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1.
J Pharmacol Exp Ther ; 367(1): 147-154, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30076263

RESUMO

Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.


Assuntos
Aminopiridinas/farmacologia , Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Disponibilidade Biológica , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley
2.
J Biol Chem ; 289(48): 33456-68, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25301950

RESUMO

HCV infection is an urgent global health problem that has triggered a drive to discover therapies that specifically target the virus. BMS-791325 is a novel direct antiviral agent specifically targeting HCV NS5B, an RNA-dependent RNA polymerase. Robust viral clearance of HCV was observed in infected patients treated with BMS-791325 in combination with other anti-HCV agents in Phase 2 clinical studies. Biochemical and biophysical studies revealed that BMS-791325 is a time-dependent, non-competitive inhibitor of the polymerase. Binding studies with NS5B genetic variants (WT, L30S, and P495L) exposed a two-step, slow binding mechanism, but details of the binding mechanism differed for each of the polymerase variants. For the clinically relevant resistance variant (P495L), the rate of initial complex formation and dissociation is similar to WT, but the kinetics of the second step is significantly faster, showing that this variant impacts the final tight complex. The resulting shortened residence time translates into the observed decrease in inhibitor potency. The L30S variant has a significantly different profile. The rate of initial complex formation and dissociation is 7-10 times faster for the L30S variant compared with WT; however, the forward and reverse rates to form the final complex are not significantly different. The impact of the L30S variant on the inhibition profile and binding kinetics of BMS-791325 provides experimental evidence for the dynamic interaction of fingers and thumb domains in an environment that supports the formation of active replication complexes and the initiation of RNA synthesis.


Assuntos
Antivirais/química , Benzazepinas/química , Hepacivirus/enzimologia , Indóis/química , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Antivirais/farmacologia , Benzazepinas/uso terapêutico , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Humanos , Indóis/uso terapêutico , Mutação de Sentido Incorreto , Ligação Proteica , RNA Replicase/química , RNA Replicase/metabolismo , RNA Viral/biossíntese , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
3.
Bioorg Med Chem Lett ; 23(6): 1622-5, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23416006

RESUMO

Synthesis and structure-activity relationship of a series of substituted piperidinyl glycine 2-cyano-4,5-methano pyrroline DPP-IV inhibitors are described. Improvement of the inhibitory activity and chemical stability of this series of compounds was respectively achieved by the introduction of bulky groups at the 4-position and 1-position of the piperidinyl glycine, leading to a series of potent and stable DPP-IV inhibitors.


Assuntos
Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Piperidinas/química , Pirrolidinas/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/síntese química , Inibidores da Dipeptidil Peptidase IV/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Relação Estrutura-Atividade , Temperatura
4.
BMC Pharmacol ; 12: 2, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22475049

RESUMO

BACKGROUND: Dipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the first phase of GLP-1 release. RESULTS: Saxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C). In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C. Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a large panel of other proteases, and have similar potency and efficacy across multiple species.Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4 inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume of addition and final concentration of substrate in these assays can change measured inhibition. We show that unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that substrate concentration was important for rapidly dissociating DPP4 inhibitors. CONCLUSIONS: Saxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid increases in substrates in vivo.


Assuntos
Adamantano/análogos & derivados , Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/metabolismo , Hipoglicemiantes/metabolismo , Adamantano/metabolismo , Algoritmos , Animais , Artefatos , Clonagem Molecular , Dipeptidases/metabolismo , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Indicadores e Reagentes , Cinética , Macaca fascicularis , Nitrilos/metabolismo , Ligação Proteica , Pirazinas/metabolismo , Pirrolidinas/metabolismo , Fosfato de Sitagliptina , Especificidade da Espécie , Triazóis/metabolismo , Vildagliptina
5.
Bioorg Med Chem Lett ; 21(22): 6909-15, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21974952
6.
Bioorg Med Chem Lett ; 21(22): 6916-24, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21782431

RESUMO

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Lactamas/química , Lactamas/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Lactamas/síntese química , Lactamas/farmacocinética , Camundongos , Modelos Moleculares , Ratos , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 21(1): 537-41, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21078556

RESUMO

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.


Assuntos
Aminas/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Indóis/síntese química , Inibidores de Proteases/química , Piridinas/síntese química , Aminas/síntese química , Aminas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/sangue , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Indóis/química , Indóis/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , Piridinas/química , Piridinas/farmacologia , Ratos , Relação Estrutura-Atividade
8.
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
9.
Eur J Pharmacol ; 593(1-3): 10-5, 2008 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-18655784

