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1.
J Am Chem Soc ; 135(18): 6838-41, 2013 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-23611635

RESUMEN

Normal cellular function, such as signal transduction, is largely controlled by the reversible phosphorylation of cellular proteins catalyzed by two major classes of enzymes, kinases and phosphatases. A misbalance in this complex and dynamic interplay leads to a variety of severe diseases, such as cancer, inflammation, or autoimmune diseases. This makes kinases as well as phosphatases equally attractive targets for therapeutic manipulation by small molecules. While the development of kinase inhibitors has resulted in several blockbuster drugs, such as imatinib, with remarkable success in the clinic and sales of many billions of U.S. dollars per year, not a single phosphatase inhibitor has yet been approved for clinical use. Similar to the kinase world, substrate-competitive phosphatase inhibitors have been developed but were not suitable for further development into clinical candidates due to their charge and limited selectivity. Research efforts, therefore, have shifted to the exploitation of allosteric sites that can regulate phosphatase activity and may enable the discovery of novel modulators of phosphatase activity with much improved pharmacological properties. However, assay systems, which enable the straightforward discovery of these inhibitor types, are missing. Here, we present a novel binding assay capable of detecting ligands of an allosteric pocket of the protein tyrosine phosphatase 1B. This assay is suitable for high-throughput screening and selectively detects ligands which bind to this unique site with a clear discrimination from substrate-competitive ligands.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Ligandos , Modelos Moleculares , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Relación Estructura-Actividad
2.
Nat Chem Biol ; 8(2): 141-3, 2011 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-22198732

RESUMEN

Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors.


Asunto(s)
Dibenzocicloheptenos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adenosina Trifosfato , Animales , Unión Competitiva , Diseño de Fármacos , Ratones , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas
3.
Nat Chem Biol ; 5(6): 394-6, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19396179

RESUMEN

Targeting kinases outside the highly conserved ATP pocket is thought to be a promising strategy for overcoming bottlenecks in kinase inhibitor research, such as limited selectivity and drug resistance. Here we report the development and application of a direct binding assay to detect small molecules that stabilize the inactive conformation of the tyrosine kinase cSrc. Protein X-ray crystallography validated the assay results and confirmed an exclusively allosteric binding mode.


Asunto(s)
Regulación Alostérica , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo
4.
Bioorg Med Chem ; 19(1): 429-39, 2011 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21130659

RESUMEN

Here we present the synthesis and biological activity of a series of 7-substituted-1-(3-bromophenylamino)isoquinoline-4-carbonitriles as inhibitors of myosin light chain kinase (MLCK) and the epidermal growth factor receptor kinase (EGFR). The inhibitory effect of these molecules was found to be dependent on the nature of the substituents at the 7-position of the isoquinoline scaffold.


Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Isoquinolinas/farmacología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Relación Estructura-Actividad
5.
Proc Natl Acad Sci U S A ; 105(51): 20251-6, 2008 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-19073914

RESUMEN

TGF-beta isoforms are key modulators of a broad range of biological pathways and increasingly are exploited as therapeutic targets. Here, we describe the crystal structures of a pan-TGF-beta neutralizing antibody, GC-1008, alone and in complex with TGF-beta3. The antibody is currently in clinical evaluation for idiopathic pulmonary fibrosis, melanoma, and renal cell cancer. GC-1008 recognizes an asymmetric binding interface across the TGF-beta homodimer with high affinity. Whereas both cognate receptors, TGF-beta-receptor types I and II, are required to recognize all 3 TGF-beta isoforms, GC-1008 has been engineered to bind with high affinity to TGF-beta1, 2, and 3 via a single interaction surface. Comparison with existing structures and models of TGF-beta interaction with its receptors suggests that the antibody binds to a similar epitope to the 2 receptors together and is therefore a structurally different but functionally identical mimic of the binding mode of both receptors.


Asunto(s)
Anticuerpos/metabolismo , Citocinas/inmunología , Imitación Molecular , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Epítopos , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta3/metabolismo
6.
J Am Chem Soc ; 132(12): 4152-60, 2010 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-20201574

RESUMEN

Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatically active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLiK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glycine-rich loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazoline-based inhibitor in p38alpha kinase. We also detected the high-affinity binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode in p38alpha, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLiK labeling strategy to detect the binding of Type I ligands that induce conformational changes in the glycine-rich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays.


