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1.
Molecules ; 26(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578831

RESUMO

Currently, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected people among all countries and is a pandemic as declared by the World Health Organization (WHO). SARS-CoVID-2 main protease is one of the therapeutic drug targets that has been shown to reduce virus replication, and its high-resolution 3D structures in complex with inhibitors have been solved. Previously, we had demonstrated the potential of natural compounds such as serine protease inhibitors eventually leading us to hypothesize that FDA-approved marine drugs have the potential to inhibit the biological activity of SARS-CoV-2 main protease. Initially, field-template and structure-activity atlas models were constructed to understand and explain the molecular features responsible for SARS-CoVID-2 main protease inhibitors, which revealed that Eribulin Mesylate, Plitidepsin, and Trabectedin possess similar characteristics related to SARS-CoVID-2 main protease inhibitors. Later, protein-ligand interactions are studied using ensemble molecular-docking simulations that revealed that marine drugs bind at the active site of the main protease. The three-dimensional reference interaction site model (3D-RISM) studies show that marine drugs displace water molecules at the active site, and interactions observed are favorable. These computational studies eventually paved an interest in further in vitro studies. Finally, these findings are new and indeed provide insights into the role of FDA-approved marine drugs, which are already in clinical use for cancer treatment as a potential alternative to prevent and treat infected people with SARS-CoV-2.


Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Domínio Catalítico , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Reposicionamento de Medicamentos , Furanos/química , Furanos/farmacologia , Humanos , Cetonas/química , Cetonas/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Inibidores de Serino Proteinase/química , Trabectedina/química , Trabectedina/farmacologia , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
2.
Nat Commun ; 11(1): 6371, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311470

RESUMO

Genetic mutations predispose the serine protease inhibitor α1-antitrypsin to misfolding and polymerisation within hepatocytes, causing liver disease and chronic obstructive pulmonary disease. This misfolding occurs via a transiently populated intermediate state, but our structural understanding of this process is limited by the instability of recombinant α1-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α1-antitrypsin at natural isotopic abundance to investigate the consequences of disease-causing mutations, and observe widespread chemical shift perturbations for methyl groups in Z AAT (E342K). By comparison with perturbations induced by binding of a small-molecule inhibitor of misfolding we conclude that they arise from rapid exchange between the native conformation and a well-populated intermediate state. The observation that this intermediate is stabilised by inhibitor binding suggests a paradoxical approach to the targeted treatment of protein misfolding disorders, wherein the stabilisation of disease-associated states provides selectivity while inhibiting further transitions along misfolding pathways.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Predisposição Genética para Doença/genética , Glicoproteínas , Humanos , Modelos Moleculares , Medicina Molecular , Mutação , Agregação Patológica de Proteínas , Conformação Proteica , Proteínas Recombinantes , Inibidores de Serino Proteinase/química
3.
An Acad Bras Cienc ; 92(2): e20200466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32556054

RESUMO

COVID-19 emerged in December 2019 in China, and since then, has disrupted global public health and changed economic paradigms. In dealing with the new Coronavirus, SARS-CoV-2, the world has not faced such extreme global fragility since the "Spanish flu" pandemic in 1918. Researchers globally are dedicating efforts to the search for an effective treatment for COVID-19. Drugs already used in a clinical setting for other pathologies have been tested as a new therapeutic approach against SARS-CoV-2, setting off a frenzy over the preliminary data of different studies. This work aims to compile and discuss the data published thus far. Despite the potential effects of some antivirals and antiparasitic against COVID-19, clinical studies must confirm real effectiveness. However, non-pharmacological approaches have proven to be the most efficient strategy to date.


Assuntos
Antibacterianos/administração & dosagem , Antiparasitários/administração & dosagem , Antivirais/administração & dosagem , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Macrolídeos/administração & dosagem , Pneumonia Viral/tratamento farmacológico , Inibidores de Serino Proteinase/administração & dosagem , Antibacterianos/química , Antibacterianos/farmacologia , Antiparasitários/química , Antiparasitários/farmacologia , Antivirais/química , Antivirais/farmacologia , Humanos , Macrolídeos/química , Macrolídeos/farmacologia , Pandemias , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia
4.
Molecules ; 25(10)2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32408547

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of -14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.


