for XEDAR. In order to understand the structural basis of this switch, we determined the X-ray crystal structures of the TNF domain of both EDA-A1 and EDA-A2 at 2.3 A and 2.2 A, respectively. While the backbone conformation around the splice difference is similar in both isoforms, the conformation of the following loop, the surface charge, and the shape of the expected receptor binding site differ significantly.