RESUMEN
The zinc- and calcium-dependent family of proteins called the MMPs (matrix metalloproteases) are collectively responsible for the degradation of the extracellular matrix. The enzymes are synthesized as zymogens, and under physiological conditions are selectively regulated by endogenous inhibitors. An imbalance between the active enzymes and their natural inhibitors leads to the accelerated destruction of connective tissue associated with the pathology of diseases such as arthritis, cancer, multiple sclerosis and cardiovascular diseases. The potential for using specific enzyme inhibitors as therapeutic agents to redress this balance has led to intensive research focused on the design, synthesis and molecular deciphering of low-molecular-mass inhibitors of this family of proteins. The design of early MMP inhibitors was based on the scissile site sequence of peptide substrates, with moieties customized to chelate the critical zinc ion at the enzymes' active site. These initial efforts were supported by X-ray and NMR data on MMP complexes, exploiting sequence and structural differences in the principal specificity pocket of the enzymes, leading to subtype-selective MMP inhibitors. This review will provide a critical appraisal of the design principles that have been utilized in generating molecules that inhibit MMPs, and explore issues relevant to obtaining clinical efficacy of MMP inhibitor-based therapies.
Asunto(s)
Diseño de Fármacos , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Animales , Simulación por Computador , Humanos , Metaloproteinasas de la Matriz/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
This review presents a summary of current approaches to extract functional information from structural data on proteins and their complexes. While structural homologs may reveal possible biochemical functions (which may be hidden at the sequence level), elucidating the exact biological role of a protein in vivo will only be possible by including other results, such as data on expression and localization.
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Bases de Datos como Asunto , Diseño de Fármacos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas/clasificación , Proteínas/genética , Relación Estructura-ActividadRESUMEN
The zinc- and calcium-dependent family of proteins called the matrix metalloproteinases are collectively responsible for the degradation of the extracellular matrix. Members of this family such as the collagenases, stromelysins and the gelatinases are involved in the routine tissue remodelling processes such as wound healing, embryonic growth and angiogenesis. Under normal circumstances the proteolytic activity of these proteins are precisely controlled at the transcriptional level, the production of the proteins in their inactive zymogen forms and also by the co-secretion of endogenous inhibitors. Imbalance between the active enzymes and their natural inhibitors leads to the accelerated destruction of connective tissue associated with the pathology of diseases such as rheumatoid and osteoarthritis. The potential for using specific enzyme inhibitors as therapeutic agents to redress this balance has led to intensive research focused on the design, syntheses and molecular structural analyses of low molecular weight inhibitors of this family of proteins. This review describes the essential structural principles and molecular interactions implicated in the innovation of matrix metalloproteinase inhibitors and discusses the features necessary for the specific inhibition of the collagenases.
Asunto(s)
Metaloproteinasas de la Matriz/química , Dominio Catalítico , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Conformación Proteica , Zinc/metabolismoRESUMEN
Over the last decade, the potential utility of matrix metalloproteinase (MMP) inhibitors as novel therapeutic agents for diseases such as arthritis, cancer and multiple sclerosis has lead to an abundance of three-dimensional structural information on this family of enzymes. This review describes the spatial features of the conserved three-dimensional scaffold of the MMPs, discusses enzyme-inhibitor interactions and relates these data to the characteristics of individual MMPs which influence the potency and selectivity of inhibitors.
RESUMEN
The family of proteins called matrix metalloproteinases (MMPs) are a class of structurally related proteins that are collectively responsible for the metabolism of extracellular matrix proteins. These zinc and calcium dependent enzymes, which include the collagenases, stromelysins and gelatinases, are involved in normal tissue remodelling processes such as wound healing, pregnancy and angiogenesis. Under physiological conditions, in addition to the regulated proteolyses of inactive precursors to the active form, the degradative nature of these enzymes are precisely controlled by endogenous inhibitors (TIMPs). The excess syntheses and production of these proteins lead to the accelerated matrix degradation associated with diseases such as arthritis, cancer and multiple sclerosis. The MMPs have therefore proved to be attractive targets for structure based drug design. The pursuit of low molecular weight inhibitors of these proteins have encouraged structural studies on several members of family, so that the molecular details of enzyme-inhibitor interactions of the MMPs have become available. These studies provide insights into the basic structural framework of the MMP superfamily and reveal characteristics which promote specificity between individual members. The analyses of the three dimensional structure of the MMPs in the context of their primary sequence and the design and selectivity of low molecular weight inhibitors of the superfamily is the subject of this review.
