RESUMEN
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
Asunto(s)
Dihidropiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirazoles/farmacología , Regulación Alostérica , Sitio Alostérico , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Islas de CpG , Cristalografía por Rayos X , Citosina/química , Citosina/metabolismo , Metilación de ADN/efectos de los fármacos , Dihidropiridinas/química , Dihidropiridinas/farmacocinética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Granulocitos/efectos de los fármacos , Granulocitos/enzimología , Granulocitos/patología , Humanos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Cinética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Modelos Moleculares , Mutación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Cultivo Primario de Células , Unión Proteica , Pirazoles/química , Pirazoles/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.
Asunto(s)
Ácido Aspártico/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inhibidores de Proteasas/química , Renina/antagonistas & inhibidores , Ácido Aspártico/química , Sitios de Unión , Cristalografía por Rayos X , Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Inhibidores de Proteasas/metabolismo , Unión Proteica , Renina/metabolismo , Relación Estructura-ActividadRESUMEN
Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular Tumoral , Cristalización , Endorribonucleasas/química , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Conformación Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.
Asunto(s)
Neoplasias Encefálicas/enzimología , Citosol/enzimología , Isocitrato Deshidrogenasa , Proteínas Mutantes , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Cristalografía por Rayos X , Glutaratos/química , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , MutaciónRESUMEN
We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 µM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 µM(-1 )s(-1) and 5.3 ± 0.5 µM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) â« k(dac) â¼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 µM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k(dac) > k(ac) â¼ k(cat), and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P(1) position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.
Asunto(s)
Aminoácidos/química , Catepsina C/química , Dipéptidos/química , Catálisis , Catepsina C/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Especificidad por SustratoRESUMEN
Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH(2), FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K(MgATP) (1.2 +/- 0.1 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)/K(FAK-tide), while k(cat) and k(cat)/K(MgATP) were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k(cat)/K(m) for MgATP but not on k(cat)/K(m) for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.
Asunto(s)
Quinasa 1 de Adhesión Focal/química , Quinasa 1 de Adhesión Focal/metabolismo , Péptidos/metabolismo , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Unión ProteicaRESUMEN
We have discovered that phenyltriazolinone is a novel and potent P1 moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P1 position revealed that the side chain of Asp189 has reoriented resulting in a novel S1 binding pocket which is larger in size to accommodate the phenyltriazolinone P1 substrate.
Asunto(s)
Anticoagulantes/síntesis química , Diseño de Fármacos , Inhibidores del Factor Xa , Isoxazoles/síntesis química , Pirazoles/síntesis química , Piridonas/síntesis química , Sulfonas/síntesis química , Anticoagulantes/química , Anticoagulantes/farmacología , Cristalografía por Rayos X , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Modelos Moleculares , Estructura Molecular , Pirazoles/química , Pirazoles/farmacología , Piridonas/química , Piridonas/farmacología , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
A novel series of 1H-indol-1-yl tertiary amine LXR agonists has been designed. Compounds from this series were potent agonists with good rat pharmacokinetic parameters. In addition, the crystal structure of an LXR agonist bound to LXRalpha will be disclosed.
Asunto(s)
Proteínas de Unión al ADN/agonistas , Indoles/síntesis química , Indoles/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Administración Oral , Animales , Colesterol/análisis , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Indoles/química , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Ratones , Conformación Molecular , Estructura Molecular , Receptores Nucleares Huérfanos , Ratas , Relación Estructura-ActividadRESUMEN
The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.
Asunto(s)
Química Farmacéutica/métodos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Diseño de Fármacos , Glucocorticoides/química , Ácido Glucurónico/química , Proteínas Inmediatas-Precoces/química , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Inhibidores de Proteínas Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Ratas , Relación Estructura-ActividadRESUMEN
Ortho-substituted biphenyl moieties are widely used in drug design. We herein report a successful use of the perpendicular conformation of the alpha-substituted phenylcyclopropyl groups to mimic the aplanar, biologically active conformation of the ortho-substituted biphenyl moieties to achieve structural diversity. This is exemplified by the design and synthesis of a series of highly potent pyrazole bicyclic-based Factor Xa (FXa) inhibitors bearing alpha-substituted phenylcyclopropyl P4 moieties. The designed perpendicular conformation was confirmed by the X-ray structure of FXa-bound compound 2r. The potential structural basis for the high FXa potency in the phenylcyclopropyl P4 analogs and their improved FXa inhibitory activities compared with the biphenyl P4 counterparts are discussed.
Asunto(s)
Compuestos de Bifenilo/química , Ciclopropanos/química , Inhibidores del Factor Xa , Factor Xa/química , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Línea Celular Tumoral , Química Farmacéutica/métodos , Diseño de Fármacos , Electrones , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Trombosis/tratamiento farmacológicoRESUMEN
Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 A crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The alphaC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a beta-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel beta-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.
Asunto(s)
Adenilil Imidodifosfato/química , Proteínas Inmediatas-Precoces/química , Proteínas Serina-Treonina Quinasas/química , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmediatas-Precoces/aislamiento & purificación , Proteínas Inmediatas-Precoces/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.
