RESUMEN
NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.
Asunto(s)
Alquinos/farmacología , Cisteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Alquinos/síntesis química , Alquinos/química , Cisteína/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasa de Factor Nuclear kappa BRESUMEN
Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.
Asunto(s)
Nucléolo Celular/efectos de los fármacos , Dactinomicina/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Proteínas Nucleares/genética , Anciano , Antibióticos Antineoplásicos/uso terapéutico , Nucléolo Celular/patología , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Nucleofosmina , Proyectos Piloto , Pronóstico , Inducción de Remisión , Terapia RecuperativaRESUMEN
Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.
Asunto(s)
Antineoplásicos/farmacología , Isoindoles/farmacología , Osteosarcoma/tratamiento farmacológico , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Estabilidad de Medicamentos , Femenino , Humanos , Isoindoles/síntesis química , Isoindoles/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover, and many of these control activities depend upon PASTA-domain containing eukaryotic-like serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few PG biosynthetic enzymes are direct kinase substrates. Here, we identify the conserved ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG biosynthesis is activated through control of ClpCP protease activity in response to signals of PG homeostasis imbalance.
Asunto(s)
Proteínas Bacterianas/metabolismo , Peptidoglicano/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Bacterianas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Muramidasa/genética , Muramidasa/metabolismo , Peptidoglicano/genética , Peptidoglicano/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Supresión Genética/genéticaRESUMEN
A growing number of approaches to "staple" α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l-cysteine with "cysteine analogues" in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53-MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l-cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d-cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data.
Asunto(s)
Cisteína/química , Fluorocarburos/química , Péptidos/química , Secuencia de Aminoácidos , Cisteína/metabolismo , Humanos , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design.
Asunto(s)
Benceno/química , Sitios de Unión , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
Regulatory T lymphocytes play a crucial role in modulating potentially self-reactive clones, and dysfunction of this cell type contributes to autoimmune disease. FOXP3 is a critical determinant of CD(4+)CD(25+)T regulatory (T(reg)) cell development and function. The aim of this study was to investigate whether genetic polymorphisms at the FOXP3 locus predispose to autoimmune endocrinopathies. Five single nucleotide polymorphisms (SNPs) and two microsatellite polymorphisms were genotyped in our Caucasian cohorts of 633 unrelated Graves' disease (GD) subjects, 104 autoimmune Addison's disease (AAD) subjects and 528 healthy controls. SNP genotyping was performed by either restriction enzyme digestion or by primer-extension-MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) assay. Microsatellites were analysed using fluorescent PCR. Case-control analysis was performed using chi(2) testing on contingency tables for allele frequency. Haplotype analysis was performed using the UNPHASED package. No evidence for disease association was found with any of the seven polymorphisms in either of the GD or AAD subjects as compared with controls (P = 0.26-0.94). Haplotype analysis found a weak evidence for the association of a minor haplotype with GD; this was not significant when corrected for multiple testing. This study has found no robust evidence that FOXP3 gene polymorphism contributes to the susceptibility to GD or AAD in the UK population.
Asunto(s)
Enfermedad de Addison/genética , Factores de Transcripción Forkhead/genética , Predisposición Genética a la Enfermedad , Enfermedad de Graves/genética , Polimorfismo Genético , Enfermedad de Addison/inmunología , Análisis Mutacional de ADN , Femenino , Genotipo , Haplotipos , Humanos , Repeticiones de Microsatélite , Estadística como Asunto , Reino UnidoRESUMEN
OBJECTIVE: A functional polymorphism at codon 55 of the small ubiquitin-like modifier-4 (SUMO4) gene (methionine to valine; M55V) has recently been associated with type 1 diabetes mellitus (T1D). We aimed to establish whether this locus also contributes towards the genetic susceptibility to Graves' disease (GD) and autoimmune Addison's disease. DESIGN: A case-control analysis was performed using genomic DNA samples from 595 unrelated white GD subjects, 104 white autoimmune Addison's disease subjects and 467 healthy white control subjects. The SUMO4 M55V single nucleotide polymorphism (SNP) was genotyped using polymerase chain reaction (PCR) amplification followed by digestion with the restriction enzyme MseI. RESULTS: There was no association of the SUMO4 M55V alleles with either GD, thyroid-associated orbitopathy or autoimmune Addison's disease when compared to controls; P = 0.28, 0.46 and 0.91, respectively, by chi2 testing. CONCLUSION: We cannot confirm a generalized role for SUMO4 in autoimmune endocrinopathy. The SUMO4 codon 55 methionine to valine polymorphism may be exclusively associated with susceptibility to T1D, or the effect of the locus in GD and Addison's disease may be much less than that found in T1D patients.
