RESUMO
The solution-based gram-scale synthesis of complex and highly potent proprotein convertase subtilisin-like/kexin type 9 (PCSK9) inhibitor 1 is presented. Construction of Northern fragment 2, followed by stepwise installation of Eastern 3, Southern 4, and Western 5 fragments, provided macrocyclic precursor 19. This intermediate was cross-linked via an intramolecular azide-alkyne click reaction, which preceded macrolactamization to afford the core framework of compound 1. Finally, coupling with poly(ethylene glycol) side-chain-based 6 gave the PCSK9 inhibitor 1.
Assuntos
Pró-Proteína Convertase 9RESUMO
Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antibacterianos/biossíntese , GMP Cíclico/análogos & derivados , Imunoglobulina G/biossíntese , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Antibacterianos/imunologia , GMP Cíclico/administração & dosagem , GMP Cíclico/imunologia , Feminino , Centro Germinativo/imunologia , Antígenos de Superfície da Hepatite B/administração & dosagem , Humanos , Imunidade Celular , Imunidade Humoral , Imunização , Imunoglobulina G/imunologia , Memória Imunológica , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Macaca mulatta , Camundongos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.
Assuntos
Janus Quinase 2/biossíntese , Janus Quinase 2/isolamento & purificação , Janus Quinase 3/biossíntese , Janus Quinase 3/isolamento & purificação , TYK2 Quinase/biossíntese , TYK2 Quinase/isolamento & purificação , Animais , Catálise , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Camundongos , Fosforilação , Estrutura Terciária de Proteína , TYK2 Quinase/antagonistas & inibidoresRESUMO
Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.
Assuntos
Janus Quinase 2/química , Janus Quinase 2/isolamento & purificação , Janus Quinase 3/química , Janus Quinase 3/isolamento & purificação , Sequência de Aminoácidos , Biocatálise , Cromatografia Líquida de Alta Pressão , Cristalização , Eletroforese em Gel de Poliacrilamida , Fermentação , Humanos , Cinética , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de ProteínaRESUMO
Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.
Assuntos
Baculoviridae/genética , Guanilato Ciclase/isolamento & purificação , Guanilato Ciclase/metabolismo , Insetos/citologia , Insetos/virologia , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/fisiologia , Técnicas de Cultura de Células , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Guanilato Ciclase/química , Humanos , Cinética , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Proteínas Recombinantes/química , Sefarose/química , Guanilil Ciclase SolúvelRESUMO
During our effort to design a receptor binding assay to aid in the elucidation of the molecular mechanism of ezetimibe, we prepared a sulfur-35 containing radioligand which exhibits improved potency over the glucuronide conjugate of ezetimibe in both native enterocyte brush border membranes and membranes from cells expressing recombinant NPC1L1. Herein, we describe the different synthetic strategies which were used to obtain this compound as well as its effectiveness in the aforementioned assay.
Assuntos
Anticolesterolemiantes/química , Azetidinas/química , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/farmacologia , Azetidinas/síntese química , Azetidinas/farmacologia , Linhagem Celular , Ezetimiba , Glucuronídeos/química , Humanos , Ligantes , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Ligação Proteica , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Radioisótopos de Enxofre/químicaRESUMO
A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.
Assuntos
Amidas/síntese química , Citidina/análogos & derivados , Citidina/síntese química , Ácidos Fosfóricos/síntese química , Pró-Fármacos/síntese química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/farmacologia , Animais , Células Cultivadas , Citidina/farmacologia , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/virologia , Humanos , Ácidos Fosfóricos/farmacologia , Pró-Fármacos/farmacologia , RNA Polimerase Dependente de RNA/metabolismo , Coelhos , Ratos , Proteínas não Estruturais Virais/metabolismoRESUMO
A very accurate and selective LC-MS/MS method was developed and validated for the quantification of 2'-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm x 2.0 mm, 3 microm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 microL/min flow rate. The 2'-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05-50 microM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5-9.1%) and accuracy (92.6-94.8%) and interday precision (CV = 9.6-11.5%) and accuracy (94.4-102.4%) as well as for recovery (82.0-112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2'-C-methyl-cytidine-triphosphate in liver tissue samples.
Assuntos
Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/análise , Fígado/química , Espectrometria de Massas em Tandem/métodos , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , Calibragem , Cromatografia Líquida/métodos , Masculino , Pró-Fármacos/administração & dosagem , Pró-Fármacos/metabolismo , Nucleosídeos de Pirimidina/administração & dosagem , Nucleosídeos de Pirimidina/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Extração em Fase Sólida/métodos , Solventes/químicaRESUMO
Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.