RESUMO

In this report we describe a novel radioligand, [(3)H](S)-2-((S)-3-Acetylamino-3-sec-butyl-2-oxo-pyrrolidin-1-yl)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-methoxy-benzylamino)-propyl]-4-phenyl-butyramide ([(3)H]BMS-599240), that exhibits robust specific binding in homogenates from cell cultures overexpressing beta-site amyloid precursor protein cleaving enzyme-1 (BACE1). Radioligand binding exhibited high affinity, K(d)=2 nM, commensurate with its inhibitory potency against BACE1. Inhibition of radioligand binding in the presence of a range of different BACE1 inhibitors exhibited the same rank order of potency as for inhibition of BACE1 enzymatic activity. BACE1-dependent binding of the radioligand was also demonstrated in mouse brain homogenates, where genetic ablation of BACE1 eliminated high affinity binding. Thus, the radioligand [(3)H]BMS-599240 is a novel tool potentially useful for evaluation of BACE1 enzyme in biological samples, and for evaluation of inhibitor binding to BACE1.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Pirrolidinonas , Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/genética , Encéfalo/citologia , Células Cultivadas , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Cinética , Ligantes , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ensaio Radioligante
10.
J Pharmacol Exp Ther ; 326(2): 502-13, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18499745

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Abeta1-40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo , Inibidores Enzimáticos/farmacologia , Fragmentos de Peptídeos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Secretases da Proteína Precursora do Amiloide/fisiologia , Peptídeos beta-Amiloides/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/fisiologia , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Knockout , Estrutura Molecular , Fragmentos de Peptídeos/sangue , Ligação Proteica , Especificidade por Substrato
11.
Arch Biochem Biophys ; 475(1): 72-9, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18455495

RESUMO

The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than approximately 1000-fold, but activated recombinant human ACC1 only by approximately 4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules. Citrate activates ACCs at lower concentrations and inhibits at higher concentrations with apparent K(d) values of 0.8+/-0.3 and 3.4+/-0.6 mM, and apparent K(i) values of 20+/-8 and 38 +/-8 mM for ACC1 and ACC2, respectively. In the absence of added citrate, both ACC1 and ACC2 were inactivated by avidin rapidly and completely. Addition of 10 mM citrate protected ACC2 from avidin inactivation; however, protection by citrate was less pronounced for ACC1. In response to citrate treatment, different aggregation patterns for the two isoforms were also observed by dynamic light scattering. Although formation of aggregates by both isoforms was sensitive to citrate, with Mg2+ and Mg-citrate addition only formation of the ACC2 aggregates showed a dependence on citrate concentration. Mass spectrometry data indicated phosphorylation of Ser79 of ACC1 (a serine known to regulate activity), and the corresponding Ser221 of ACC2. Taken together, these data suggest that recombinant human ACC1 and ACC2 are differentially activated by citrate, most likely through conformational changes leading to aggregation, with ACC2 being more sensitive to this activator.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Ácido Cítrico/farmacologia , Acetil-CoA Carboxilase/genética , Animais , Baculoviridae/genética , Relação Dose-Resposta a Droga , Drosophila/citologia , Drosophila/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Luz , Fosforilação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Relação Estrutura-Atividade
12.
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 , Ligação de Hidrogênio , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína
13.
Bioorg Med Chem Lett ; 17(23): 6476-80, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17937986

RESUMO

The synthesis and structure-activity relationships of novel dipeptidyl peptidase IV inhibitors replacing the classical cyanopyrrolidine P1 group with other small nitrogen heterocycles are described. A unique potency enhancement was achieved with beta-branched natural and unnatural amino acids, particularly adamantylglycines, linked to a (2S,3R)-2,3-methanopyrrolidine based scaffold.


Assuntos
Dipeptídeos/química , Inibidores da Dipeptidil Peptidase IV , Inibidores da Dipeptidil Peptidase IV/química , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Nitrilos/química , Nitrilos/farmacologia , Relação Estrutura-Atividade
14.
Biochemistry ; 46(5): 1423-31, 2007 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-17260972

RESUMO

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.


Assuntos
Glucoquinase/metabolismo , Adenilil Imidodifosfato/metabolismo , Catálise , Glucose/metabolismo , Humanos , Cinética , Ligantes , Palmitoil Coenzima A/metabolismo , Ligação Proteica , Conformação Proteica
15.
Bioorg Med Chem Lett ; 16(6): 1731-4, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16376077

RESUMO

A series of seco-prolinenitrile-containing dipeptides were synthesized and assayed as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV, a promising new target for treatment of type 2 diabetes. The inhibitors described herein assess the minimum structural requirements at P1 for this enzyme, resulting in the identification of inhibitors with low nM potency.


Assuntos
Adenosina Desaminase/química , Dipeptídeos , Dipeptidil Peptidase 4/química , Inibidores Enzimáticos , Glicoproteínas/química , Nitrilos , Prolina/química , Compostos de Anilina/metabolismo , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Nitrilos/síntese química , Nitrilos/química , Nitrilos/farmacologia
16.
Arch Biochem Biophys ; 445(1): 9-18, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16364232

RESUMO

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.