Asunto(s)
Matriz Extracelular/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Secuencia de Consenso , Femenino , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neovascularización Fisiológica , Embarazo , Estructura Secundaria de Proteína , Alineación de Secuencia , Cicatrización de HeridasRESUMEN
The herpes simplex virus VP16 protein functions as a potent transcriptional activator and targets DNA sites with the consensus TAATGARAT present in all the viral immediate-early gene promoters. To do so, VP16 directs assembly of a multiprotein complex involving two cellular proteins, host cell factor (HCF) and the Oct-1 DNA-binding transcription factor. To investigate the importance of specific protein-protein interactions to formation of this VP16-induced complex (VIC), we used oligopeptides to prevent VIC assembly. Linear and cyclic peptides corresponding to a region of VP16 previously implicated in complex formation were potent inhibitors of VIC assembly. To further characterize the protein interactions involved, we cloned a human cDNA encoding the minimal VP16 interaction domain of HCF, containing amino acids 1 to 380 [HCF (1-380)]. The REHAYS-based peptides active in preventing VIC assembly were found to specifically block binding of VP16 to HCF (1-380), without affecting VP16-Oct-1 binding. The inhibitory activity of these VP16 peptides was strictly sequence specific for the EHAY residues. Site-directed mutagenesis of the HCF (1-380) domain revealed residues E102 and K105 to be critical determinants in support of VIC formation. Alteration of a single residue in HCF, K105, was shown to virtually abolish complex assembly. Interestingly however, none of the HCF mutants that were impaired in their ability to support complex formation exhibited defects in direct VP16 binding, supporting loss of function at a higher order in complex assembly.
Asunto(s)
Proteínas de Unión al ADN , Proteína Vmw65 de Virus del Herpes Simple/fisiología , Proteínas/fisiología , Secuencia de Aminoácidos , Proteínas de Homeodominio/metabolismo , Factor C1 de la Célula Huésped , Humanos , Datos de Secuencia Molecular , Mutación , Factor 1 de Transcripción de Unión a Octámeros , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
N-terminal analysis of aggrecan fragments lost from bovine nasal cartilage cultured in the presence of recombinant human interleukin 1alpha revealed a predominant ARGSVIL sequence with an additional ADLEX sequence. Production of the ARGSVIL-containing fragments has been attributed to the action of a putative proteinase, aggrecanase. The minor sequence (ADLEX) corresponds to a new reported cleavage product; comparison of this sequence with the available partial sequence of bovine aggrecan indicates that this is the product of a cleavage occurring towards the C-terminus of the protein. Matrix metalloproteinase (MMP) inhibitors inhibited aggrecan loss from bovine nasal explants incubated in the presence of recombinant human interleukin 1alpha. A strong correlation between inhibition of aggrecan metabolism and inhibition of stromelysin 1 (MMP 3) (r=0.93) suggests a role for stromelysin or a stromelysin-like enzyme in cartilage aggrecan metabolism. However, the compounds were approx. 1/1000 as potent in inhibiting aggrecan loss from the cartilage explants as they were in inhibiting stromelysin. There was little or no correlation between inhibition of aggrecan metabolism and inhibition of gelatinase B (MMP 9) or inhibition of collagenase 1 (MMP 1). Studies with collagenase inhibitors with a range of potencies showed a correlation between inhibition of collagenase activity and inhibition of collagen degradation in the cartilage explant assay. This indicates that in interleukin 1alpha-driven bovine nasal cartilage destruction, stromelysin (or a closely related enzyme) is involved in aggrecan metabolism, whereas collagenase is principally responsible for collagen degradation.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas de la Matriz Extracelular , Metaloendopeptidasas/antagonistas & inhibidores , Tabique Nasal/metabolismo , Proteoglicanos/metabolismo , Agrecanos , Animales , Bovinos , Proteoglicanos Tipo Condroitín Sulfato/química , Colágeno/metabolismo , Humanos , Interleucina-1/farmacología , Lectinas Tipo C , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Mapeo Peptídico , Proteoglicanos/químicaRESUMEN
It is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X-ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O-His-O "catalytic triad" and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions.