Asunto(s)
Colorantes Fluorescentes/química , Glicina/química , Inhibidores de Proteínas Quinasas , Cristalografía por Rayos X , Glicina/antagonistas & inhibidores , Glicina/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología
7.
Chembiochem ; 11(18): 2557-66, 2010 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-21080395

RESUMEN

Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.


Asunto(s)
Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Espectrometría de Fluorescencia/métodos , Animales , Pollos , Cristalografía por Rayos X , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Mutación , Neoplasias/tratamiento farmacológico , Proteínas Quinasas/química , Proteínas Quinasas/genética , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/química , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
8.
J Virol ; 83(7): 3365-73, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19153241

RESUMEN

TRIM5alpha is a retrovirus restriction factor in the host cell cytoplasm that blocks infection before provirus establishment. Restriction activity requires capsid (CA)-specific recognition by the PRYSPRY domain of TRIM5alpha. To better understand the restriction mechanism, nine charge-cluster-to-triple-alanine mutants in the TRIM5alpha PRYSPRY domain were assessed for CA-specific restriction activity. Five mutants distributed along the TRIM5alpha PRYSPRY primary sequence disrupted restriction activity against N-tropic murine leukemia virus and equine infectious anemia virus. Modeling of the TRIM5alpha PRYSPRY domain based on the crystal structures of PRYSPRY-19q13.4.1, GUSTAVUS, and TRIM21 identified a surface patch where disruptive mutants clustered. All mutants in this patch retained CA-binding activity, a reticular distribution in the cytoplasm, and steady-state protein levels comparable to those of the wild type. Residues in the essential patch are conserved in TRIM5alpha orthologues and in closely related paralogues. The same surface patch in the TRIM18 and TRIM20 PRYSPRY domains is the site of mutants causing Opitz syndrome and familial Mediterranean fever. These results indicate that, in addition to CA-specific binding, the PRYSPRY domain possesses a second function, possibly binding of a cofactor, that is essential for retroviral restriction activity by TRIM5alpha.


Asunto(s)
Proteínas de la Cápside/metabolismo , Proteínas Portadoras/metabolismo , Virus de la Anemia Infecciosa Equina/inmunología , Virus de la Leucemia Murina/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Factores de Restricción Antivirales , Sitios de Unión , Proteínas Portadoras/genética , Línea Celular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
9.
J Am Chem Soc ; 131(37): 13286-96, 2009 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-19572644

RESUMEN

Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less conserved allosteric sites would be expected to offer new opportunities for scaffold development. Because no high-throughput method was previously available, we developed a fluorescence-based kinase binding assay for identifying and characterizing ligands which stabilize the inactive kinase conformation. Here, we present a description of the development and validation of this assay using the serine/threonine kinase p38alpha. By covalently attaching fluorophores to the activation loop of the kinase, we were able to detect conformational changes and measure the K(d), k(on), and k(off) associated with the binding and dissociation of ligands to the allosteric pocket. We report the SAR of a synthesized focused library of pyrazolourea derivatives, a scaffold known to bind with high affinity to the allosteric pocket of p38alpha. Additionally, we used protein X-ray crystallography together with our assay to examine the binding and dissociation kinetics to characterize potent quinazoline- and quinoline-based type II inhibitors, which also utilize this binding pocket in p38alpha. Last, we identified the b-Raf inhibitor sorafenib as a potent low nanomolar inhibitor of p38alpha and used protein X-ray crystallography to confirm a unique binding mode to the inactive kinase conformation.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Bencenosulfonatos/farmacología , Unión Competitiva , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Estabilidad de Enzimas , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sorafenib , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
10.
J Am Chem Soc ; 131(51): 18478-88, 2009 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-19950957

RESUMEN

Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Cristalografía por Rayos X , Estabilidad de Enzimas , Humanos , Ligandos , Proteína Quinasa 14 Activada por Mitógenos/química , Unión Proteica , Conformación Proteica , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad
11.
Bioorg Med Chem ; 16(7): 3482-8, 2008 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-18316192

RESUMEN

Resistance to kinase-targeted cancer drugs has recently been linked to a single point mutation in the ATP binding site of the kinase. In EGFR, the crucial Thr790 gatekeeper residue is mutated to a Met and prevents reversible ATP competitive inhibitors from binding. Irreversible 4-(phenylamino)quinazolines have been shown to overcome this drug resistance and are currently in clinical trials. In order to obtain a detailed structural understanding of how irreversible inhibitors overcome drug resistance, we used Src kinase as a model system for drug resistant EGFR-T790M. We report the first crystal structure of a drug resistant kinase in complex with an irreversible inhibitor. This 4-(phenylamino)quinazoline inhibits wild type and drug resistant EGFR in vitro at low nM concentrations. The co-crystal structure of drug resistant cSrc-T338M kinase domain provides the structural basis of this activity.


