RESUMO
Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca(2+)-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) (Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy.
Assuntos
Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Animais , Antígenos de Superfície , Humanos , Neuroglia/metabolismo , Neurotransmissores/metabolismoAssuntos
Carboxipeptidases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Animais , Encéfalo/enzimologia , Lesões Encefálicas/prevenção & controle , Modelos Animais de Doenças , Glutamato Carboxipeptidase II , Ataque Isquêmico Transitório/tratamento farmacológico , Rim/enzimologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Compostos Organofosforados/farmacologia , Compostos Organofosforados/uso terapêuticoRESUMO
Aspartic endopeptidases (plasmepsins) have been implicated in the degradation of hemoglobin in the erythrocytic stage of infection by Plasmodium falciparum. To develop new targets for drug development, these enzymes have been isolated and cloned, expressed, and studied structurally and enzymatically. This study expands this approach to two other species of the malarial parasite, P. vivax and P. malariae. Expression of cloned genes from these species, utilizing methodology similar to that employed in the original reports on the enzymes from P. falciparum, has provided active enzymes for analysis by kinetic methods. We describe here studies of three enzymes, plasmepsin II from P. falciparum, and one plasmepsin from both P. vivax and P. malariae, utilizing oligopeptide substrates and low-molecular-weight inhibitors. These analyses provide new information on the properties of distinct regions of the active site cleft; such data can suggest strategies for drug design to inhibit these critical enzymes of the parasite.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Plasmodium malariae/enzimologia , Plasmodium vivax/enzimologia , Animais , Clonagem Molecular , Escherichia coli/genética , Cinética , Modelos Moleculares , Oligopeptídeos/metabolismo , Inibidores de Proteases , Proteínas Recombinantes/antagonistas & inibidores , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k(cat)/Km ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.
Assuntos
Catepsina D/metabolismo , Corantes Fluorescentes/síntese química , Oligopeptídeos/síntese química , Catepsina D/química , Corantes Fluorescentes/metabolismo , Humanos , Ligação de Hidrogênio , Fígado/enzimologia , Modelos Moleculares , Naftalenossulfonatos , Oligopeptídeos/metabolismo , Conformação Proteica , Espectrometria de Fluorescência/métodos , Especificidade por Substrato , p-Dimetilaminoazobenzeno/análogos & derivadosRESUMO
Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D-pepstatin A complex, a series of statine-containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P2t) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side-chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264-276). In addition, the effects of single side-chain modifications were often nonadditive. Structure-activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.
Assuntos
Aminoácidos/química , Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Inibidores de Proteases/farmacologia , Aminoácidos/metabolismo , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , TermodinâmicaRESUMO
HIV-1 and HIV-2 proteinases (PR) are responsible for the processing of viral polyproteins, a step that is crucial for the formation of infectious virus particles. PR represents one of the most important targets for antiviral chemotherapy. Inhibitors of HIV-1 PR usually exhibit a 10- to 100-fold weaker affinity for HIV-2 PR. In order to design subnanomolar inhibitors for both HIV-1 and HIV-2 PRs, we prepared a series of compounds varying in the type of scissile bond replacement as well as in the P1, P1', and P2' side chains. While inhibitors containing reduced amide, hydroxyethylamine and statine isosteres had Ki values in the range of 10(-10)-10(-9) M against HIV-1 PR; their activities against HIV-2 PR were several orders of magnitude lower. Glutamic acid was identified to be the optimal P2' residue for both PRs. HIV-2 PR was shown to be more sensitive to P2' Glu-->Gln replacement. Using this data set we were able to design and prepare hydroxyethylene isostere containing inhibitors that were equipotent against both PRs.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Oligopeptídeos/farmacologia , Sítios de Ligação , Desenho de Fármacos , Escherichia coli/genética , Inibidores da Protease de HIV/química , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/químicaRESUMO
HIV-1 and HIV-2 proteases (PR) which play the key role in the formation of infectious viral particles offer a target for inhibitors that could block the maturation step. Inhibitors o HIV-1 PR exhibit mostly 1-2 orders of magnitude weaker affinity for HIV-2 PR. The subsite specificity study of the HIV-1 and HIV-2 proteases performed with inhibitors varying in the type of nonhydrolysable bonds and amino acid residues in the P1, P1'and P2'positions has led us to the design of inhibitors with 2S,4S and 2R,4S stereomeres of the hydroxyethylene isostere and Glu or Gln in the P2'positions. These compounds inhibit HIV-1 and HIV-2 proteases in vitro in subnanomolar concentrations and exhibit the activity in tissue culture.
Assuntos
Fármacos Anti-HIV/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Oligopeptídeos/farmacologia , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/uso terapêutico , Células COS , Desenho de Fármacos , Etilenos , Produtos do Gene gag/biossíntese , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , HIV-1/genética , HIV-1/fisiologia , Humanos , Oligopeptídeos/química , Proteínas Recombinantes/biossíntese , Saquinavir/química , Saquinavir/farmacologia , Saquinavir/uso terapêutico , Estereoisomerismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.
