RESUMEN
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Histonas/química , Isoxazoles/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Biotinilación , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Isoxazoles/síntesis química , Isoxazoles/farmacología , Cinética , Ligandos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Docilidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , metaminobenzoatos/síntesis química , metaminobenzoatos/química , metaminobenzoatos/farmacologíaRESUMEN
Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP(C)) into a ß-sheet-rich disease-causing isoform (PrP(Sc)) is the key molecular event in the formation of PrP(Sc) aggregates. The molecular mechanisms underlying the PrP(C)-to-PrP(Sc) conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP(Sc) in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23-230)] and N-terminally truncated recPrP(89-230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP(C) into PrP(Sc).
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Proteínas PrPC/química , Proteínas PrPC/inmunología , Proteínas PrPSc/química , Animales , Cromatografía Liquida , Ratones , Modelos Moleculares , Proteínas PrPC/genética , Proteínas PrPSc/genética , Proteínas Recombinantes/química , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
The MCL1 gene is frequently amplified in cancer and codes for the antiapoptotic protein myeloid cell leukemia 1 (MCL1), which confers resistance to the current standard of care. Therefore, MCL1 is an attractive anticancer target. Here we describe BRD-810 as a potent and selective MCL1 inhibitor and its key design principle of rapid systemic clearance to potentially minimize area under the curve-driven toxicities associated with MCL1 inhibition. BRD-810 induced rapid cell killing within 4 h in vitro but, in the same 4-h window, had no impact on cell viability or troponin I release in human induced pluripotent stem cell-derived cardiomyocytes, even at suprapharmacologic concentrations. In vivo BRD-810 induced efficacy in xenograft hematological and solid tumor models despite the short residence time of BRD-810 in plasma. In totality, our data support the hypothesis that short-term inhibition of MCL1 with BRD-810 can induce apoptosis in tumor cells while maintaining an acceptable safety profile. We, therefore, intend to advance BRD-810 to clinical trials.
RESUMEN
Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation.
Asunto(s)
Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Tirosina , Fosforilación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Ligandos , Membrana CelularRESUMEN
Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.
Asunto(s)
Encéfalo/efectos de los fármacos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Enfermedades por Prión/patología , Pirimidinas/farmacología , Anilidas/farmacología , Anilidas/uso terapéutico , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Endopeptidasa K/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Ratones , Modelos Moleculares , Neuroblastoma/patología , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas PrPC/análisis , Enfermedades por Prión/tratamiento farmacológico , Conformación Proteica/efectos de los fármacos , Pirimidinas/uso terapéutico , Silicio , Estadísticas no Paramétricas , Factores de Tiempo , Transfección/métodosRESUMEN
Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.
Asunto(s)
Proteínas PrPSc/antagonistas & inhibidores , Animales , Calorimetría , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Hidrólisis , Inmunohistoquímica , Cinética , Ratones , Proteínas PrPSc/metabolismoRESUMEN
The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bencimidazoles/química , Bencimidazoles/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Diseño de Fármacos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Bencimidazoles/farmacocinética , Proteínas Portadoras/química , Proteínas de Unión al ADN , Femenino , Humanos , Metilación , Ratones , Simulación del Acoplamiento Molecular , Proteínas Nucleares/química , Unión ProteicaRESUMEN
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.
Asunto(s)
Didrogesterona/farmacología , Piperazinas/farmacología , Pirazoles/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Sitios de Unión , Factor de Transcripción COUP II/antagonistas & inhibidores , Factor de Transcripción COUP II/fisiología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/fisiología , Genes Reporteros , Células HeLa , Humanos , Concentración 50 Inhibidora , Ligandos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Unión Proteica , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
Making new ligands for a given protein by in situ ligation of building blocks (or fragments) is an attractive method. However, it suffers from inherent limitations, such as the limited number of available chemical reactions and the low information content of usual chemical library deconvolution. Here, we describe a focused screening of adenosine derivatives using X-ray crystallography. We discovered an unexpected and biocompatible chemical reactivity and have simultaneously identified the mode of binding of the resulting products. We observed that the NAD kinase from Listeria monocytogenes (LmNADK1) can promote amide formation between 5'-amino-5'-deoxyadenosine and carboxylic acid groups. This unexpected reactivity allowed us to bridge in situ two adenosine derivatives to fully occupy the active NAD site. This guided the design of a close analog showing micromolar inhibition of two human pathogenic NAD kinases and potent bactericidal activity against Staphylococcus aureus in vitro.
Asunto(s)
Adenosina/análogos & derivados , Antibacterianos/síntesis química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Staphylococcus aureus/efectos de los fármacos , Adenosina/síntesis química , Adenosina/química , Adenosina/farmacología , Secuencias de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Biocatálisis , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Listeria monocytogenes/enzimología , Modelos Moleculares , Unión Proteica , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg(2+) ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.
Asunto(s)
Proteínas Bacterianas/química , Listeria monocytogenes/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Secuencias de Aminoácidos/genética , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Cristalografía por Rayos X , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Listeria monocytogenes/genética , Magnesio/química , Magnesio/metabolismo , Mutación Missense , NAD/química , NAD/metabolismo , Fosfofructoquinasa-1/química , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato/genéticaRESUMEN
The MabA protein from Mycobacterium tuberculosis is a validated drug target. Previous structural studies of this protein showed dynamic behaviour in the catalytic site and described motion between an open 'active' holo form (with NADP) and a closed 'inactive' apo form (without NADP). Here, a mutation (G139A) is reported that leads to complete protein inactivation and freezes the catalytic site into its closed form, even in the presence of the cofactor. This observation suggests a new way to develop anti-MabA drugs via protein stabilization of the 'inactive' form.