Asunto(s)
Enfermedad de Graves/genética , Polimorfismo de Nucleótido Simple , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Enfermedad de Addison/genética , Estudios de Casos y Controles , Codón/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Metionina/genética , Valina/genéticaRESUMEN
The genetic basis for Graves' disease remains largely unknown, but significant linkage to microsatellite markers on 20q11 suggests that this region harbors a susceptibility gene. One obvious candidate gene at this 20q11 locus is CD40, which encodes a B-cell-surface receptor that is involved in T-cell to B-cell signaling and is implicated in control of T-cell autoreactivity. In addition, an allele of a single nucleotide polymorphism (SNP) in the Kozak consensus sequence of the 5' untranslated region of CD40 exon 1 has been reported to show modest evidence for association with Graves' disease. We have investigated the role of this 5' untranslated region (5' UTR) in Graves' disease susceptibility in our cohort of 451 unrelated white subjects with Graves' disease and 446 healthy controls. The CD40 5'UTR SNP (C --> T, position -1) was polymerase chain reaction (PCR)amplified and genotyped using the restriction enzyme NcoI. The frequency of the C allele was 74.8% in Graves' probands compared to 75.1% in controls (not significant [NS]). We find no evidence to support allelic association with Graves' disease at this CD40 SNP, despite the adequate power of the study. We are unable to confirm a role for CD40 in Graves' disease pathogenesis in our U.K. population, however, further studies involving larger patient cohorts and a saturated SNP marker map are required to resolve this issue.
Asunto(s)
Antígenos CD40/genética , Enfermedad de Graves/genética , Polimorfismo de Nucleótido Simple , Regiones no Traducidas 5'/genética , Linfocitos B/fisiología , Estudios de Casos y Controles , Genotipo , Humanos , Repeticiones de MicrosatéliteRESUMEN
The immunodysregulation, polyendocrinopathy, enteropathy syndrome (IPEX), is a rare disorder of immune regulation resulting in multiple autoimmune disorders, which demonstrates X-linked recessive inheritance. The disease gene, FOXP3, was identified in 2001, and several mutations within this gene have since been described in patients with IPEX. We used linkage analysis, mutational screening of the FOXP3 gene, human leukocyte antigen typing, and analysis of X-chromosome inactivation to investigate 2 kindreds (21 subjects in total) with 4 male infants (3 now deceased) and 1 girl affected by IPEX. In 1 family a novel FOXP3 mutation was identified in the proband, with a single base deletion at codon 76 of exon 2, leading to a frameshift, which predicted a truncated protein product (108 residues vs. 431 in wild type). In the second family, the FOXP3 locus was excluded by recombination, and mutational analysis of the gene was negative. The affected girl from this family was shown to have human leukocyte antigen DR2 and DR6 alleles and random X-chromosome inactivation in peripheral blood mononuclear cells. Our analysis has elucidated the molecular basis of IPEX in one family and has, for the first time, provided evidence for an autosomal locus, suggesting genetic heterogeneity in this syndrome.
Asunto(s)
Proteínas de Unión al ADN/genética , Heterogeneidad Genética , Enfermedades del Sistema Inmune/genética , Enfermedades Intestinales/genética , Poliendocrinopatías Autoinmunes/genética , Secuencia de Bases/genética , Estudios de Casos y Controles , ADN/genética , Análisis Mutacional de ADN , Compensación de Dosificación (Genética) , Femenino , Factores de Transcripción Forkhead , Ligamiento Genético , Humanos , Lactante , Recién Nacido , Escala de Lod , Masculino , Mutación , Linaje , SíndromeRESUMEN
The product of the deleted in colorectal carcinoma (DCC) gene has a role in apoptosis and is a positional candidate for IDDM6, the putative chromosome 18q12-q23 autoimmune disease locus. We hypothesised that a nonconservative substitution (DCC 201 R --> G; nucleotide (nt) 601 C --> G), located in an extracellular immunoglobulin-like domain of DCC, is an aetiological determinant of autoimmunity. We tested this hypothesis by genetically testing the nt 601 C --> G polymorphism for association with three autoimmune phenotypes in a large population-based case-control study. There was no evidence for association of DCC nt 601 C --> G with autoimmune disease in cohorts comprising 2253 subjects with rheumatoid arthritis, type I diabetes and Graves' disease, and 2225 control subjects, from New Zealand and the United Kingdom. Furthermore, using the transmission disequilibrium test, there was no significant evidence for biased transmission of the nt 601 C --> G polymorphism to probands within a 382 family type I diabetes affected sibpair cohort from the United Kingdom. Thus, the DCC 201 R --> G polymorphism does not appreciably influence risk of developing the autoimmune diseases tested.