RESUMEN
When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.
Asunto(s)
Ribosomas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Anticuerpos/metabolismo , Biocatálisis , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Estabilidad Proteica , Reproducibilidad de los Resultados , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidoresRESUMEN
Accurate ranking of compounds with regards to their binding affinity to a protein using computational methods is of great interest to pharmaceutical research. Physics-based free energy calculations are regarded as the most rigorous way to estimate binding affinity. In recent years, many retrospective studies carried out both in academia and industry have demonstrated its potential. Here, we present the results of large-scale prospective application of the FEP+ method in active drug discovery projects in an industry setting at Merck KGaA, Darmstadt, Germany. We compare these prospective data to results obtained on a new diverse, public benchmark of eight pharmaceutically relevant targets. Our results offer insights into the challenges faced when using free energy calculations in real-life drug discovery projects and identify limitations that could be tackled by future method development. The new public data set we provide to the community can support further method development and comparative benchmarking of free energy calculations.
Asunto(s)
Descubrimiento de Drogas , Ligandos , Estudios Prospectivos , Estudios Retrospectivos , TermodinámicaRESUMEN
Mcl-1 is an anti-apoptotic protein overexpressed in hematological malignancies and several human solid tumors. Small molecule inhibition of Mcl-1 would offer an effective therapy to Mcl-1 mediated resistance. Subsequently, it has been the target of extensive research in the pharmaceutical industry. The discovery of a novel class of Mcl-1 small molecule inhibitors is described beginning with a simple biaryl sulfonamide hit derived from a high through put screen. A medicinal chemistry effort aided by SBDD generated compounds capable of disrupting the Mcl-1/Bid protein-protein interaction in vitro. The crystal structure of the Mcl-1 bound ligand represents a unique binding mode to the BH3 binding pocket where binding affinity is achieved, in part, through a sulfonamide oxygen/Arg263 interaction. The work highlights the some of the key challenges in designing effective protein-protein inhibitors for the Bcl-2 class of proteins.
Asunto(s)
Descubrimiento de Drogas , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Asunto(s)
Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Animales , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Piperidinas/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/síntesis química , Pirimidinas/síntesis química , Especificidad por Sustrato , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previously we reported the discovery of CRA-898 (1), a diazine indole acetic acid containing CRTH2 antagonist. This compound had good in vitro and in vivo potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. However, it showed low oral exposure in nonrodent safety species (dogs and monkeys). In the current paper, we wish to report our efforts to understand and improve the poor PK in nonrodents and development of a new isoquinolinone subseries that led to identification of a new development candidate, CRA-680 (44). This compound was efficacious in both a house dust mouse model of allergic lung inflammation (40 mg/kg qd) as well as a guinea pig allergen challenge model of lung inflammation (20 mg/kg bid).
Asunto(s)
Acetatos/química , Hipersensibilidad/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Quinolonas/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Humanos , Quinolonas/química , Células Th2RESUMEN
New classes of CRTH2 antagonists, the pyridazine linker containing indole acetic acids, are described. The initial hit 1 had good potency but poor permeability, metabolic stability, and PK. Initial optimization led to compounds of type 2 with low oxidative metabolism but poor oral bioavailability. Poor permeability was identified as a liability for these compounds. Addition of a linker between the indole and diazine moieties afforded a series with good potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. 32 was identified as the development track candidate. It was potent in cell based, binding, and whole blood assays and exhibited good PK profile. It was efficacious in mouse models of contact hypersensitivity (1 mg/kg b.i.d.) and house dust (20 mg/kg q.d.) when dosed orally. In sheep asthma, administration at 1 mg/kg iv completely blocked the LAR and AHR and attenuated the EAR phase.
Asunto(s)
Hipersensibilidad/tratamiento farmacológico , Ácidos Indolacéticos/síntesis química , Piridazinas/síntesis química , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Broncoconstricción/efectos de los fármacos , Células CACO-2 , Forma de la Célula/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Dermatitis por Contacto/tratamiento farmacológico , Dermatitis por Contacto/inmunología , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Hipersensibilidad/inmunología , Inmunoensayo , Ácidos Indolacéticos/farmacocinética , Ácidos Indolacéticos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Permeabilidad , Piridazinas/farmacocinética , Piridazinas/farmacología , Pyroglyphidae/inmunología , Ratas , Ovinos , Relación Estructura-ActividadAsunto(s)
Arginina/química , Inhibidores Enzimáticos/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/farmacología , Relación Estructura-ActividadRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes. This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane permeability of PTP1B inhibitors.
Asunto(s)
Modelos Moleculares , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Tiofenos/síntesis química , Animales , Sitios de Unión , Células CACO-2 , Dominio Catalítico , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Semivida , Hepatocitos , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Estructura Molecular , Fosfotirosina/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/química , Ratas , Ratas Sprague-Dawley , Solubilidad , Relación Estructura-Actividad , Tiofenos/farmacocinética , Tiofenos/farmacología , Distribución TisularRESUMEN
The following account describes our systematic effort to replace one of the carboxylate groups of our diacid thiophene PTP1B inhibitors. Active hits were validated using enzymatic assays before pursuing efforts to improve the potency. Only when the C2 carboxylic acid was replaced with another ionizable functional group was reversible and competitive inhibition retained. Use of a tetrazole ring or 1,2,5-thiadiazolidine-3-one-1,1-dioxide as a carboxylate mimetic led to the discovery of two unique starting series that showed improved permeability (PAMPA) and potency of the order of 300nM. The SAR from these efforts underscores some of the major challenges in developing small molecule inhibitors for PTP1B.
Asunto(s)
Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Tiofenos/farmacología , Ácidos/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/clasificación , Humanos , Estructura Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
A series of monocyclic thiophenes was designed and synthesized as PTP1B inhibitors. Guided by X-ray co-crystal structural information and computational modeling, rational design led to key interactions with Asp48 and improved inhibitory potency against PTP1B.