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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Exerc Immunol Rev ; 26: 80-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32139350

RESUMO

An increasing body of evidence suggests that age-related immune changes and chronic inflammation contribute to cancer development. Recognizing that exercise has protective effects against cancer, promotes immune function, and beneficially modulates inflammation with ageing, this review outlines the current evidence indicating an emerging role for exercise immunology in preventing and treating cancer in older adults. A specific focus is on data suggesting that muscle- derived cytokines (myokines) mediate anti-cancer effects through promoting immunosurveillance against tumourigenesis or inhibiting cancer cell viability. Previous studies suggested that the exercise-induced release of myokines and other endocrine factors into the blood increases the capacity of blood serum to inhibit cancer cell growth in vitro. However, little is known about whether this effect is influenced by ageing. Prostate cancer is the second most common cancer in men. We therefore examined the effects of serum collected before and after exercise from healthy young and older men on the metabolic activity of androgen-responsive LNCaP and androgen-unresponsive PC3 prostate cancer cells. Exercise-conditioned serum collected from the young group did not alter cell metabolic activity, whereas post-exercise serum (compared with pre-exercise serum) from the older men inhibited the metabolic activity of LNCaP cancer cells. Serum levels of candidate cancer-inhibitory myokines oncostatin M and osteonectin increased in both age groups following exercise. Serum testosterone increased only in the younger men postexercise, potentially attenuating inhibitory effects of myokines on the LNCaP cell viability. The data from our study and the evidence in this review suggest that mobilizing serum factors and immune cells may be a key mechanism of how exercise counteracts cancer in the older population.


Assuntos
Envelhecimento , Exercício Físico , Sistema Imunitário , Oncostatina M/sangue , Osteonectina/sangue , Neoplasias da Próstata/prevenção & controle , Idoso , Linhagem Celular Tumoral , Humanos , Masculino
3.
J Cell Mol Med ; 24(6): 3724-3738, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32065471

RESUMO

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola-forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down-regulation of caveola-forming proteins (caveolin-1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola-forming proteins up-regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down-regulation of caveola-forming proteins impaired this response and prevented hyperosmolarity-induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola-forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix-degrading enzyme production, mesenchymal phenotype and invasion. Caveola-forming proteins mediate, at least in part, the pro-invasive response of GBM to pressure. This may represent a novel target in GBM treatment.

4.
Mar Drugs ; 17(12)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842369

RESUMO

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.


Assuntos
Cnidários/classificação , Serina Proteases/efeitos dos fármacos , Inibidores de Serino Proteinase/farmacologia , Animais , Bovinos , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Simulação por Computador , Humanos , Simulação de Dinâmica Molecular , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/isolamento & purificação , Tripsina/efeitos dos fármacos , Tripsina/metabolismo , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/farmacologia
5.
Biochemistry ; 58(21): 2524-2533, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31058493

RESUMO

Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel ß-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.


Assuntos
Calicreínas/química , Peptídeos Cíclicos/química , Proteínas de Plantas/química , Inibidores de Serino Proteinase/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Ciclização , Escherichia coli/metabolismo , Humanos , Ligação de Hidrogênio , Calicreínas/antagonistas & inibidores , Calicreínas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Proteínas de Plantas/farmacologia , Ligação Proteica , Conformação Proteica em Folha beta , Inibidores de Serino Proteinase/farmacologia , Spodoptera/citologia , Spodoptera/metabolismo , Transfecção
6.
J Neurooncol ; 143(2): 207-220, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949900

RESUMO

INTRODUCTION: Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15 months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. METHODS: The mRNA expression of caveola-forming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. RESULTS: Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal samples and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.


Assuntos
Neoplasias Encefálicas/patologia , Caveolina 1/metabolismo , Glioblastoma/patologia , Proteínas de Ligação a RNA/metabolismo , Animais , Biomarcadores Tumorais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Células Cultivadas , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Prognóstico , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética
7.
J Med Chem ; 62(7): 3696-3706, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30888159

RESUMO

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with a sequence and structure similar to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors. Here, we focus on the P5' residue in the Bowman-Birk inhibitory loop and produce a library of SFTI variants to characterize the P5' specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5' sequence diversity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have divergent specificities that can be harnessed in engineering studies. By optimizing the P5' residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at 7 of the scaffold's 14 residues retains a similar structure to SFTI-1. These findings provide new insights into P5' specificity requirements for the Bowman-Birk inhibitory loop.