Asunto(s)
Inhibidores del Factor Xa , Fibrinolíticos/síntesis química , Pirazoles/síntesis química , Piridonas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Coagulación Sanguínea/efectos de los fármacos , Cristalografía por Rayos X , Perros , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacología , Humanos , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Pirazoles/farmacocinética , Pirazoles/farmacología , Piridonas/farmacocinética , Piridonas/farmacología , Conejos , Relación Estructura-ActividadRESUMEN
A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.
RESUMEN
Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.
Asunto(s)
Compuestos de Anilina/síntesis química , Proteínas de Unión al ADN/agonistas , Maleimidas/síntesis química , Receptores Citoplasmáticos y Nucleares/agonistas , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Genes Reporteros , Histona Acetiltransferasas , Humanos , Ligandos , Receptores X del Hígado , Luciferasas/genética , Maleimidas/química , Maleimidas/farmacología , Modelos Moleculares , Estructura Molecular , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Coactivador 1 de Receptor Nuclear , Receptores Nucleares Huérfanos , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
As part of an ongoing effort to prepare orally active factor Xa inhibitors using structure-based drug design techniques and molecular recognition principles, a systematic study has been performed on the pharmacokinetic profile resulting from replacing the benzamidine in the P1 position with less basic benzamidine mimics or neutral residues. It is demonstrated that lowering the pK(a) of the P1 ligand resulted in compounds (3-benzylamine, 15a; 1-aminoisoquinoline, 24a; 3-aminobenzisoxazole, 23a; 3-phenylcarboxamide, 22b; and 4-methoxyphenyl, 22a) with improved pharmacokinetic features mainly as a result of decreased clearance, increased volume of distribution, and enhanced oral absorption. This work resulted in a series of potent and orally bioavailable factor Xa inhibitors that ultimately led to the discovery of SQ311, 24a. SQ311, which utilizes a 1-aminoisoquinoline as the P1 ligand, inhibits factor Xa with a K(i) of 0.33 nM and demonstrates both good in vivo antithrombotic efficacy and oral bioavailability.
Asunto(s)
Anticoagulantes/síntesis química , Anticoagulantes/farmacología , Benzamidinas/farmacología , Inhibidores del Factor Xa , Guanidinas/farmacología , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Animales , Anticoagulantes/farmacocinética , Sitios de Unión/efectos de los fármacos , Disponibilidad Biológica , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Enlace de Hidrógeno , Indicadores y Reactivos , Absorción Intestinal , Imitación Molecular , Conejos , Relación Estructura-Actividad , Trombina/químicaRESUMEN
Factor Xa, a serine protease, is at the critical juncture between the intrinsic and extrinsic pathways of the coagulation cascade. Inhibition of factor Xa has the potential to provide effective treatment for both venous and arterial thrombosis. We recently described a series of meta-substituted phenylpyrazoles that are highly potent, selective, and orally bioavailable factor Xa inhibitors. In this paper we report our efforts to further optimize the selectivity profile of our factor Xa inhibitors with a series of ortho- and/or para-substituted phenylpyrazole derivatives. The most potent compounds display sub-nanomolar inhibition constants for factor Xa and show greater than 1000-fold selectivity against other serine proteases. These compounds are also effective in a rabbit model of arteriovenous shunt thrombosis. Optimization of this series led to the preclinical development of DPC602, a 2-(aminomethyl)phenylpyrazole analogue, as a highly potent, selective, and orally bioavailable factor Xa inhibitor.
Asunto(s)
Inhibidores del Factor Xa , Pirazoles/síntesis química , Administración Oral , Animales , Derivación Arteriovenosa Quirúrgica , Disponibilidad Biológica , Cristalografía por Rayos X , Perros , Factor Xa/química , Humanos , Pirazoles/farmacocinética , Pirazoles/farmacología , Conejos , Relación Estructura-Actividad , Trombosis/prevención & controlRESUMEN
The identification of indeno[1,2-c]pyrazol-4-ones as inhibitors of cyclin-dependent kinases (CDKs) has led to the discovery of a series of novel and potent compounds. Herein, we report the effects of substitutions at C3 of the indeno[1,2-c]pyrazol-4-one core with alkyls, heterocycles, and substituted phenyls. Substitutions at the para position of the phenyl ring at C3 were generally well-tolerated; however, larger groups were generally inactive. For alkyls directly attached to C3, longer chain substituents were not tolerated; however, shorter alkyl groups and cyclic alkyls were acceptable. In general, the heterocycles at C3 gave the most potent analogues. One such heterocycle, 24j, was examined in detail and was determined to have a biological profile consistent with CDK inhibition. An X-ray crystal structure of one of the alkyl compounds, 13q, complexed with CDK2 was determined and showed the inhibitor residing in the adenosine 5'-triphosphate pocket of the enzyme.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Proteínas Proto-Oncogénicas , Pirazoles/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , División Celular/efectos de los fármacos , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/química , Pirazoles/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.
RESUMEN
VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 A resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a pi-pi interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/química , Piperazinas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Recombinantes/química , Aurora Quinasas , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , Cristalografía , Cristalografía por Rayos X , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.