Assuntos
Azetidinas/metabolismo , Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Animais , Azetidinas/química , Células CACO-2 , Linhagem Celular , Clonagem Molecular , Cães , Ezetimiba , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Reprodutibilidade dos Testes , Sitosteroides/metabolismo , Sulfonamidas/química , Transfecção , Trítio , beta-Lactamas/metabolismoRESUMO
Comprehensive and accurate identification of structural variations (SVs) from next generation sequencing data remains a major challenge. We develop FusorSV, which uses a data mining approach to assess performance and merge callsets from an ensemble of SV-calling algorithms. It includes a fusion model built using analysis of 27 deep-coverage human genomes from the 1000 Genomes Project. We identify 843 novel SV calls that were not reported by the 1000 Genomes Project for these 27 samples. Experimental validation of a subset of these calls yields a validation rate of 86.7%. FusorSV is available at https://github.com/TheJacksonLaboratory/SVE .
Assuntos
Algoritmos , Genoma Humano , Variação Estrutural do Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , SoftwareRESUMO
A series of beta-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogues led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide (6), a potent, orally active DPP-4 inhibitor (IC(50) = 6.3 nM) with excellent selectivity, oral bioavailability in preclinical species, and in vivo efficacy in animal models. Compound 6 was selected for further characterization as a potential new treatment for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Hipoglicemiantes/síntese química , Fenilalanina/análogos & derivados , Inibidores de Proteases/síntese química , Triazóis/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , Cristalografia por Raios X , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Proteínas Musculares/antagonistas & inibidores , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Coelhos , Canais de Sódio , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologiaRESUMO
Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.
Assuntos
Antivirais/síntese química , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Ribonucleosídeos/síntese química , Adenosina Desaminase/química , Administração Oral , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Linhagem Celular , Estabilidade de Medicamentos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação , Purina-Núcleosídeo Fosforilase/química , RNA Viral/biossíntese , Ratos , Ribonucleosídeos/química , Ribonucleosídeos/farmacocinética , Relação Estrutura-AtividadeRESUMO
We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.
RESUMO
We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.
RESUMO
Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.
Assuntos
Bioquímica/métodos , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Análise de Sequência/métodos , Acilação , Colorimetria , Formiatos/metabolismo , Hidrolases/metabolismo , Isotiocianatos/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Piroglutamil-Peptidase I/metabolismo , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/isolamento & purificação , Soluções , Anidridos Succínicos/metabolismoRESUMO
Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.
Assuntos
Benzimidazóis/metabolismo , Inibidores Enzimáticos/metabolismo , Janus Quinase 3/química , Piridonas/metabolismo , Pirimidinas/metabolismo , Pirróis/metabolismo , TYK2 Quinase/química , Sítios de Ligação , Calorimetria , Humanos , Janus Quinase 3/metabolismo , Cinética , Modelos Moleculares , Piperidinas , Ligação Proteica , Estrutura Terciária de Proteína , TYK2 Quinase/metabolismoRESUMO
Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.
Assuntos
Proteínas ADAM/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/isolamento & purificação , Catálise , Domínio Catalítico , Ativação Enzimática , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de ProteínaRESUMO
OBJECTIVE: Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala(271)/Val(272) scissile bond, generating an approximately 30-kd fragment with the new C-terminus VRAA(271) and an approximately 50-85-kd fragment with the new N-terminus (272)VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. METHODS: Fibronectin-degrading activity in human OA chondrocyte-conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond (269)RAA downward arrowVal(272), and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA(271) and (272)VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. RESULTS: Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. CONCLUSION: The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA(271) and (272)VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.
Assuntos
Proteínas ADAM/metabolismo , Proteínas ADAM/farmacologia , Alanina/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Fibronectinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Osteoartrite do Joelho/metabolismo , Idoso de 80 Anos ou mais , Células Cultivadas , Condrócitos/patologia , Meios de Cultivo Condicionados/farmacologia , Epitopos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Serina Endopeptidases/metabolismoRESUMO
The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.
Assuntos
Amidas/metabolismo , Amidas/uso terapêutico , Antivirais/metabolismo , Citidina/análogos & derivados , Hepatite C/tratamento farmacológico , Ácidos Fosfóricos/metabolismo , Ácidos Fosfóricos/uso terapêutico , Pró-Fármacos/metabolismo , Pró-Fármacos/uso terapêutico , Amidas/farmacologia , Amidas/toxicidade , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/toxicidade , Linhagem Celular , Citidina/metabolismo , Citidina/farmacologia , Citidina/uso terapêutico , Citidina/toxicidade , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Ácidos Fosfóricos/farmacologia , Ácidos Fosfóricos/toxicidade , Polifosfatos/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/toxicidade , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.