Assuntos
Aminoácidos/metabolismo , Serina Proteases/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Quimotripsina/metabolismo , Fator XIIa/metabolismo , Humanos , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Trombina/metabolismo , Tripsina/metabolismo
8.
PLoS One ; 14(1): e0210842, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30668585

RESUMO

Engagement of an extended ß-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like ß-sheet to enzyme inhibition. Here we report the crystal structure of an simplified ß-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by ß-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.


Assuntos
Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Tripsina/química , Motivos de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Helianthus/química , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia
9.
ACS Med Chem Lett ; 9(12): 1258-1262, 2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30613336

RESUMO

Kallikrein-related peptidase 4 (KLK4) is a serine protease that has putative intracellular and extracellular functions in prostate cancer progression. Here we show that MCoTI-II, a 34-amino acid cyclic peptide found in the seeds of red gac (Momordica cochinchinensis), is an inhibitor of KLK4. By grafting a preferred KLK4 cleavage sequence into MCoTI-II, we produced a highly potent KLK4 inhibitor (K i = 0.1 nM) that displayed 100,000-fold selectivity over related KLKs and the ability to penetrate cells. Additionally, by substituting positively charged noncontact residues in this compound, we produced a potent and selective KLK4 inhibitor that does not penetrate cells. The inhibitors were shown to be nontoxic to human cells and stable in human serum. These KLK4 inhibitors provide useful chemical tools to further define the role(s) of both intracellular and extracellular KLK4 in prostate cancer cell lines and disease models.

10.
Bioorg Med Chem Lett ; 27(14): 3096-3100, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28545975

RESUMO

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Quinazolinonas/química , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/síntese química , Cinamatos/química , Cinamatos/toxicidade , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Quinazolinas/síntese química , Quinazolinas/química , Quinazolinas/toxicidade , Quinazolinonas/farmacologia , Rad51 Recombinase/metabolismo , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
11.
J Invest Dermatol ; 137(2): 430-439, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27697464

RESUMO

Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (Ki = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis.


Assuntos
Epiderme/metabolismo , Calicreínas/antagonistas & inibidores , Calicreínas/metabolismo , Humanos , Biblioteca de Peptídeos
12.
Sci Rep ; 6: 35385, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27767076

RESUMO

The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.


Assuntos
Calicreínas/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Regulação da Expressão Gênica , Helianthus , Humanos , Ligação de Hidrogênio , Metais/química , Simulação de Dinâmica Molecular , Níquel/química , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Serina Proteases/química , Tripsina/química
13.
J Med Chem ; 59(15): 7287-92, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27434175

RESUMO

Thrombosis is a leading cause of morbidity and mortality associated with cardiovascular diseases. Inhibition of factor XIIa (FXIIa) provides thrombus protection without bleeding complications. Here, we defined the extended substrate specificity of FXIIa and its close homologue factor Xa and used these data, together with inhibitor-based and structure-guided methods, to engineer selective FXIIa inhibitors based on Momordica cochinchinensis trypsin inhibitor-II.


Assuntos
Proteínas Sanguíneas/farmacologia , Desenho de Fármacos , Fator XIIa/antagonistas & inibidores , Momordica/química , Oligopeptídeos/farmacologia , Proteínas Sanguíneas/síntese química , Proteínas Sanguíneas/química , Relação Dose-Resposta a Droga , Fator XIIa/metabolismo , Humanos , Simulação de Dinâmica Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Relação Estrutura-Atividade
14.
Biol Chem ; 397(12): 1237-1249, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-26894578

RESUMO

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.


Assuntos
Domínio Catalítico , Calicreínas/antagonistas & inibidores , Calicreínas/metabolismo , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Engenharia de Proteínas , Sequência de Aminoácidos , Calicreínas/química , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Ligação Proteica , Especificidade por Substrato
15.
J Med Chem ; 58(20): 8257-68, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26393374

RESUMO

Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact ß-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2' specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2' preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.


