RESUMEN
A series of indazoles have been discovered as KHK inhibitors from a pyrazole hit identified through fragment-based drug discovery (FBDD). The optimization process guided by both X-ray crystallography and solution activity resulted in lead-like compounds with good pharmaceutical properties.
Asunto(s)
Descubrimiento de Drogas , Fructoquinasas/antagonistas & inhibidores , Indazoles/farmacología , Pirazoles/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Indazoles/síntesis química , Indazoles/química , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.
Asunto(s)
Inhibidores del Factor Xa , Indazoles/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Células CACO-2 , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Factor Xa/química , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Indazoles/química , Indazoles/farmacología , Microsomas Hepáticos/enzimología , Modelos Moleculares , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , TermodinámicaRESUMEN
We screened the ligand-binding domain of estrogen-related receptor (ERR) gamma in ThermoFluor, in an effort to develop chemical tools and decipher the biology of this orphan nuclear receptor. Several ligands were found to stabilize thermodynamically the protein. Amongst the ligands were bisphenol A (BPA) and 4-chloro-3-methyl phenol (ClCH3Ph). These ligands were further characterized and found to be competitive for 4-hydroxytamoxifen (4OHT) binding, a known reported antagonist ligand for ERRgamma, but functionally they did not enhance or disrupt affinity of the receptor for co-activator peptides. The preservation of the constitutive active conformation of the receptor in the presence of these two ligands was confirmed upon the determination of the co-crystal structures. The structures of BPA and ClCH3Ph were determined to a resolution of 2.1 and 2.3A, respectively, and the antagonist 4OHT was refined to 2.5A resolution. In the presence of BPA and ClCH3Ph the receptor maintained the transcriptional active conformation as reported previously for the apo-protein in the presence of a co-activator peptide fragment. In addition the ERRgamma-BPA structure identifies an interaction between the phenolic-OH and the side chain of N346. The preservation of the constitutive active conformation of the receptor in the presence of the small phenol compounds suggest that the biological activity of the receptor might be regulated by a natural occurring ligand.
Asunto(s)
Fenoles/farmacología , Receptores Citoplasmáticos y Nucleares/química , Receptores de Estrógenos/química , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Estructura Terciaria de Proteína/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores de Estrógenos/agonistas , Receptores de Estrógenos/antagonistas & inhibidores , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identified as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These compounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth factor-induced receptor phosphorylation in SK-BR-3 cells (IC(50)=54 nM) and cellular proliferation in vitro (IC(50)=14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of EGFR with a close analog (17) was determined and validated our design rationale.
Asunto(s)
Química Farmacéutica/métodos , Receptores ErbB/antagonistas & inhibidores , Hidrazonas/química , Hidrazonas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/síntesis química , Receptor ErbB-2/antagonistas & inhibidores , Animales , Diseño de Fármacos , Humanos , Hidrazonas/farmacología , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Oximas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC(50) values in the nanomolar range. Structure-activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design rationale.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Oximas/farmacología , Pirimidinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Oximas/síntesis química , Oximas/química , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.
Asunto(s)
Inhibidores de la Angiogénesis/química , Fragmentos de Péptidos/química , Plasminógeno/química , Secuencia de Aminoácidos , Angiostatinas , Cristalización , Cristalografía por Rayos X , Humanos , Kringles , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Pichia , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
We describe here a method using protein crystallography as the sole detection tool for fragment-based lead discovery. The methodology consists of iterative design, synthesis, and X-ray crystallographic screening of three libraries of compounds. Target-specific compound design, by way of active site electron density in the presence of a bound fragment hit and the intentional lack of solution activity bias form the basis of our approach. We provide an example of this alternative fragment-based drug design (FBDD) method, detailing results from a campaign using ketohexokinase to generate a unique lead series with promising drug-like properties.
Asunto(s)
Cristalografía por Rayos X , Diseño de Fármacos , Electrones , Bibliotecas de Moléculas Pequeñas , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Dominio Catalítico/efectos de los fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Fructoquinasas/antagonistas & inhibidores , Fructoquinasas/química , Fructosa/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Sulfatos/químicaRESUMEN
Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.
RESUMEN
Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 µM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.