Assuntos
Betacoronavirus/enzimologia , Desenho de Fármacos , Serina Endopeptidases/química , Inibidores de Serino Proteinase/química , Bibliotecas de Moléculas Pequenas , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Gabexato/análogos & derivados , Gabexato/química , Gabexato/metabolismo , Gabexato/farmacologia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/metabolismo , Inibidores de Serino Proteinase/farmacologia
5.
FEBS Open Bio ; 10(6): 995-1004, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32374074

RESUMO

A novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or 2019 novel coronavirus] has been identified as the pathogen of coronavirus disease 2019. The main protease (Mpro , also called 3-chymotrypsin-like protease) of SARS-CoV-2 is a potential target for treatment of COVID-19. A Mpro homodimer structure suitable for docking simulations was prepared using a crystal structure (PDB ID: 6Y2G; resolution 2.20 Å). Structural refinement was performed in the presence of peptidomimetic α-ketoamide inhibitors, which were previously disconnected from each Cys145 of the Mpro homodimer, and energy calculations were performed. Structure-based virtual screenings were performed using the ChEMBL database. Through a total of 1 485 144 screenings, 64 potential drugs (11 approved, 14 clinical, and 39 preclinical drugs) were predicted to show high binding affinity with Mpro . Additional docking simulations for predicted compounds with high binding affinity with Mpro suggested that 28 bioactive compounds may have potential as effective anti-SARS-CoV-2 drug candidates. The procedure used in this study is a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that may significantly shorten the clinical development period with regard to drug repositioning.


Assuntos
Betacoronavirus/enzimologia , Quimases/metabolismo , Infecções por Coronavirus/metabolismo , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Preparações Farmacêuticas/metabolismo , Pneumonia Viral/metabolismo , Inibidores de Serino Proteinase/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/efeitos dos fármacos , Domínio Catalítico , Quimases/antagonistas & inibidores , Quimases/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cristalização , Bases de Dados de Compostos Químicos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Preparações Farmacêuticas/química , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Inibidores de Serino Proteinase/química , Proteínas Virais/química
6.
BMC Mol Cell Biol ; 21(1): 38, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32450796

RESUMO

BACKGROUND: Detailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides. RESULTS: Monitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other. CONCLUSIONS: Our reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using 19F NMR spectroscopy.


Assuntos
Vírus da Dengue/enzimologia , Espectroscopia de Ressonância Magnética , Serina Proteases/química , Inibidores de Serino Proteinase/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Vírus da Dengue/genética , Flúor/química , Hidrogênio/química , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Isótopos de Nitrogênio/química , Conformação Proteica , Serina Proteases/genética , Proteínas não Estruturais Virais/química , Água/química
7.
J Biotechnol ; 313: 11-17, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32126268

RESUMO

TLC-Bioautography is a fast and effective method for assessing the inhibitory effect of compounds present in plant extracts against microbial species. However, this method has a hidden, currently underutilized potential for evaluating the presence of inhibitory compounds against selected enzymes. The aim of this work was to design a functional TLC-Bioautography method for the evaluation of protease inhibitors present in plant extracts. The method is based on the hydrolysis of Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BApNA) by α-chymotrypsin as a representative serine protease to produce coloured para-nitroaniline (pNA). Derivatization of pNA with both sodium nitrite and N-(1-naphthyl) ethylenediamine (NPED) leads to the formation of a pink azo dye. This step improves the resolution of active compounds on the chromatogram, which appear as light spots on a pink background. The developed method was tested for the analysis of protease inhibitors in different plant materials such as grape pomace from Vitis vinifera, Picea abies bark, Hippophae rhamnoides berries, Hordeum sativum bran, Triticum aestivum bran and Avena sativa bran. Plant extracts, which could not be analysed by a commonly used spectrophotometric method due to interference, were assessed by this method.


Assuntos
Quimotripsina/antagonistas & inibidores , Hippophae/química , Picea/química , Extratos Vegetais/química , Inibidores de Serino Proteinase/isolamento & purificação , Vitis/química , Benzoilarginina Nitroanilida/metabolismo , Cromatografia , Frutas/química , Hidrólise , Casca de Planta/química , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia
8.
Future Med Chem ; 12(2): 147-159, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031024

RESUMO

Aim: We applied genetic programming approaches to understand the impact of descriptors on inhibitory effects of serine protease inhibitors of Mycobacterium tuberculosis (Mtb) and the discovery of new inhibitors as drug candidates. Materials & methods: The experimental dataset of serine protease inhibitors of Mtb descriptors was optimized by genetic algorithm (GA) along with the correlation-based feature selection (CFS) in order to develop predictive models using machine-learning algorithms. The best model was deployed on a library of 918 phytochemical compounds to screen potential serine protease inhibitors of Mtb. Quality and performance of the predictive models were evaluated using various standard statistical parameters. Result: The best random forest model with CFS-GA screened 126 anti-tubercular agents out of 918 phytochemical compounds. Also, genetic programing symbolic classification method is optimized descriptors and developed an equation for mathematical models. Conclusion: The use of CFS-GA with random forest-enhanced classification accuracy and predicted new serine protease inhibitors of Mtb, which can be used for better drug development against tuberculosis.