Asunto(s)
Resistencia a Medicamentos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Cristalografía por Rayos X , Receptores ErbB/química , Receptores ErbB/genética , Clorhidrato de Erlotinib , Modelos Moleculares , Estructura Molecular , Mutación/genética , Inhibidores de Proteínas Quinasas/síntesis química , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad
12.
FEBS Lett ; 580(1): 99-106, 2006 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-16364311

RESUMEN

We determined the first structure of PRYSPRY, a domain found in over 500 different proteins, involved in innate immune signaling, cytokine signaling suppression, development, cell growth and retroviral restriction. The fold encompasses a 7-stranded and a 6-stranded antiparallel beta-sheet, arranged in a beta-sandwich. In the crystal, PRYSPRY forms a dimer where the C-terminus of an acceptor molecule binds to the concave surface of a donor molecule, which represents a putative interaction site. Mutations in the PRYSPRY domains of Pyrin, which are responsible for familial Mediterranean fever, map on the putative PRYSPRY interaction site.


Asunto(s)
Enfermedades Autoinmunes , Proteínas del Citoesqueleto/química , Inmunidad Innata , Mutación , Transducción de Señal , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Dimerización , Humanos , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Unión Proteica , Estructura Terciaria de Proteína , Pirina , Retroviridae/química , Retroviridae/genética , Retroviridae/inmunología , Transducción de Señal/inmunología , Homología Estructural de Proteína
13.
J Med Chem ; 58(17): 6844-63, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26275028

RESUMEN

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.


Asunto(s)
Antineoplásicos/química , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Bases de Datos de Compuestos Químicos , Diseño de Fármacos , Receptores ErbB/genética , Humanos , Cinética , Neoplasias Pulmonares , Modelos Moleculares , Conformación Molecular , Mutación , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Bibliotecas de Moléculas Pequeñas , Solubilidad , Relación Estructura-Actividad , Familia-src Quinasas/química , Familia-src Quinasas/genética
14.
J Med Chem ; 57(10): 4252-62, 2014 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-24754677

RESUMEN

Discoidin domain-containing receptors (DDRs) exhibit a unique mechanism of action among the receptor tyrosine kinases (RTKs) because their catalytic activity is induced by extracellular collagen binding. Moreover, they are essential components in the assimilation of extracellular signals. Recently, DDRs were reported to be significantly linked to tumor progression in breast cancer by facilitating the processes of invasion, migration, and metastasis. Here, we report the successful development of a fluorescence-based, direct binding assay for the detection of type II and III DFG-out binders for DDR2. Using sequence alignments and homology modeling, we designed a DDR2 construct appropriate for fluorescent labeling. Successful assay development was validated by sensitive detection of a reference DFG-out binder. Subsequent downscaling led to convenient application to high-throughput screening formats. Screening of a representative compound library identified high-affinity DDR2 ligands validated by orthogonal activity-based assays, and a subset of identified compounds was further investigated with respect to DDR1 inhibition.


Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores Mitogénicos/antagonistas & inhibidores , Receptores con Dominio Discoidina , Diseño de Fármacos , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Ligandos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/química , Receptores Mitogénicos/metabolismo , Relación Estructura-Actividad
15.
ACS Chem Biol ; 8(1): 58-70, 2013 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-23249378

RESUMEN

The modulation of kinase function has become an important goal in modern drug discovery and chemical biology research. In cancer-targeted therapies, kinase inhibitors have been experiencing an upsurge, which can be measured by the increasing number of kinase inhibitors approved by the FDA in recent years. However, lack of efficacy, limited selectivity, and the emergence of acquired drug resistance still represent major bottlenecks in the clinic and challenge inhibitor development. Most known kinase inhibitors target the active kinase and are ATP competitive. A second class of small organic molecules, which address remote sites of the kinase and stabilize enzymatically inactive conformations, is rapidly moving to the forefront of kinase inhibitor research. Such allosteric modulators bind to sites that are less conserved across the kinome and only accessible upon conformational changes. These molecules are therefore thought to provide various advantages such as higher selectivity and extended drug target residence times. This review highlights various strategies that have been developed to utilizing exclusive structural features of kinases and thereby modulating their activity allosterically.


Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/química , Regulación Alostérica , Unión Competitiva , Humanos , Conformación Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/metabolismo
16.
J Mol Biol ; 425(22): 4455-67, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23911552

RESUMEN

Brassinosteroid signaling kinases (BSKs) are plant-specific receptor-like cytoplasmic protein kinases involved in the brassinosteroid signaling pathway. Unlike common protein kinases, they possess a naturally occurring alanine residue at the "gatekeeper" position, as well as other sequence variations. How BSKs activate downstream proteins such as BSU1, as well as the structural consequences of their unusual sequential features, was unclear. We crystallized the catalytic domain of BSK8 and solved its structure by multiple-wavelength anomalous dispersion phasing methods to a resolution of 1.5Å. In addition, a co-crystal structure of BSK8 with 5-adenylyl imidodiphosphate (AMP-PNP) revealed unusual conformational arrangements of the nucleotide phosphate groups and catalytic key motifs, typically not observed for active protein kinases. Sequential analysis and comparisons with known pseudokinase structures suggest that BSKs represent constitutively inactive protein kinases that regulate brassinosteroid signal transfer through an allosteric mechanism.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas Quinasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Dominio Catalítico , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/metabolismo
17.
Nat Chem ; 5(1): 21-8, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23247173

RESUMEN

Fragment-based ligand and drug discovery predominantly employs sp(2)-rich compounds covering well-explored regions of chemical space. Despite the ease with which such fragments can be coupled, this focus on flat compounds is widely cited as contributing to the attrition rate of the drug discovery process. In contrast, biologically validated natural products are rich in stereogenic centres and populate areas of chemical space not occupied by average synthetic molecules. Here, we have analysed more than 180,000 natural product structures to arrive at 2,000 clusters of natural-product-derived fragments with high structural diversity, which resemble natural scaffolds and are rich in sp(3)-configured centres. The structures of the cluster centres differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by means of protein X-ray crystallography and the identification of novel stabilizers of inactive conformations of p38α MAP kinase and of inhibitors of several phosphatases.


Asunto(s)
Productos Biológicos/química , Descubrimiento de Drogas , Ligandos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Algoritmos , Cristalografía por Rayos X , Concentración 50 Inhibidora , Estructura Molecular , Quinonas/química , Quinonas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/farmacología
18.
J Med Chem ; 56(1): 241-53, 2013 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-23270382

RESUMEN

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.


Asunto(s)
Adenosina Trifosfato/metabolismo , Antiinflamatorios no Esteroideos/síntesis química , Dibenzocicloheptenos/síntesis química , Modelos Moleculares , Factor de Necrosis Tumoral alfa/sangre , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adenosina Trifosfato/química , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Sitios de Unión , Dibenzocicloheptenos/química , Dibenzocicloheptenos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Lipopolisacáridos/farmacología , Unión Proteica , Solubilidad , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/química
19.
J Med Chem ; 56(14): 5757-72, 2013 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-23773153

RESUMEN

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.


Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Resistencia a Antineoplásicos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Mutación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-kit/genética , Relación Estructura-Actividad
20.
Eur J Med Chem ; 48: 1-15, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22154891

RESUMEN

In this paper, we present the structure-based design, synthesis and biological activity of N-pyrazole, N'-thiazole-ureas as potent inhibitors of p38α mitogen-activated protein kinase (p38α MAPK). Guided by complex crystal structures, we employed the initially identified N-aryl, N'-thiazole urea scaffold and introduced key structural elements that allowed the formation of novel hydrogen bonding interactions within the allosteric site of p38α, resulting in potent type III inhibitors. [4-(3-tert-Butyl-5-{[(1,3-thiazol-2-ylamino)carbonyl]amino}-1H-pyrazol-1-yl)-phenyl]acetic acid 18c was found to be the most potent compound within this series and inhibited p38α activity with an IC(50) of 135 ± 21 nM. Its closest analog, ethyl [4-(3-tert-butyl-5-{[(1,3-thiazol-2-ylamino)carbonyl]amino}-1H-pyrazol-1-yl)phenyl]acetate 18b, effectively inhibited p38α mediated phosphorylation of the mitogen activated protein kinase activated protein kinase 2 (MK2) in HeLa cells.


Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/síntesis química , Tiazoles/síntesis química , Urea/análogos & derivados , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirazoles/farmacología , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Urea/síntesis química , Urea/química , Urea/farmacología , Difracción de Rayos X
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