Asunto(s)
Éteres/síntesis química , Hipoglucemiantes/síntesis química , Receptores de Estrógenos/metabolismo , Tiazolidinedionas/síntesis química , Administración Oral , Animales , Unión Competitiva , Disponibilidad Biológica , Cristalografía por Rayos X , Diabetes Mellitus/tratamiento farmacológico , Perros , Éteres/farmacocinética , Éteres/farmacología , Femenino , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Insulina/sangre , Resistencia a la Insulina , Ligandos , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Obesidad/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/genética , Relación Estructura-Actividad , Tiazolidinedionas/farmacocinética , Tiazolidinedionas/farmacología , Triglicéridos/sangre , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Chymases (EC 3.4.21.39) are mast cell serine proteinases that are variably expressed in different species and, in most cases, display either chymotryptic or elastolytic substrate specificity. Given that chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, and cardiovascular disorders, it is important to understand interspecies differences of the enzymes as well as the behavior of inhibitors with them. We have expressed chymases from humans, macaques, dogs, sheep (MCP2 and MCP3), guinea pigs, and hamsters (HAM1 and HAM2) in baculovirus-infected insect cells. The enzymes were purified and characterized with kinetic constants by using chromogenic substrates. We evaluated in vitro the potency of five nonpeptide inhibitors, originally targeted against human chymase. The inhibitors exhibited remarkable cross-species variation of sensitivity, with the greatest potency observed against human and macaque chymases, with K(i) values ranging from approximately 0.4 to 72nM. Compounds were 10-300-fold less potent, and in some instances ineffective, against chymases from the other species. The X-ray structure of one of the potent phosphinate inhibitors, JNJ-18054478, complexed with human chymase was solved at 1.8A resolution to further understand the binding mode. Subtle variations in the residues in the active site that are already known to influence chymase substrate specificity can also strongly affect the compound potency. The results are discussed in the context of selecting a suitable animal model to study compounds ultimately targeted for human chymase.
Asunto(s)
Quimasas/metabolismo , Animales , Baculoviridae/metabolismo , Cricetinae , Perros , Cobayas , Humanos , Macaca , Mastocitos/enzimología , Mastocitos/metabolismo , Serina Proteasas , Ovinos , Especificidad por Sustrato , Rayos XRESUMEN
A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.
Asunto(s)
Fructoquinasas/antagonistas & inhibidores , Indazoles/síntesis química , Modelos Moleculares , Piperidinas/síntesis química , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Electrones , Humanos , Técnicas In Vitro , Indazoles/química , Indazoles/farmacocinética , Masculino , Microsomas Hepáticos/metabolismo , Estructura Molecular , Piperidinas/química , Piperidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.
Asunto(s)
Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos H3/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Tetrahidroisoquinolinas/síntesis química , Tetrahidroisoquinolinas/farmacología , Animales , Cristalografía por Rayos X , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Semivida , Humanos , Indicadores y Reactivos , Modelos Moleculares , Conformación Molecular , Norepinefrina/metabolismo , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinética , Relación Estructura-Actividad , Tetrahidroisoquinolinas/farmacocinéticaRESUMEN
The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.
Asunto(s)
Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Proteasas/farmacología , Animales , Antineoplásicos/farmacología , Sitios de Unión , Glucemia/metabolismo , Células CACO-2 , Dipeptidil Peptidasa 4/metabolismo , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Cinética , Ratones , Modelos Químicos , Modelos Moleculares , Ratas , Relación Estructura-ActividadRESUMEN
Angiostatin is an inhibitor of angiogenesis, the process by which new blood vessels are formed from pre-existing ones. The fact that tumor growth and metastasis dissemination are angiogenesis-dependent processes has awakened interest in angiogenesis inhibitors as anticancer treatment drugs. Angiostatin, a potent angiogenesis inhibitor, is currently in phase I clinical trials. Human angiostatin containing the first three kringle domains of plasminogen (K1-3) has been crystallized and high-resolution data were collected. The crystals belong to the P4(1)2(1)2 space group, with unit-cell parameters a = b = 56.94, c = 192.97 A. A data set to a resolution of 1.75 A with an overall R(merge) and I/sigma(I) of 7% and 19.5, respectively, was obtained.
Asunto(s)
Fragmentos de Péptidos/química , Plasminógeno/química , Angiostatinas , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Fragmentos de Péptidos/genética , Plasminógeno/genética , Conformación ProteicaRESUMEN
Branching enzyme catalyzes the formation of the branch points in glycogen and starch by cleavage of the alpha-1,4 link and its subsequent transfer to the alpha-1,6 position. This paper reports the crystallization and preliminary structural studies of an amino-terminally truncated branching enzyme from Escherichia coli. High-resolution diffracting crystals were obtained and a complete native data set to a resolution of 2.3 A was collected. These crystals belong to the P2(1) space group, with unit-cell parameters a = 91.44, b = 102.58, c = 185.41 A, beta = 91.38 degrees. A native data set with 99.6% completeness, an overall R(merge) of 0.086 and I/sigma(I) of 10.43 was obtained.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/química , Escherichia coli/enzimología , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Cristalización , Cristalografía por Rayos X , Conformación Proteica , Selenometionina/químicaRESUMEN
Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.