RESUMO
The full three-dimensional structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent, sulfonamide hydroxamic acid inhibitor (CGS 27023) has been determined by NMR spectroscopy. The results reveal a core domain for the protein consisting of three alpha-helices and five beta-sheet strands with an overall tertiary fold similar to the catalytic domains of other matrix metalloproteinase family members. The S1' pocket, which is the major site of hydrophobic binding interaction, was found to be a wide cleft spanning the length of the protein and presenting facile opportunity for inhibitor extension deep into the pocket. Comparison with the reported X-ray structure of collagenase-3 showed evidence of flexibility for the loop region flanking the S1' pocket in both NMR and X-ray data. This flexibility was corroborated by NMR dynamics studies. Inhibitor binding placed the methoxy phenyl ring in the S1' pocket with the remainder of the molecule primarily solvent-exposed. The binding mode for this inhibitor was found to be similar with respect to stromelysin-1 and collagenase-1; however, subtle comparative differences in the interactions between inhibitor and enzyme were observed for the three MMPs that were consistent with their respective binding potencies.
Assuntos
Colagenases/química , Inibidores Enzimáticos/química , Ácidos Hidroxâmicos/química , Metaloproteinase 1 da Matriz/química , Metaloproteinase 3 da Matriz/química , Sulfonamidas/química , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Espectroscopia de Ressonância Magnética , Metaloproteinase 13 da Matriz , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
The tertiary structure of a unique C5a receptor antagonist was determined by two-dimensional NMR spectroscopy. The core domain of this 8-kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)-C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C-terminal D-Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D-Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.
Assuntos
Antígenos CD/fisiologia , Complemento C5a/metabolismo , Receptores de Complemento/antagonistas & inibidores , Receptores de Complemento/fisiologia , Sequência de Aminoácidos , Antígenos CD/metabolismo , Complemento C5a/genética , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Receptor da Anafilatoxina C5a , Receptores de Complemento/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes involved in the degradation and remodeling of extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMP activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. Thus, MMP inhibitors are expected to be useful for the treatment of these disorders. This article reviews briefly the biochemistry of MMPs and evidence for their pathogenic roles using molecular biology approaches. Biomolecular structures used in the design of MMP inhibitors are thoroughly covered. Major emphasis is on recently published potent, small molecular weight MMP inhibitors and their pharmacological properties. Finally, available clinical results of compounds in development are summarized.
Assuntos
Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases , Sequência de Aminoácidos , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/uso terapêutico , Conformação ProteicaRESUMO
A novel inhibitor of reverse transcriptase was studied by solid-state NMR. Three phases of the compound were examined which included the dihydrate and two anhydrous polymorphs (Form I and Form III). By correlating (1)H and (13)C solution NMR with the solid-state (13)C NMR CP/MAS and CPPI spectral editing experiments, comparative (13)C assignments were made for each phase. Polymorphs of Form I and Form III and the dihydrate were easily distinguished based upon chemical shift patterns of the carbon resonances. The (1)H spin-lattice relaxation times were also measured for each phase which provided information on the mobility and relative crystallinity. The (13)C ssNMR spectrum of Form I showed the presence of a minor component identified as the dihydrate. Weight/percent quantitation of major and minor components in Form I was obtained from integrated intensities of a 50:50 mixture containing weighed amounts of Form I and the pure dihydrate. Comparison of the ssNMR and X-ray powder diffraction techniques is discussed.
Assuntos
Azepinas/química , Espectroscopia de Ressonância Magnética , Piridinas/química , Inibidores da Transcriptase Reversa/química , Tecnologia Farmacêutica/métodos , Cristalização , Cristalografia por Raios X , Estrutura Molecular , Difração de Pó , Água/químicaRESUMO
Glucose metabolism of human red blood cells was investigated using carbon-13 nuclear magnetic resonance spectroscopy under both oxygenated and nonoxygenated conditions. These results show that under oxygenated conditions reversal of 3-phosphoglyceraldehyde to glucose is in competition with its catabolism to lactate.
Assuntos
Glicemia/metabolismo , Eritrócitos/metabolismo , Espectroscopia de Ressonância Magnética , Humanos , Técnicas In Vitro , Oxigênio/fisiologiaRESUMO
31P NMR spectroscopy was used to monitor the cardiac energy metabolism in hypothyroid rat hearts. Differential alterations in phosphocreatine and inorganic phosphate levels were observed upon treatment of hypothyroid animals with DT4 and LT4, while both agents were equipotent in reducing cholesterol. These results show potential for NMR spectroscopy as a technique to determine therapeutic selectivity.