Asunto(s)
Algoritmos , Sitios de Unión , Enzimas/química , Conformación Proteica , Bases de Datos Factuales , Endopeptidasas/química , Esterasas/químicaRESUMEN
Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits proinflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu4-Leu5-Arg6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe17, Phe2l, Ile22, and Leu43. These two receptor-binding regions are separated by over 20 A in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-8/metabolismo , Neutrófilos/fisiología , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calcio/sangre , Membrana Celular/inmunología , Clonación Molecular , Epítopos/análisis , Epítopos/metabolismo , Humanos , Interleucina-8/biosíntesis , Interleucina-8/química , Interleucina-8/genética , Isoleucina , Leucina , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neutrófilos/inmunología , Fenilalanina , Estructura Secundaria de Proteína , Receptores de Interleucina-8A , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
In rheumatoid and osteoarthritis, degradation of articular cartilage is mediated by the matrix metalloproteinases collagenase, stromelysin and gelatinase. The key event in this process is the cleavage of triple helical collagen by collagenase. We have determined the crystal structure of the catalytic domain of human recombinant fibroblast collagenase complexed with a synthetic inhibitor at 2.2 A resolution. The protein fold is similar to the amino termini of the zinc endopeptidases astacin thermolysin and elastase despite a lack of primary sequence homology. The conformation of the bound inhibitor provides a molecular basis for the design of inhibitors of collagenase and other matrix metalloproteinases. Such inhibitors should be useful in the treatment of a variety of diseases including arthritis and cancer.
Asunto(s)
Colagenasas/química , Inhibidores de la Metaloproteinasa de la Matriz , Secuencia de Aminoácidos , Artritis Reumatoide/metabolismo , Sitios de Unión , Cartílago/metabolismo , Catálisis , Colágeno/química , Colágeno/metabolismo , Colagenasas/genética , Fibroblastos/enzimología , Humanos , Técnicas In Vitro , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Osteoartritis/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Zinc/químicaRESUMEN
The hydrogen bonding of bovine ribonuclease A derived from the high resolution X-ray structure has been studied in detail. Correlations have been examined for main-chain-main-chain hydrogen bond angles, torsion angles and distances, respectively. Differences are found consistently for correlations associated with alpha-helix and beta-sheet, respectively. Ten of the 124 side-chains have four or more hydrogen bond contacts; two, including Glu-101, have five or more. Three potential C = O---H, three N---X and three potential side-chain H-bonds fail to form. A search for highly inaccessible buried residues resulted in nine outstanding examples, all of which are conserved across 38 known mammalian ribonuclease A sequences, indicating the importance of these residues for structural stability. Of the two histidines in the active site, His-12 has five hydrogen bonds and His-119 three. The conformational space accessible to these two catalytically important residues studied by means of simple non-bonded contact energy calculations confirms the existence of two alternative, interchangeable locations for His-119, while His-12 is locked in a local energy minimum.
