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1.
ACS Med Chem Lett ; 10(10): 1486-1491, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31620238

RESUMO

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1, and a series of modifications led to optimized compound 13. Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose.

2.
J Med Chem ; 62(20): 8973-8995, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31318208

RESUMO

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

3.
J Med Chem ; 62(20): 8953-8972, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31314518

RESUMO

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

4.
ACS Med Chem Lett ; 10(3): 383-388, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30891145

RESUMO

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

5.
J Biomol NMR ; 68(4): 237-247, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28711957

RESUMO

An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.


Assuntos
Marcação por Isótopo/métodos , Aminoácidos/análise , Aminoácidos/química , Animais , Baculoviridae , Antígenos CD4/biossíntese , Antígenos CD4/química , Antígenos CD4/isolamento & purificação , Isótopos de Carbono , Meios de Cultura/análise , Meios de Cultura/química , Quinase 1 de Adesão Focal/biossíntese , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/isolamento & purificação , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Proteínas , Células Sf9 , Spodoptera
6.
Acta Crystallogr D Struct Biol ; 72(Pt 5): 658-74, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27139629

RESUMO

The cytokine TGF-ß modulates a number of cellular activities and plays a critical role in development, hemostasis and physiology, as well as in diseases including cancer and fibrosis. TGF-ß signals through two transmembrane serine/threonine kinase receptors: TGFßR1 and TGFßR2. Multiple structures of the TGFßR1 kinase domain are known, but the structure of TGFßR2 remains unreported. Wild-type TGFßR2 kinase domain was refractory to crystallization, leading to the design of two mutated constructs: firstly, a TGFßR1 chimeric protein with seven ATP-site residues mutated to their counterparts in TGFßR2, and secondly, a reduction of surface entropy through mutation of six charged residues on the surface of the TGFßR2 kinase domain to alanines. These yielded apo and inhibitor-bound crystals that diffracted to high resolution (<2 Å). Comparison of these structures with those of TGFßR1 reveal shared ligand contacts as well as differences in the ATP-binding sites, suggesting strategies for the design of pan and selective TGFßR inhibitors.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
7.
Anal Biochem ; 501: 56-65, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26874021

RESUMO

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Transaminases/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/enzimologia , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ácido Cinurênico/metabolismo , Espectrometria de Massas/métodos , Espectrometria de Fluorescência/métodos , Transaminases/metabolismo
8.
J Biol Chem ; 290(17): 11061-74, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25762719

RESUMO

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.


Assuntos
Modelos Moleculares , Transdução de Sinais , Linfócitos T/enzimologia , TYK2 Quinase/química , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Estrutura Terciária de Proteína , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , TYK2 Quinase/genética
9.
J Am Soc Mass Spectrom ; 25(12): 2093-102, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25223306

RESUMO

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.ᅟ


Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Medição da Troca de Deutério , Receptores ErbB/antagonistas & inibidores , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína
10.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 2): 173-81, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24637750

RESUMO

Tau-tubulin kinase 1 (TTBK1) is a dual-specificity (serine/threonine and tyrosine) kinase belonging to the casein kinase 1 superfamily. TTBK1 is a neuron-specific kinase that regulates tau phosphorylation. Hyperphosphorylation of tau is implicated in the pathogenesis of Alzheimer's disease. Two kinase-domain constructs of TTBK1 were expressed in a baculovirus-infected insect-cell system and purified. The purified TTBK1 kinase-domain proteins were crystallized using the hanging-drop vapor-diffusion method. X-ray diffraction data were collected and the structure of TTBK1 was determined by molecular replacement both as an apo structure and in complex with a kinase inhibitor.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Animais , Baculoviridae/genética , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Células Sf9 , Especificidade por Substrato
11.
J Med Chem ; 57(5): 1855-79, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24397558

RESUMO

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.


Assuntos
Antivirais/farmacologia , Benzazepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Antivirais/química , Antivirais/farmacocinética , Benzazepinas/química , Benzazepinas/farmacocinética , Cães , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Indóis/química , Indóis/farmacocinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Ratos , Relação Estrutura-Atividade
12.
J Biomol Screen ; 18(9): 1072-83, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24062352

RESUMO

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.


Assuntos
Descoberta de Drogas , Drogas em Investigação/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Interações de Medicamentos , Drogas em Investigação/química , Drogas em Investigação/metabolismo , Corantes Fluorescentes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/química , Relação Estrutura-Atividade
13.
Chem Biol Drug Des ; 78(5): 739-48, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21883956

RESUMO

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 Å resolution and PP2 at 1.9 Å resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-ordered protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.


Assuntos
Cromossomos Humanos X , Proteínas Tirosina Quinases/química , Tirosina Quinase da Agamaglobulinemia , Sítios de Ligação , Cristalografia por Raios X , Dasatinibe , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/química , Tiazóis/química , Quinases da Família src/química , Quinases da Família src/metabolismo
14.
Biochem J ; 436(2): 331-9, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21410432

RESUMO

CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.