Assuntos
Anticolesterolemiantes/uso terapêutico , Dextrotireoxina/uso terapêutico , Coração/efeitos dos fármacos , Hipotireoidismo/tratamento farmacológico , Miocárdio/metabolismo , Fosfatos/metabolismo , Tiroxina/uso terapêutico , Trifosfato de Adenosina/análise , Animais , Colesterol/sangue , Metabolismo Energético , Hipotireoidismo/diagnóstico , Espectroscopia de Ressonância Magnética , Masculino , Fosfocreatina/análise , Ratos , Ratos EndogâmicosRESUMO
The full three-dimensional structure of the catalytic domain of human stromelysin-1 (SCD) complexed to a novel and potent, nonpeptidic inhibitor has been determined by nuclear magnetic resonance spectroscopy (NMR). To accurately mimic assay conditions, the structure was obtained in Tris buffer at pH 6.8 and without the presence of organic solvent. The results showed that the major site of enzyme-inhibitor interaction occurs in the S1' pocket whereas portions of the inhibitor that occupy the shallow S2' and S1 pockets remained primarily solvent exposed. Because this relatively small inhibitor could not deeply penetrate stromelysin's long narrow hydrophobic S1' pocket, the enzyme was found to adopt a dramatic fold in the loop region spanning residues 221-231, allowing occupation of the solvent-accessible S1' channel by the enzyme itself. This remarkable conformational fold at the enzyme binding site resulted in constriction of the S1' loop region about the inhibitor. Examination of the tertiary structure of the stromelysin-inhibitor complex revealed few hydrogen-bonding or hydrophobic interactions between the inhibitor and enzyme that can contribute to overall binding energy; hence the resultant compact structure may in part account for the relatively high potency exhibited by this inhibitor.
Assuntos
Domínio Catalítico , Inibidores Enzimáticos/química , Ácidos Hidroxâmicos/química , Metaloproteinase 3 da Matriz/química , Inibidores de Metaloproteinases de Matriz , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Substâncias Macromoleculares , Metaloproteinase 3 da Matriz/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
We have developed a 1H NMR technique to selectively edit the spectrum of perfused liver for specific resonances of metabolites that occur in low concentration. The method employs selective DANTE pulses, which avoid exciting the water signal and at the same time control the J modulation effect in the homonuclear spin-echo experiment. By difference spectroscopy, we have suppressed the background signals from lipids and water and have resolved the CH3 resonance of lactate at 1.33 ppm. Moreover, the technique is highly selective and allows us to select the CH3 resonance of alanine at 1.47 ppm in the presence of the CH3 resonance of lactate at 1.33 ppm, even though the latter was much larger before editing. We have applied this technique to study the metabolic effect of ethanol in perfused mouse liver and have observed that the rate of formation of lactate from pyruvate is increased by a factor of 2.8 when ethanol is added.
Assuntos
Fígado/metabolismo , Animais , Etanol/metabolismo , Cinética , Lactatos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Masculino , Camundongos , Perfusão , Piruvatos/metabolismo , Ácido PirúvicoRESUMO
15N NMR spectra were taken of 15N-enriched tRNA extracted from bakers yeast; ammonium sulfate was used as a nitrogen source. The increase in the degree of denaturation of tRNA, which occurs with increase in temperature from 30 degrees C to 70 degrees C, resulted in no large changes in 15N chemical shifts at acidic and neutral pH but quite pronounced changes in proton-15N nuclear Overhauser effects.
Assuntos
RNA de Transferência , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , RNA Fúngico , Saccharomyces cerevisiaeRESUMO
The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1' pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2' and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.
Assuntos
Ácidos Hidroxâmicos , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/química , Pirazinas , Sítios de Ligação , Colagenases/metabolismo , Simulação por Computador , Fibroblastos/enzimologia , Humanos , Espectroscopia de Ressonância Magnética , Metaloproteinase 3 da Matriz/metabolismo , Modelos Moleculares , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica , SulfonamidasRESUMO
The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an alpha-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel alpha-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor.
Assuntos
Complemento C5a/química , Espectroscopia de Ressonância Magnética/métodos , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
The progression of carbon tetrachloride-induced liver damage, determined by 31P NMR spectroscopy, was compared with selected serum enzyme and histological changes in rats. ATP levels declined as early as 8 h post-CCl4 administration, with partial recovery observed at 168 h. The results show that ATP reduction correlates with necrosis. In addition, early decline in ATP occurring prior to significant hepatocellular necrosis indicates abnormal energy metabolism.