Assuntos
Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Animais , Sítios de Ligação , Células COS , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , Modelos Moleculares , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Calf chymosin molecules exist in the two alternative structural forms: the first one has S1 and S3 binding pockets occluded by its own Tyr77 residue (the self-inhibited form); the second has these pockets free for a substrate binding (the active form). The preliminary incubation of the enzyme with a pentapeptide corresponding to the histidine-proline cluster of the specific substrate kappa-casein results in a 200-fold increase of the hydrolysis rate for the enzyme 'slow substrate'. The result suggests that the cluster is an allosteric effector that promotes the conversion of the enzyme into the active form. These data provide the experimental ground for the explanation of chymosin specificity towards kappa-casein.
Assuntos
Caseínas/metabolismo , Quimosina/química , Quimosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caseínas/química , Caseínas/genética , Bovinos , Cristalografia por Raios X , Hidrólise , Técnicas In Vitro , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Inhibitors of HIV-1 protease represent a new class of antiretroviral compounds. Here, we report the design and synthesis of two novel C2 symmetry-based inhibitors, MP-134 and MP-167, specifically targeted against HIV-1 variants with reduced sensitivity to another related protease inhibitor, A-77003. In addition, we describe the in vitro selection of viral variants with reduced sensitivity of these two protease inhibitors. An isoleucine-to-valine substitution at residue 84 (I84V) of the HIV-1 protease confers resistance to MP-134, whereas a glycine-to-valine substitution at residue 48 (G48V) confers resistance to MP-167. Testing other protease inhibitors against these variants has revealed specific overlapping patterns of resistance among these agents. These findings have important implications in the design of combination regimens using multiple protease inhibitors and underscore the need to develop non-cross-resistant compounds to be used toward this goal.
Assuntos
Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular , Desenho de Fármacos , Resistência Microbiana a Medicamentos , Protease de HIV/química , Protease de HIV/efeitos dos fármacos , Inibidores da Protease de HIV/química , HIV-1/enzimologia , HIV-1/genética , Humanos , Compostos de Metilureia/farmacologia , Dados de Sequência Molecular , Estrutura Molecular , Piridinas/farmacologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Valina/análogos & derivadosRESUMO
Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico/química , Oligopeptídeos/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Sequência de Bases , Dicroísmo Circular , Gráficos por Computador , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pepsina A/química , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , SuínosRESUMO
A new method of synthesizing ortho-methylated phenylalanines has been developed. Phenylalanines with at least one free ortho-position undergo a Pictet-Spengler cyclization with formaldehyde followed by hydrogenolytic splitting of the endocyclic benzylic C--N bond of 1,2,3,4-tetrahydroisoquinolines and afford corresponding ortho-methyl derivatives. Repeating this reaction sequence on the ortho-substituted phenylalanines yielded ortho,ortho-disubstituted derivatives, and para-substituted phenylalanines yielded ortho,para-disubstituted analogs. Our modified method of cyclization preserved the configuration at the chiral center: hydrogenolysis, on the other hand, led to racemization. Incorporation of the methylated phenylalanines into position 2 of oxytocin led to, in the case of the D-isomers, potent uterotonic inhibitors.
Assuntos
Hidrogênio/química , Isoquinolinas/química , Oligopeptídeos/síntese química , Ocitocina/análogos & derivados , Fenilalanina/química , Tetra-Hidroisoquinolinas , Sequência de Aminoácidos , Animais , Feminino , Metilação , Dados de Sequência Molecular , Estrutura Molecular , Ocitocina/química , Conformação Proteica , Ratos , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Útero/efeitos dos fármacosAssuntos
Escherichia coli/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Protease de HIV/genética , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genéticaRESUMO
A series of 47 N-truncated reduced bond inhibitors, systematically modified at individual positions (P1, P'1, P'2, P'3, and P'4), were synthesized and used to map the subsite preferences of HIV-1 protease. The tight binding inhibitor of HIV-1 protease t-butoxycarbonyl-Phe-[CH2NH]Phe-Glu-Phe-NH2 (Ki = 0.2 nM) was chosen as the parent structure for further modifications. The P'2 glutamic acid was found to fit well into the S'2 subsite of the protease. The conformational restriction of any phenylalanine residue or saturation of more than one phenylalanine side chain in P'1 or P'3 lead is to a large Ki increase. Introduction of tyrosine in the P1 position improves the binding by an order of magnitude. The S'4 subsite of the protease was shown to accommodate large structural changes in the inhibitor at this position. Therefore P'4 may serve as an ideal region for further modification in order to improve bioavailability of this type of compound. An improved method of direct comparison of tight binding inhibitors with subnanomolar Ki values has been described.
Assuntos
Inibidores da Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/enzimologia , Sítios de Ligação , Protease de HIV/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Truncation of a peptide substrate in the N-terminus and replacement of its scissile amide bond with a non-cleavable reduced bond results in a potent inhibitor of HIV-1 protease. A series of such inhibitors has been synthesized, and S2-S3' subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P1' and Glu in P2'. The S3' pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2' position yields a tight-binding inhibitor of HIV-1 protease, Boc-Phe-[CH2-NH]-Phe-Glu-Phe-OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a Ki of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1-P1' linkage for proper binding. A new type of peptide bond mimetic, N-hydroxylamine -CH2-N(OH)-, has been synthesized. Binding of hydroxylamino inhibitor of HIV-1 protease is further improved with respect to reduced-bond inhibitor.