Assuntos
Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/farmacologia , Sequência de Aminoácidos , Coagulação Sanguínea/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Fibrinólise/efeitos dos fármacos , Calicreínas/antagonistas & inibidores , Calicreínas/química , Modelos Moleculares , Peptídeos Cíclicos/química , Engenharia de Proteínas
16.
Biochem J ; 469(2): 243-53, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25981970

RESUMO

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.


Assuntos
Calicreínas/antagonistas & inibidores , Peptídeos Cíclicos/química , Proteínas de Plantas/química , Humanos , Calicreínas/química , Peptídeos Cíclicos/genética , Proteínas de Plantas/genética , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
17.
Proteomics Clin Appl ; 8(5-6): 389-402, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24677727

RESUMO

Preserving the integrity of the skin's outermost layer (the epidermis) is vital for humans to thrive in hostile surroundings. Covering the entire body, the epidermis forms a thin but impenetrable cellular cordon that repels external assaults and blocks escape of water and electrolytes from within. This structure exists in a perpetual state of regeneration where the production of new cellular subunits at the base of the epidermis is offset by the release of terminally differentiated corneocytes from the surface. It is becoming increasingly clear that proteases hold vital roles in assembling and maintaining the epidermal barrier. More than 30 proteases are expressed by keratinocytes or infiltrating immune cells and the activity of each must be maintained within narrow limits and confined to the correct time and place. Accordingly, over- or under-exertion of proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening. This review explores the current state of knowledge on the involvement of proteases in skin diseases and the latest findings from proteomic and transcriptomic studies focused on uncovering novel (patho)physiological roles for these enzymes.


Assuntos
Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Dermatopatias/enzimologia , Animais , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Peptídeo Hidrolases/genética , Inibidores de Proteases/metabolismo , Dermatopatias/patologia
18.
Trends Mol Med ; 20(3): 166-78, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24380647

RESUMO

Recent findings from the clinic and the laboratory have transformed the way proteases and their inhibitors are perceived in the outermost layer of the skin, the epidermis. It now appears that an integrated proteolytic network operates within the epidermis, comprising more than 30 enzymes that carry out a growing list of essential functions. Equally, defective regulation or execution of protease-mediated processes is emerging as a key contributor to diverse human skin pathologies, and in recent years the number of diseases attributable to aberrant proteolytic activity has more than doubled. Here, we survey the different roles of proteases in epidermal homeostasis (from processing enzymes to signalling molecules) and explore the spectrum of rare and common human skin disorders where proteolytic pathways are dysregulated.


Assuntos
Peptídeo Hidrolases/metabolismo , Dermatopatias/enzimologia , Diferenciação Celular , Epiderme/enzimologia , Homeostase , Humanos , Queratinócitos/metabolismo , Inibidores de Proteases/metabolismo , Proteólise , Pele/enzimologia , Pele/patologia , Dermatopatias/patologia
19.
Mol Cell Endocrinol ; 382(2): 899-908, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24239616

RESUMO

Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3ß-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.


Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/genética , Norpregnenos/farmacologia , Receptores Androgênicos/genética , Androgênios/química , Androgênios/metabolismo , Biotransformação , Linhagem Celular Tumoral , Di-Hidrotestosterona/química , Di-Hidrotestosterona/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Acetato de Medroxiprogesterona/química , Acetato de Medroxiprogesterona/farmacologia , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Norpregnanos/metabolismo , Norpregnenos/química , Norpregnenos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade
20.
Biopolymers ; 100(5): 510-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24078181

RESUMO

Potent and specific enzyme inhibition is a key goal in the development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclized peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein-related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through the use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry. This approach identified a single dominant inhibitor species with molecular weight of 1562.4 Da, which is consistent with the SFTI variant SFTI-WCTF. Once synthesized individually this inhibitor showed an IC50 of 173.9 ± 7.6 nM against chromogenic substrates and could block protein proteolysis. Molecular modeling analysis suggested that selection of SFTI-WCTF was driven by specific aromatic interactions and stabilized by an enhanced internal hydrogen bonding network. This approach provides a robust and rapid route to inhibitor selection and design.


Assuntos
Helianthus , Inibidores da Tripsina , Helianthus/química , Ligação de Hidrogênio , Calicreínas , Peptídeos Cíclicos/química , Tripsina/química , Inibidores da Tripsina/química
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