Assuntos
Adamantano/análogos & derivados , Dipeptídeos/química , Dipeptidil Peptidase 4/química , Inibidores Enzimáticos/química , Adamantano/química , Catálise , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ressonância Magnética Nuclear Biomolecular , Solventes
17.
J Med Chem ; 48(15): 5025-37, 2005 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-16033281

RESUMO

Efforts to further elucidate structure-activity relationships (SAR) within our previously disclosed series of beta-quaternary amino acid linked l-cis-4,5-methanoprolinenitrile dipeptidyl peptidase IV (DPP-IV) inhibitors led to the investigation of vinyl substitution at the beta-position of alpha-cycloalkyl-substituted glycines. Despite poor systemic exposure, vinyl-substituted compounds showed extended duration of action in acute rat ex vivo plasma DPP-IV inhibition models. Oxygenated putative metabolites were prepared and were shown to exhibit the potency and extended duration of action of their precursors in efficacy models measuring glucose clearance in Zucker(fa/fa) rats. Extension of this approach to adamantylglycine-derived inhibitors led to the discovery of highly potent inhibitors, including hydroxyadamantyl compound BMS-477118 (saxagliptin), a highly efficacious, stable, and long-acting DPP-IV inhibitor, which is currently undergoing clinical trials for treatment of type 2 diabetes.


Assuntos
Adamantano/análogos & derivados , Adamantano/síntese química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptídeos/síntese química , Dipeptidil Peptidase 4/metabolismo , Glicina/análogos & derivados , Glicina/síntese química , Hipoglicemiantes/síntese química , Inibidores de Proteases/síntese química , Adamantano/farmacologia , Animais , Disponibilidade Biológica , Glicemia/análise , Diabetes Mellitus Tipo 2/fisiopatologia , Dipeptídeos/farmacologia , Teste de Tolerância a Glucose , Glicina/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Insulina/sangue , Masculino , Camundongos , Camundongos Obesos , Microssomos Hepáticos/metabolismo , Nitrilos/síntese química , Nitrilos/farmacologia , Prolina/análogos & derivados , Prolina/síntese química , Prolina/farmacologia , Inibidores de Proteases/farmacologia , Ratos , Ratos Zucker , Estereoisomerismo , Relação Estrutura-Atividade
18.
Bioorg Med Chem Lett ; 15(18): 3992-5, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16046120

RESUMO

Dipeptidyl peptidase IV (DPP4) is a multifunctional type II transmembrane serine peptidase which regulates various physiological processes, most notably plasma glucose homeostasis by cleaving peptide hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Inhibition of DPP4 is a potentially valuable therapy for type 2 diabetes. Synthesis and structure-activity relationships of a series of substituted diprolyl nitriles are described, leading to the identification of compound 1 with a measured DPP4 K(i) of 3.6 nM.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Nitrilos/farmacologia , Inibidores de Proteases/farmacologia , Sítios de Ligação , Ciclização , Dipeptidil Peptidase 4/química , Modelos Moleculares , Estrutura Molecular , Nitrilos/química , Inibidores de Proteases/química , Estrutura Terciária de Proteína
19.
Arch Biochem Biophys ; 436(2): 367-76, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15797249

RESUMO

Dipeptidyl peptidase-IV is a cell surface protease which plays an important role in glucose homeostasis through proteolytic inactivation of incretin hormones, primarily glucagon like peptide-1 (GLP-1). Substrate N-terminal amino acid (S2-S1) specificity is rather clearly defined, while no substantial information is available on the significance of amino acid interactions towards the C-terminus after the scissile bond (so called prime S1'-S4' or distant S5'-S28' sites). In the present study the increasing length of the peptide towards prime sites (S1'-S4') resulted in approximately 7-fold decrease in Km. Moreover, the Km for GLP-1 cleavage was comparable to that of an S2-S4' peptide, suggesting that few, if any, important enzyme-substrate interactions occur beyond the active site. Effect of substrate length on kcat was less obvious, but kcat/Km showed an increasing trend when His-Ala-pNA (representing the natural two N-terminal residues) was compared to GLP-1. To probe the impact of increasing substrate length on the free energy of activation (as has been suggested for elastase and chymotrypsin) we performed temperature studies. To adequately interpret thermodynamic data we sought to understand what steps limit the kcat expression. Steady-state parameters of the reactions catalyzed by serine proteases are composed of microscopic constants describing binding, acylation, and deacylation steps. Viscosity and pre-steady-state studies suggested that His-Ala-pNA cleavage is limited in the deacylation half-reaction, most likely the product release step. Thus, the free energy of activation, as calculated from the Eyring equation, is underestimated (at least for His-Ala-pNA) and the effect of substrate length on the acylation step (and transition-state stabilization) could not be unambiguously assessed.


Assuntos
Dipeptidil Peptidase 4/química , Sítios de Ligação , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Glucagon/química , Peptídeo 1 Semelhante ao Glucagon , Humanos , Hidrólise , Cinética , Modelos Químicos , Fragmentos de Peptídeos/química , Ligação Proteica , Precursores de Proteínas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , Temperatura , Termodinâmica , Fatores de Tempo
20.
Arch Biochem Biophys ; 410(2): 307-16, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12573291

RESUMO

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.


Assuntos
Ácido Aspártico Endopeptidases/química , Proteínas Recombinantes/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Células CHO , Catálise , Domínio Catalítico , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cricetinae , Relação Dose-Resposta a Droga , Drosophila , Endopeptidases , Escherichia coli/metabolismo , Glicosilação , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Luz , Bicamadas Lipídicas/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Fatores de Tempo
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