Assuntos
Trifosfato de Adenosina/sangue , Fígado/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Alanina Transaminase/sangue , Animais , Argininossuccinato Liase/sangue , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono/toxicidade , Fígado/patologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
The functions of the C5a anaphylatoxin are expressed through its interaction with a cell-surface receptor with seven transmembrane helices. The interaction of C5a with the receptor has been explained by a two-site model whereby recognition and effector sites on C5a bind, respectively, to recognition and effector domains on the receptor, leading to receptor activation (Chenoweth, D. E., and Hugli, T. E. (1980) Mol. Immunol. 17, 151-161. In addition, the extracellular N-terminal region of the C5a receptor has been implicated as the recognition domain for C5a, responsible for approximately 50% of the binding energy of the C5a-receptor complex (Mery, L., and Boulay, F. (1994) J. Biol. Chem. 269, 3457-3463; DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol. Chem. 269, 14446-14450). In this work, the interactions of C5a with the N-terminal domain of the C5a receptor were examined by use of recombinant human C5a molecules and peptide fragments M1NSFN5YTTPD10YGHYD15DKDTL20DLNTP25VDKTS30NTLR(hC5aRF-1-34), acetyl-HYD15DKDTL20DLNTP25VDKTS30NTLR (hC5aRF-13-34), and acetyl-TL20DLNTP25VDKTS30N-amide (hC5aRF-19-31) derived from human C5a receptor. Binding induced resonance perturbations in the NMR spectra of the receptor fragments and the C5a molecules indicated that the isolated Nterminal domain or residues 1-34 of the C5a receptor retain specific binding to C5a and to a C5a analog devoid of the agonistic C-terminal tail in the intact C5a. Residues of C5a perturbed by the binding of the receptor peptides are localized within the helical core of the C5a structure, in agreement with the results from functional studies employing mutated C5a and intact receptor molecules. All three receptor peptides, hC5aRF-1-34, hC5aRF-13-34, and hC5aRF-19-31, responded to the binding of C5a through the 21-30 region containing either hydrophobic, polar, or positively charged residues such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30. The 21-30 segment of all three receptor fragments was found to have a partially folded conformation in solution, independent of residues 1-18. These results indicate that a short peptide sequence, or residues 21-30, of the C5a receptor N terminus may constitute the binding domain for the recognition site on C5a.
Assuntos
Anafilatoxinas/metabolismo , Antígenos CD/metabolismo , Complemento C5a/metabolismo , Receptores de Complemento/metabolismo , Sequência de Aminoácidos , Antígenos CD/química , Antígenos CD/genética , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Receptor da Anafilatoxina C5a , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The conformations of cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-) in dimethyl sulfoxide-d6 (DMSO-d6) and water were determined using two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Comparisons were made between conformations of the cyclic pentapeptide in both solvents. The NMR study revealed that, while the backbone remained relatively unchanged in both solvents, the side-chains adopted distinctly different orientations in DMSO-d6 vs. H2O. A modeling study, minus NOE constraints, produced a set of low-energy conformers possessing agreement in backbone conformation with the NMR-derived structures; however, lowest-energy conformers did not have this agreement. These results show that different solvents can significantly affect the preferred side-chain conformation of small cyclic peptides in solution. This finding will impact the selection of solvent when determining structures for use as templates in rational drug design.
Assuntos
Dimetil Sulfóxido/química , Modelos Moleculares , Peptídeos Cíclicos/química , Conformação Proteica , Água/química , Sequência de Aminoácidos , Fenômenos Químicos , Físico-Química , Antagonistas dos Receptores de Endotelina , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Soluções , Solventes/química , TermodinâmicaRESUMO
The transferred nuclear Overhauser effect has been used to determine the biologically active conformations of two stromelysin inhibitors. Both inhibitors used in this study were hydroxamic acids generated via chemical synthesis. These structures, representing the conformation of each inhibitor bound to stromelysin, superimposed with excellent agreement. The study also provided information on the shape and orientation of the S2' and S1' pockets of the enzyme relative to thermolysin. Comparisons were made between stromelysin and thermolysin inhibitors to critically examine thermolysin as a template for stromelysin-inhibitor design. The enzyme-bound conformations of these stromelysin inhibitors were determined for use as a template in conformationally restricted drug design.