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1.
J Med Chem ; 59(13): 6455-69, 2016 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-27305487

RESUMEN

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma.


Asunto(s)
Aminopiridinas/farmacología , Antineoplásicos/farmacología , Carcinoma Endometrioide/tratamiento farmacológico , Descubrimiento de Drogas , Neoplasias Endometriales/tratamiento farmacológico , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Administración Oral , Regulación Alostérica/efectos de los fármacos , Aminopiridinas/administración & dosificación , Aminopiridinas/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Carcinoma Endometrioide/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Endometriales/patología , Femenino , Humanos , Imidazoles/administración & dosificación , Imidazoles/química , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-Actividad
2.
J Med Chem ; 55(11): 5291-310, 2012 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-22533986

RESUMEN

This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.


Asunto(s)
Adenosina Trifosfato/fisiología , Antineoplásicos/síntesis química , Imidazoles/síntesis química , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/síntesis química , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Imidazoles/química , Imidazoles/farmacología , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Conformación Proteica , Piridinas/química , Piridinas/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Relación Estructura-Actividad
3.
J Biol Chem ; 286(23): 20666-76, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-21454604

RESUMEN

A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Neoplasias Pulmonares/enzimología , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/química , Pirrolidinonas/química , Quinolinas/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Dominio Catalítico , Ensayos Clínicos Fase III como Asunto , Cristalografía por Rayos X , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirrolidinonas/uso terapéutico , Quinolinas/uso terapéutico
4.
J Biol Chem ; 286(23): 20677-87, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-21454610

RESUMEN

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.


Asunto(s)
Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Adenosina Trifosfato/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética
5.
Rapid Commun Mass Spectrom ; 23(1): 12-22, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19051226

RESUMEN

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, beta-lapachone) is an anticancer agent, currently in multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. This study focuses on in vitro metabolism in cryopreserved hepatocytes from mice, rats, dogs and humans using [(14)C]-labeled ARQ 501. Metabolite profiles were characterized using liquid chromatography/mass spectrometry combined with an accurate radioactivity counter. Ion trap mass spectrometry was employed for further structural elucidation. A total of twelve metabolites were detected in the mammalian hepatocytes studied; all of which but one were generated from phase II conjugation reactions. Ten of the observed metabolites were produced by conjugations occurring at the reduced ortho-quinone carbonyl groups of ARQ 501. The metabolite profiles revealed that glucuronidation was the major biotransformation pathway in mouse and human hepatocytes. Monosulfation was the major pathway in dog, while, in rat, it appears glucuronidation and sulfation pathways contributed equally. Three major metabolites were found in rats: monoglucuronide M1, monosulfate M6, and glucuronide-sulfate M9. Two types of diconjugation metabolites were formed by attachment of the second glycone to an adjacent hydroxyl or to an existing glycone. Of the diconjugation metabolites, glucosylsulfate M10, diglucuronide M5, and glucuronide-glucoside M11 represent rarely observed phase II metabolites in mammals. The only unconjugated metabolite was generated through hydrolysis and was observed in rat, dog and human hepatocytes. ARQ 501 appeared less stable in human hepatocytes than in those of other species. To further elucidate the metabolism of ARQ 501 in extrahepatic sites, its metabolism in human kidney, lung and intestine cells was also studied, and only monoglucuronide M1 was observed in all the cell types examined.


Asunto(s)
Antineoplásicos/metabolismo , Cromatografía Liquida/métodos , Hepatocitos/metabolismo , Espectrometría de Masas/métodos , Naftoquinonas/metabolismo , Animales , Células Cultivadas , Perros , Glucósidos/química , Glucósidos/metabolismo , Glucuronatos/química , Glucuronatos/metabolismo , Humanos , Hidrólisis , Mucosa Intestinal/metabolismo , Marcaje Isotópico , Riñón/metabolismo , Pulmón/metabolismo , Metabolómica/métodos , Ratones , Ratas , Sulfatos/química , Sulfatos/metabolismo
6.
Bioorg Med Chem ; 16(10): 5635-43, 2008 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-18424157

RESUMEN

ARQ 501 (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b] pyran-5,6-dione), a synthetic version of beta-Lapachone, is a promising anti-cancer agent currently in multiple Phase II clinical trials. Promising anti-cancer activity was observed in Phase I and Phase II trials. Metabolism by red blood cells of drugs is an understudied area of research and the metabolites arising from oxidative ring opening (M2 and M3), decarbonylation/ring contraction (M5), and decarbonylation/oxidation (M4 and M6) of ARQ 501 offer a unique opportunity to provide insight into these metabolic processes. Since these metabolites were not detected in in vitro incubations of ARQ 501 with liver microsomes and were structurally diverse, confirmation by chemical synthesis was considered essential. In this report, we disclose the synthetic routes employed and the characterization of the reference standards for these blood metabolites as well as additional postulated structures, which were not confirmed as metabolites.


