RESUMO
We report a low molecular weight inhibitor of alpha-amylases based on a linear peptidic scaffold designed de novo through the use of combinatorial chemistry. The inhibitory motif denoted PAMI (peptide amylase inhibitor) was selected by using L-peptide libraries and was fine-tuned by the introduction of unnatural modifications. PAMI specifically inhibits glycoside hydrolases of family 13. Its interaction with porcine pancreatic alpha-amylase was characterized by inhibition kinetics, fluorescence competition assays with natural alpha-amylase inhibitors, and isothermal titration calorimetry. We demonstrate that the critical amino acid residues in PAMI are shared with those in the macromolecular proteinaceous inhibitors that, however, bind to alpha-amylases through a spatially scattered set of intermolecular contacts. Thus, natural molecular evolution as well as combinatorial evolution selected the same alpha-amylase binding determinants for completely different spatial frameworks.
Assuntos
Inibidores Enzimáticos/química , Ligantes , Mimetismo Molecular , Biblioteca de Peptídeos , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cinética , Estrutura Molecular , SuínosRESUMO
The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.
Assuntos
Inseticidas/química , Inseticidas/farmacologia , Phaseolus/química , Lectinas de Plantas/farmacologia , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Fungos/enzimologia , Caracois Helix/enzimologia , Insetos/enzimologia , Ácaros/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oligoquetos/enzimologia , Lectinas de Plantas/química , Ligação Proteica , Especificidade por SubstratoRESUMO
The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of alpha-amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mite's gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops.
Assuntos
Acarbose/farmacologia , Inibidores Enzimáticos/farmacologia , Inseticidas , Ácaros/enzimologia , alfa-Amilases/antagonistas & inibidores , Animais , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/ultraestrutura , Ativação Enzimática , Concentração de Íons de HidrogênioRESUMO
Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.
Assuntos
Nicotiana/enzimologia , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Precursores de Proteínas/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Ciclopentanos/farmacologia , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oxilipinas , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Nicotiana/metabolismoRESUMO
Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.
Assuntos
Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Sequência de Aminoácidos , Catepsina H , Catepsinas/antagonistas & inibidores , Catepsinas/química , Dicroísmo Circular , Cisteína Endopeptidases/química , Ativação Enzimática , Precursores Enzimáticos/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.