Assuntos
Indóis/antagonistas & inibidores , Indóis/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Pirazóis/antagonistas & inibidores , Pirazóis/química , Sequência de Aminoácidos , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/metabolismo , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/metabolismo , Pirazóis/metabolismo
15.
J Med Chem ; 53(15): 5620-8, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20684603

RESUMO

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.


Assuntos
Inibidores da Dipeptidil Peptidase IV , Hipoglicemiantes/síntese química , Imidazóis/síntese química , Pirimidinas/síntese química , Animais , Domínio Catalítico , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/química , Cães , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Imidazóis/farmacocinética , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Obesos , Modelos Moleculares , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
16.
J Med Chem ; 51(17): 5330-41, 2008 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-18690676

RESUMO

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.


Assuntos
Antineoplásicos/síntese química , Fosfotransferases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridonas/farmacologia , Receptores de Fatores de Crescimento/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-met , Piridonas/síntese química , Relação Estrutura-Atividade , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores
17.
Acta Crystallogr D Biol Crystallogr ; D64(Pt 7): 705-10, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18566506

RESUMO

The crystal structure of unphosphorylated p38alpha MAP kinase complexed with a representative pyrrolotriazine-based inhibitor led to the elucidation of the high-affinity binding mode of this class of compounds at the ATP-binding site. The ligand binds in an extended conformation, with one end interacting with the adenine-pocket hinge region, including a hydrogen bond from the carboxyl O atom of Met109. The other end of the ligand interacts with the hydrophobic pocket of the binding site and with the backbone N atom of Asp168 in the DFG activation loop. Addition of an extended benzylmorpholine group forces the DFG loop to flip out of position and allows the ligand to make additional interactions with the protein.


Assuntos
Anilidas/química , Benzamidas/química , Proteína Quinase 14 Ativada por Mitógeno/química , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Pirróis/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Modelos Moleculares , Ligação Proteica
18.
J Am Soc Mass Spectrom ; 19(6): 841-54, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18400511

RESUMO

Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed. Here we demonstrate that mass spectrometry analysis of the time course of limited proteolysis reactions provides new information that is self-sufficient to identify all proteolytic fragments. The method uses a non-specific protease like subtilisin and exploits information contained in the time-resolved dataset such as: increased likelihood of identifying larger fragments generated during initial proteolysis solely by their masses, additivity of the masses of two mutually exclusive sequence regions that generate the full-length molecule (or an already assigned subfragment), and analyses of the proteolytic subfragment patterns that are facilitated by having established the initial cleavage sites. We show that the identities of the observed proteolytic fragments can be determined by LC/MS alone because enough constraints exist in the time-resolved dataset. For a medium-sized protein, it takes about 8 h to complete the study, a significant improvement over the traditional SDS-PAGE and N-terminal sequencing method, which usually takes several days. We illustrate this method with application to the catalytic domain of mitogen-activated protein kinase-activated protein kinase-2, and compare the results with N-terminal sequencing data and the known X-ray crystal structure.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Proteína Quinase 1 Ativada por Mitógeno/química , Peptídeo Hidrolases/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Ativação Enzimática , Dados de Sequência Molecular , Fatores de Tempo
19.
Protein Sci ; 17(2): 240-50, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18227430

RESUMO

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.


Assuntos
Adamantano/análogos & derivados , Dipeptídeos/química , Dipeptidil Peptidase 4/química , Adamantano/química , Adamantano/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Ligações de Hidrogênio , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína
20.
Cancer Res ; 66(11): 5790-7, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16740718

RESUMO

Chronic myeloid leukemia (CML) is caused by the constitutively activated tyrosine kinase breakpoint cluster (BCR)-ABL. Current frontline therapy for CML is imatinib, an inhibitor of BCR-ABL. Although imatinib has a high rate of clinical success in early phase CML, treatment resistance is problematic, particularly in later stages of the disease, and is frequently mediated by mutations in BCR-ABL. Dasatinib (BMS-354825) is a multitargeted tyrosine kinase inhibitor that targets oncogenic pathways and is a more potent inhibitor than imatinib against wild-type BCR-ABL. It has also shown preclinical activity against all but one of the imatinib-resistant BCR-ABL mutants tested to date. Analysis of the crystal structure of dasatinib-bound ABL kinase suggests that the increased binding affinity of dasatinib over imatinib is at least partially due to its ability to recognize multiple states of BCR-ABL. The structure also provides an explanation for the activity of dasatinib against imatinib-resistant BCR-ABL mutants.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/química , Pirimidinas/química , Tiazóis/química , Animais , Benzamidas , Cristalografia por Raios X , Dasatinibe , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Humanos , Mesilato de Imatinib , Modelos Moleculares , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Tiazóis/metabolismo , Tiazóis/farmacologia
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