Asunto(s)
Naftoquinonas/síntesis química , Naftoquinonas/metabolismo , Eritrocitos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética/métodos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Naftoquinonas/química , Estereoisomerismo
7.
Drug Metab Dispos ; 36(4): 641-8, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18180274

RESUMEN

3,4-Dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (ARQ 501; beta-lapachone) showed promising anticancer activity in phase I clinical trials as monotherapy and in combination with cytotoxic drugs. ARQ 501 is currently in multiple phase II clinical trials. In vitro incubation in fresh whole blood at 37 degrees C revealed that ARQ 501 is stable in plasma but disappears rapidly in whole blood. Our data showed that extensive metabolism in red blood cells (RBCs) was mainly responsible for the rapid disappearance of ARQ 501 in whole blood. By comparison, covalent binding of ARQ 501 and/or its metabolites to whole blood components was a minor contributor to the disappearance of this compound. Sequestration of intact ARQ 501 in RBCs was not observed. Cross-species metabolite profiles from incubating [(14)C]ARQ 501 in freshly drawn blood were characterized using a liquid chromatography-mass spec-trometry-accurate radioactivity counter. The results show that ARQ 501 was metabolized more rapidly in mouse and rat blood than in dog, monkey, and human blood, with qualitatively similar metabolite profiles. Six metabolites were identified in human blood using ultra-high performance liquid chromatography/time-of-flight mass spectrometry, and the postulated structure of five metabolites was confirmed using synthetic standards. We conclude that the primary metabolic pathway of ARQ 501 in human blood involved oxidation of the two adjacent carbonyl groups to produce dicarboxylic and monocarboxylic metabolites, elimination of a carbonyl group to form a ring-contracted metabolite, and lactonization to produce two metabolites with a pyrone ring to form a ring-contracted metabolite. Metabolism by RBCs may play a role in clearance of ARQ 501 from the blood compartment in cancer patients.


Asunto(s)
Naftoquinonas/sangre , Animales , Perros , Cromatografía de Gases y Espectrometría de Masas/métodos , Haplorrinos , Humanos , Ratones , Naftoquinonas/química , Naftoquinonas/metabolismo , Unión Proteica , Ratas , Especificidad de la Especie
8.
Eur J Med Chem ; 43(5): 1081-4, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-17707553

RESUMEN

The reaction of iproplatin with reduced glutathione at different mole ratios yielded cis-di(isopropylamine)chloro-glutathionatoplatinum(II), not the expected cis-dichloro- species, indicating a mode of action of this anticancer agent that is different from that of cis-diamminedichloroplatinum(II).


Asunto(s)
Antineoplásicos/química , Glutatión/química , Compuestos Organoplatinos/química , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray
10.
Pharm Res ; 19(2): 124-31, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11883638

RESUMEN

PURPOSE: The purpose of this work is to evaluate the extent of the binding of cisplatin (cis-diamminedichloroplatinum(II)) to DNA in the presence and absence of biological thiols, glutathione, and cysteine, and to test the hypothesis whether the platinum-thiol complexes can serve as a drug reservoir for subsequent binding to DNA. METHODS: Reactions of cisplatin (50 microM to 1.0 mM) with calf thymus DNA (870 microM to 6.75 mM) in the presence and absence of glutathione and cysteine (0 to 10 mM) were carried out at pH 4.4, 7.0, and 7.3. Following the reactions, the DNA was enzymatically digested with nucleases, separated by RP HPLC, and analyzed to determine the extent of DNA binding. The method was independently verified by proton NMR measurements. RESULTS: At neutral pH, and equimolar concentrations of DNA and thiols, only a very small amount of platinum (<5%) was coordinated to DNA, and most of the platinum was coordinated to the thiols. At pH 4.4, binding to DNA was dominant over the binding to thiols. No conversion of platinum-thiol to platinum-DNA complexes was observed up to 7 days of incubation. CONCLUSION: At physiological pH, the cisplatin was exclusively coordinated to biological thiols and platinum-DNA was a minor adduct. Data presented in this paper does not support the "drug reservoir" hypothesis.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Cisplatino/química , Cisplatino/farmacología , Platino (Metal)/química , Compuestos de Sulfhidrilo/química , Cromatografía Líquida de Alta Presión , Cisteína/química , ADN de Cadena Simple/química , Hidrólisis , Espectroscopía de Resonancia Magnética , Espectrofotometría Ultravioleta
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