RESUMEN
We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50â¯=â¯28â¯nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50â¯=â¯13â¯nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50â¯=â¯25â¯nM) and LnCaP-Vancouver prostate cells (IC50â¯=â¯66â¯nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Imidazoles/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/síntesis química , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Imidazoles/administración & dosificación , Imidazoles/síntesis química , Ratones , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Relación Estructura-ActividadRESUMEN
Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. ASIC gating is modulated by divalent cations as well as small molecules; however, the molecular determinants of gating modulation by divalent cations are not well understood. Previously, we identified two small molecules that bind to ASIC1a at a novel site in the acidic pocket and modulate ASIC1 gating in a manner broadly resembling divalent cations, raising the possibility that these small molecules may help to illuminate the molecular determinants of gating modulation by divalent cations. Here, we examined how these two groups of modulators might interact as well as mutational effects on ASIC1a gating and its modulation by divalent cations. Our results indicate that binding of divalent cations to an acidic pocket site plays a key role in gating modulation of the channel.
Asunto(s)
Canales Iónicos Sensibles al Ácido , Protones , Cationes Bivalentes/metabolismo , Canales Iónicos Sensibles al Ácido/metabolismo , MutaciónRESUMEN
Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.
Asunto(s)
Canales Iónicos Sensibles al Ácido/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/genética , Canales Iónicos Sensibles al Ácido/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetulus , Cinética , Potenciales de la Membrana , Moduladores del Transporte de Membrana/química , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , TaquifilaxisRESUMEN
The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.
Asunto(s)
Benzodiazepinonas/farmacología , Doxorrubicina/farmacología , Proteínas Proto-Oncogénicas c-mdm2/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Desnudos , Complejos Multiproteicos , Mutación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Asunto(s)
Benzodiazepinas/síntesis química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacología , Sitios de Unión , Línea Celular Tumoral , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Imitación Molecular , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2 , Estereoisomerismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Asunto(s)
Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/química , Moduladores de Receptores de Cannabinoides/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Endocannabinoides , Glicéridos/química , Glicéridos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Electricidad EstáticaRESUMEN
A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.
Asunto(s)
Ingeniería de Proteínas , Proteínas Tirosina Quinasas Receptoras/síntesis química , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cristalización , Citoplasma/química , Citoplasma/genética , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/síntesis química , Proteínas Mutantes Quiméricas/genética , Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Alineación de Secuencia , SpodopteraRESUMEN
MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4-10mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1-370, removal of the C-terminal autoinhibitory peptide in 1-338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1-338 by p38alpha further increased activity 12-fold. A putative constitutively active mutant, 1-370/T222E/T334E, was 6.3-fold less active than phosphorylated 1-370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg(2+)-dependent. Residues 1-40 were required for high-affinity binding of ADP, ATPgammaS, staurosporine, and K252a. A mutation M138A rendered 1-370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 microM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.
Asunto(s)
Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/química , Sitios de Unión , Activación Enzimática , Estabilidad de Enzimas , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/análisis , Isoenzimas/química , Ligandos , Unión Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Relación Estructura-Actividad , Temperatura , TermodinámicaRESUMEN
Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).
Asunto(s)
Benzodiazepinas/química , Diseño de Fármacos , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Alquilación , Animales , Benzodiazepinas/síntesis química , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Ácidos Pentanoicos/química , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/química , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/químicaRESUMEN
A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.
Asunto(s)
Benzodiazepinas/síntesis química , Benzodiazepinas/farmacología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Técnicas Químicas Combinatorias , Polarización de Fluorescencia , Humanos , Concentración 50 Inhibidora , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2 , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.