RESUMEN
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/terapia , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/antagonistas & inhibidores , Triptasas/inmunología , Adolescente , Regulación Alostérica/inmunología , Animales , Línea Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ConejosRESUMEN
Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.
Asunto(s)
Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactamas/farmacología , Animales , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Lactamas/química , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic ß-tryptase inhibitors, but its unique activation mechanism is less well-explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N terminus into its "activation pocket," indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric ß-tryptase mutant (I99C*/Y75A/Y37bA, where C* is cysteinylated Cys-99) cannot form a dimer or tetramer, yet it is active but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each ß-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.
Asunto(s)
Heparina/metabolismo , Péptido Hidrolasas/metabolismo , Regiones Promotoras Genéticas , Multimerización de Proteína , Triptasas/genética , Triptasas/metabolismo , Regulación Alostérica , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Subunidades de Proteína , Triptasas/químicaRESUMEN
Receptor-interacting protein kinase 1 (RIPK1), a key component of the cellular necroptosis pathway, has gained recognition as an important therapeutic target. Pharmacologic inhibition or genetic inactivation of RIPK1 has shown promise in animal models of disease ranging from acute ischemic conditions, chronic inflammation, and neurodegeneration. We present here a class of RIPK1 inhibitors that is distinguished by a lack of a lipophilic aromatic group present in most literature inhibitors that typically occupies a hydrophobic back pocket of the protein active site. Despite not having this ubiquitous feature of many known RIPK1 inhibitors, we were able to obtain compounds with good potency, kinase selectivity, and pharmacokinetic properties in rats. The use of the lipophilic yet metabolically stable pentafluoroethyl group was critical to balancing the potency and properties of optimized analogs.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Humanos , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Relación Estructura-ActividadRESUMEN
Targeting the interaction with or displacement of the 'right' water molecule can significantly increase inhibitor potency in structure-guided drug design. Multiple computational approaches exist to predict which waters should be targeted for displacement to achieve the largest gain in potency. However, the relative success of different methods remains underexplored. Here, we present a comparison of the ability of five water prediction programs (3D-RISM, SZMAP, WaterFLAP, WaterRank, and WaterMap) to predict crystallographic water locations, calculate their binding free energies, and to relate differences in these energies to observed changes in potency. The structural cohort included nine Bruton's Tyrosine Kinase (BTK) structures, and nine bromodomain structures. Each program accurately predicted the locations of most crystallographic water molecules. However, the predicted binding free energies correlated poorly with the observed changes in inhibitor potency when solvent atoms were displaced by chemical changes in closely related compounds.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/química , Simulación por Computador , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Agua/química , Cristalografía por Rayos X , Ligandos , Unión Proteica , Dominios Proteicos , Programas Informáticos , Solventes/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
Asunto(s)
Plasticidad de la Célula/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Piridonas/farmacología , Tiofenos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Piridonas/química , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.
Asunto(s)
Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , TYK2 Quinasa/metabolismoRESUMEN
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
Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like ß-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Péptidos/farmacología , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células CHO , Dominio Catalítico , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetulus , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/genética , Humanos , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Proteína C/química , Proteína C/genética , Proteína C/metabolismo , Ingeniería de Proteínas , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Furanos/farmacología , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Animales , Sitios de Unión , Cristalografía por Rayos X , Perros , Receptores ErbB/química , Clorhidrato de Erlotinib/farmacología , Furanos/síntesis química , Furanos/química , Furanos/farmacocinética , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Indazoles/síntesis química , Indazoles/química , Indazoles/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Mutación Puntual , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacocinética , RatasRESUMEN
BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridazinas/química , Piridazinas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacología , Tiofenos/química , Tiofenos/farmacología , Agammaglobulinemia Tirosina Quinasa , Animales , Perros , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Tirosina Quinasas/metabolismo , Piridazinas/metabolismo , Piridazinas/farmacocinética , Pirimidinonas/metabolismo , Pirimidinonas/farmacocinética , Ratas , Tiofenos/metabolismo , Tiofenos/farmacocinéticaRESUMEN
The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.
Asunto(s)
Genes Relacionados con las Neoplasias/genética , Mutación/genética , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/genética , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Humanos , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/genética , MAP Quinasa Quinasa 4/genética , Masculino , Neoplasias/enzimología , Neoplasias/patología , Neoplasias Ováricas/clasificación , Neoplasias Ováricas/genética , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Proteínas Quinasas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Complejo Antígeno-Anticuerpo/química , Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Especificidad de Anticuerpos , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión de Anticuerpos , Dominio Catalítico , Línea Celular Tumoral , Mapeo Epitopo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Melanoma/enzimología , Melanoma/metabolismo , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.
Asunto(s)
Oligopéptidos/farmacología , Proproteína Convertasas/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Proproteína Convertasa 9 , Proproteína Convertasas/química , Proproteína Convertasas/genética , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (410 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Células de Riñón Canino Madin DarbyRESUMEN
Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi's [J. Med. Chem.2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem.2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem.2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450's 2C9 and 2C19. Lowering the logD by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.
Asunto(s)
Descubrimiento de Drogas , Compuestos Heterocíclicos/farmacología , Imidazoles/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HCT116 , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Humanos , Imidazoles/síntesis química , Imidazoles/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazinas/síntesis química , Pirazinas/química , Relación Estructura-ActividadRESUMEN
There is evidence that small molecule inhibitors of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signaling cascade, could represent a novel asthma therapeutic class. Moreover, given the expected chronic dosing regimen of any asthma treatment, highly selective as well as potent inhibitors would be strongly preferred in any potential therapeutic. Here we report hit-to-lead optimization of a series of indazoles that demonstrate sub-nanomolar inhibitory potency against ITK with strong cellular activity and good kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of the complexes.
Asunto(s)
Descubrimiento de Drogas , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/síntesis química , Indazoles/química , Células Jurkat , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM).
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Pironas/síntesis química , Pironas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2.