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1.
J Biol Chem ; 290(33): 20360-73, 2015 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-26134571

RESUMEN

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl ß,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.


Asunto(s)
Quinasa 4 del Receptor Acoplado a Proteína-G/química , Quinasa 4 del Receptor Acoplado a Proteína-G/metabolismo , Hipertensión/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Quinasa 4 del Receptor Acoplado a Proteína-G/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
2.
J Biol Chem ; 286(13): 11218-25, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21247903

RESUMEN

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.


Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/química , Animales , Línea Celular , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Fosforilación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Spodoptera , Relación Estructura-Actividad , Tirosina Quinasa c-Mer
3.
J Biol Chem ; 286(8): 6433-48, 2011 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-21118801

RESUMEN

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.


Asunto(s)
Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Dominio Catalítico/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora
4.
J Biol Chem ; 285(7): 4587-94, 2010 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19864428

RESUMEN

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3'-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3'-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.


Asunto(s)
Proteínas Quinasas S6 Ribosómicas 70-kDa/química , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Estaurosporina/metabolismo , Ultracentrifugación
5.
Biochemistry ; 48(21): 4488-96, 2009 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-19284778

RESUMEN

BACE-1 (beta-site amyloid precursor protein cleaving enzyme), a prominent target in Alzheimer's disease drug discovery efforts, was surveyed using Tethering technology to discover small molecule fragment ligands that bind to the enzyme active site. Screens of a library of >15000 thiol-containing fragments versus a panel of BACE-1 active site cysteine mutants under redox-controlled conditions revealed several novel amine-containing fragments that could be selectively captured by subsets of the tethering sites. For one such hit class, defined by a central aminobenzylpiperidine (ABP) moiety, X-ray crystal structures of BACE mutant-disulfide conjugates revealed that the fragment bound by engaging both catalytic aspartates with hydrogen bonds. The affinities of ABP fragments were improved by structure-guided chemistry, first for conjugation as thiol-containing fragments and then for stand-alone, noncovalent inhibition of wild-type (WT) BACE-1 activity. Crystallography confirmed that the inhibitors bound in exactly the same mode as the disulfide-conjugated fragments that were originally selected from the screen. The ABP ligands represent a new type of nonpeptidic BACE-1 inhibitor motif that has not been described in the aspartyl protease literature and may serve as a starting point for the development of BACE-1-directed Alzheimer's disease therapeutics.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Biocatálisis , Dominio Catalítico , Cisteína , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Mutación , Péptidos/química , Piperidinas/química , Piperidinas/metabolismo , Relación Estructura-Actividad
6.
Bioorg Med Chem Lett ; 19(17): 4993-5, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19640712

RESUMEN

During our ongoing efforts to develop a small molecule inhibitor targeting the beta-amyloid cleaving enzyme (BACE-1), we discovered a class of compounds bearing an aminoimidazole motif. Initial optimization led to potent compounds that have high Pgp efflux ratios. Crystal structure-aided design furnished conformationally constrained compounds that are both potent and have relatively low Pgp efflux ratios. Computational studies performed after these optimizations suggest that the introduction of the constraint enhances potency via additional hydrophobic interactions rather than conformational restriction.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Imidazoles/química , Inhibidores de Proteasas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Imidazoles/síntesis química , Imidazoles/farmacología , Conformación Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína
7.
Bioorg Med Chem Lett ; 19(11): 2977-80, 2009 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19409780

RESUMEN

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK(a) of these inhibitors and the pH of the BACE-1 enzyme binding assay.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Concentración de Iones de Hidrógeno , Unión Proteica , Relación Estructura-Actividad
8.
Bioorg Med Chem Lett ; 19(4): 1240-4, 2009 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-19155174

RESUMEN

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.


Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinazolinas/síntesis química , Quinazolinas/farmacología , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Humanos , Conformación Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Quinazolinas/química
9.
Protein Sci ; 16(12): 2626-35, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17965187

RESUMEN

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.


Asunto(s)
Adenosina Trifosfato/análogos & derivados , Purinas/química , Proteínas Quinasas S6 Ribosómicas 90-kDa/química , Estaurosporina/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Purinas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Estaurosporina/metabolismo
10.
J Biol Chem ; 283(50): 34864-72, 2008 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-18922802

RESUMEN

Prostasin (also called channel activating protease-1 (CAP1)) is an extracellular serine protease implicated in the modulation of fluid and electrolyte regulation via proteolysis of the epithelial sodium channel. Several disease states, particularly hypertension, can be affected by modulation of epithelial sodium channel activity. Thus, understanding the biochemical function of prostasin and developing specific agents to inhibit its activity could have a significant impact on a widespread disease. We report the expression of the prostasin proenzyme in Escherichia coli as insoluble inclusion bodies, refolding and activating via proteolytic removal of the N-terminal propeptide. The refolded and activated enzyme was shown to be pure and monomeric, with kinetic characteristics very similar to prostasin expressed from eukaryotic systems. Active prostasin was crystallized, and the structure was determined to 1.45 A resolution. These apoprotein crystals were soaked with nafamostat, allowing the structure of the inhibited acyl-enzyme intermediate structure to be determined to 2.0 A resolution. Comparison of the inhibited and apoprotein forms of prostasin suggest a mechanism of regulation through stabilization of a loop which interferes with substrate recognition.