Asunto(s)
Ribonucleasa Pancreática , Sitios de Unión , Cristalografía , Histidina , Enlace de Hidrógeno , Oxígeno , Conformación Proteica , Agua , Rayos XRESUMEN
The alpha-helix defined in 1951 by Pauling et al. on the basis of model building and X-ray fibre diffraction data has 3.65 residues per turn (n) achieved with planar peptides, torsion angles of phi = -48 degrees and psi = -57 degrees and hydrogen bonds which are close to linear. Although X-ray analyses of proteins have confirmed the general correctness of the model for the helix, recent high resolution (1.7-1.0 A) diffraction studies have shown that the parameters described by Pauling et al. and later by Perutz and Arnott and Wonacott are not a good description of the alpha-helices in globular proteins, where the mean values of phi, psi are usually close to -63 degrees, -42 degrees. Here we show that these values arise as a mean of two significantly different classes in amphipathic helices depending on whether the peptide carbonyl oxygen is hydrogen bonded to a solvent or polar side-chain atom. The hydrogen bonds made by the hydrophilic carbonyls to the NH groups within helices are longer and less linear than those involving hydrophobic carbonyls. We also show that these effects are associated with a significant curvature of helices in globular proteins. For example, the alpha-helix in avian pancreatic peptide (aPP) has a radius of curvature of approximately 70 A. These results are of significance in the packing of helices in fibrous and globular proteins, in the calculation of their dipole moments, solvent accessibilities and internal energies, and in the theoretical estimation of spectroscopic properties such as circular dichroism and Raman scattering.
Asunto(s)
Polipéptido Pancreático , Conformación Proteica , SolventesRESUMEN
The substrate specificity of pancreatic ribonuclease A is discussed in light of observations based on accurate X-ray structure analysis of several enzyme-nucleotide complexes. A hypothesis for protein-nucleic acid recognition is presented which proposes that: (a) pyrimidine bases in RNA are recognised by ribonuclease due to the charge complementarity of two groups (the amide nitrogen and the side chain oxygen (OG) of threonine 45) of the protein and relevant atoms in the heterocyclic base (O2 and N3 in pyrimidine nucleotides); (b) interaction of the protein with the ribose moiety of the nucleotides is non-specific; and (c) conformational flexibility in the region of the scissile P-O bond is provided by different locations of the phosphoryl oxygens, rather than by an overall translation of the phosphate moiety.
Asunto(s)
ARN/metabolismo , Ribonucleasa Pancreática/metabolismo , Animales , Bovinos , Hidrólisis , Conformación de Ácido Nucleico , Páncreas/enzimología , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
The modified purine nucleotide 8-oxo-guanosine-2'-phosphate binds at the pyrimidine binding site of ribonuclease-A. The O8-2'GMP inhibitor is in a syn conformation, with an intramolecular hydrogen bond between the N-3 atom of the base and the O-5' atom of the ribose. The essential groups of the protein involved in base recognition are O gamma 45 and N-45, which form hydrogen bonds to the five-membered ring of the heterocyclic base. Mobility of enzyme side-chains (viz. Lys41, Lys66, His119) close to the catalytic cleft of the protein allows conformational flexibility in the substrate binding region of ribonuclease-A. Inhibitor binding alters the solvent structure of the protein but the overall shape of the enzyme is not effected.
Asunto(s)
Nucleótidos de Guanina , Guanosina Monofosfato , Ribonucleasa Pancreática , Animales , Sitios de Unión , Bovinos , Guanosina Monofosfato/análogos & derivados , Sustancias Macromoleculares , Conformación Molecular , Páncreas/enzimología , Conformación Proteica , Ribonucleasa Pancreática/antagonistas & inhibidores , Difracción de Rayos XRESUMEN
The results of X-ray analyses of three ribonuclease-A-nucleotide complexes, at 2.3 A, are reported. A modified purine mononucleotide, 8-oxo-guanosine 2'-phosphate in a syn conformation, binds at the pyrimidine-binding site of the catalytic cleft. Solvent molecules are expelled from the active site due to inhibitor binding. The positions of the side-chains of the active-site residues Gln-11, His-12 and Thr-45 are well defined and do not alter on inhibitor binding. The mobility of Lys-41 is greatly reduced in the protein-nucleotide complexes and the terminal amino group interacts directly with the 2'-phosphate group of the nucleotides. In the complex of the enzyme with a modified pyrimidine, cytidine-N(3)-oxide 2'-phosphate, His-119 is stabilised in the minor site of the native protein [Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38, 2210-2217], while in the protein-purine derivative the imidazole group is located in the major site. Inhibitor binding induces movements in the side-chains of Lys-7 and Lys-66 which also modify the conformation of the active-site cleft of ribonuclease A.