Asunto(s)
Hipertensión/metabolismo , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Apoproteínas/química , Benzamidinas , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Guanidinas/química , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Renaturación de Proteína , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Especificidad por Sustrato
11.
Bioorg Med Chem Lett ; 17(3): 823-7, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17107793

RESUMEN

Several simple scoring methods were examined for 2 series of beta-secretase (BACE-1) inhibitors to identify a docking/scoring protocol which could be used to design BACE-1 inhibitors in a drug discovery program. Both the PLP1 score and MMFFs interaction energy (E(inter)) performed as well or better than more computationally intensive methods for a set of substrate-based inhibitors, while the latter performed well for both sets of inhibitors.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Conformación Molecular
12.
Bioorg Med Chem Lett ; 17(4): 1117-21, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17112725

RESUMEN

BACE-1 is a flexible enzyme with experimentally determined motion in the flap region, the catalytic aspartates, and the 10s loop. Four in-house crystallographically determined complexes of tertiary carbinamine inhibitors revealed 10s loop motion in the S(3) pocket. These X-ray structures were used to correlate K(i) values, which span over five orders of magnitude, with the calculated interaction energy, using the Merck Molecular Force Field for a series of 19 tertiary carbinamine inhibitors.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Ácido Aspártico/farmacología , Sitios de Unión , Catálisis , Fenómenos Químicos , Química Física , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ligandos , Conformación Molecular , Relación Estructura-Actividad , Termodinámica
13.
Bioorg Med Chem Lett ; 17(21): 5831-5, 2007 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-17827011

RESUMEN

This letter describes replacements for the P3 amide moiety present in previously reported tertiary carbinamine macrolactones. Although P-gp efflux issues associated with these amide-macrolactones were solved and full brain penetration was measured in one case, potency was compromised in the process.


Asunto(s)
Aminas/farmacocinética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/metabolismo , Inhibidores Enzimáticos/farmacocinética , Cristalografía por Rayos X , Modelos Moleculares
14.
Bioorg Med Chem Lett ; 17(6): 1788-92, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17257835

RESUMEN

A series of low-molecular weight 2,6-diamino-isonicotinamide BACE-1 inhibitors containing an amine transition-state isostere were synthesized and shown to be highly potent in both enzymatic and cell-based assays. These inhibitors contain a trans-S,S-methyl cyclopropane P(3) which bind BACE-1 in a 10s-loop down conformation giving rise to highly potent compounds with favorable molecular weight and moderate to high susceptibility to P-glycoprotein (P-gp) efflux.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Niacinamida/síntesis química , Niacinamida/farmacología , Animales , Baculoviridae/efectos de los fármacos , Baculoviridae/enzimología , Disponibilidad Biológica , Células Cultivadas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Peso Molecular , Niacinamida/farmacocinética , Ratas , Relación Estructura-Actividad
15.
Bioorg Med Chem Lett ; 17(21): 5989-94, 2007 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-17804227

RESUMEN

The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).


Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinolonas/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Quinolonas/química , Relación Estructura-Actividad
16.
Bioorg Med Chem Lett ; 16(3): 641-4, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16263281

RESUMEN

We have synthesized and evaluated a series of conformationally biased P3 amide replacements based on an isophthalamide lead structure. The studies resulted in the identification of the beta-secretase inhibitor 7m which has an in vitro IC(50)=35 nM. The synthesis and biological activities of these compounds are described.


Asunto(s)
Amidas/química , Endopeptidasas/metabolismo , Inhibidores de Proteasas/síntesis química , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide , Diseño de Fármacos , Concentración 50 Inhibidora , Modelos Moleculares , Ácidos Ftálicos/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad
17.
Bioorg Med Chem Lett ; 16(23): 6049-53, 2006 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-16978863

RESUMEN

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.


Asunto(s)
Indazoles/química , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Humanos , Indazoles/metabolismo , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-Actividad
18.
Biochemistry ; 44(27): 9430-40, 2005 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-15996097

RESUMEN

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.


Asunto(s)
Aldehídos/química , Inhibidores de Proteínas Quinasas/química , Pirroles/química , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/química , Aldehídos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Borohidruros/química , Línea Celular , Cristalografía por Rayos X , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Pirroles/metabolismo , Receptor de Insulina/metabolismo , Bases de Schiff/química , Relación Estructura-Actividad
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