Assuntos
Mycobacterium tuberculosis/enzimologia , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Aprendizado de Máquina , Modelos Moleculares , Serina Proteases/genética , Inibidores de Serino Proteinase/química
10.
J Med Chem ; 63(2): 816-826, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31855419

RESUMO

Chymase is a serine protease that is predominantly expressed by mast cells and has key roles in immune defense and the cardiovascular system. This enzyme has also emerged as a therapeutic target for cardiovascular disease due to its ability to remodel cardiac tissue and generate angiotensin II. Here, we used the nature-derived cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1) as a template for designing novel chymase inhibitors. The key binding contacts of SFTI-1 were optimized by combining a peptide substrate library screen with structure-based design, which yielded several variants with potent activity. The lead variant was further modified by replacing the P1 Tyr residue with para-substituted Phe derivatives, generating new inhibitors with improved potency (Ki = 1.8 nM) and higher selectivity over closely related enzymes. Several variants were shown to block angiotensin I cleavage in vitro, highlighting their potential for further development and future evaluation as pharmaceutical leads.


Assuntos
Quimases/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Substituição de Aminoácidos , Angiotensina II/biossíntese , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fenilalanina/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Tirosina/química
11.
Toxins (Basel) ; 11(12)2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31817486

RESUMO

Elastase is a globular glycoprotein and belongs to the chymotrypsin family. It is involved in several inflammatory cascades on the basis of cleaving the important connective tissue protein elastin, and is strictly regulated to a balance by several endogenous inhibitors. When elastase and its inhibitors are out of balance, severe diseases will develop, especially those involved in the cardiopulmonary system. Much attention has been attracted in seeking innovative elastase inhibitors and various advancements have been taken on clinical trials of these inhibitors. Natural functional peptides from venomous animals have been shown to have anti-protease properties. Here, we identified a kazal-type serine protease inhibitor named ShSPI from the cDNA library of the venom glands of Scolopendra hainanum. ShSPI showed significant inhibitory effects on porcine pancreatic elastase and human neutrophils elastase with Ki values of 225.83 ± 20 nM and 12.61 ± 2 nM, respectively. Together, our results suggest that ShSPI may be an excellent candidate to develop a drug for cardiopulmonary diseases.


Assuntos
Venenos de Artrópodes , Inibidores de Serino Proteinase , Animais , Artrópodes , Biblioteca Gênica , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Plasma/química , Dobramento de Proteína , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/metabolismo
12.
Cells ; 8(12)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817705

RESUMO

Alpha 1-antitrypsin deficiency (AATD) is the most common genetic cause of liver disease in children and is associated with early-onset chronic liver disease in adults. AATD associated liver injury is caused by hepatotoxic retention of polymerized mutant alpha 1-antitrypsin molecules within the endoplasmic reticulum. Currently, there is no curative therapy for AATD. In this study, we selected small molecules with the potential to bind mutant alpha 1-antitrypsin (Z-variant) to inhibit its accumulation in hepatocytes. We used molecular docking to select candidate compounds that were validated in cell and animal models of disease. A crystal structure of polymerized alpha 1-antitrypsin molecule was used as the basis for docking 139,735 compounds. Effects of the top scoring compounds were investigated in a cell model that stably expresses Z-variant alpha 1-antitrypsin and in PiZ mice expressing Z-variant human alpha 1-antitrypsin (Z-hAAT), encoded by SERPINA1*E342K. 4','5-(Methylenedioxy)-2-nitrocinnamic acid was predicted to bind cleaved alpha 1-antitrypsin at the polymerization interface, and observed to co-localize with Z-hAAT, increase Z-hAAT degradation, inhibit intracellular accumulation of Z-hAAT, and alleviate liver fibrosis.


Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Inibidores de Serino Proteinase/farmacologia , alfa 1-Antitripsina/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Multimerização Proteica , Inibidores de Serino Proteinase/química , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , alfa 1-Antitripsina/química
13.
Cell Chem Biol ; 26(11): 1559-1572.e9, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543462

RESUMO

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Hepcidinas/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Inibidores de Serino Proteinase/química , Benzotiazóis/química , Sítios de Ligação , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Proteínas Ligadas por GPI/metabolismo , Proteína da Hemocromatose/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Peptidomiméticos , Proteólise/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/metabolismo , Inibidores de Serino Proteinase/farmacologia , Regulação para Cima/efeitos dos fármacos
14.
Bioorg Med Chem Lett ; 29(20): 126675, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31521475

RESUMO

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.


Assuntos
Benzamidinas/química , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , Inibidores de Serino Proteinase/química , Sequência de Aminoácidos , Benzamidinas/farmacologia , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Humanos , Isomerismo , Modelos Moleculares , Estrutura Molecular , Mutação , Ligação Proteica , Inibidor de Serinopeptidase do Tipo Kazal 5/genética , Inibidores de Serino Proteinase/farmacologia , Relação Estrutura-Atividade
15.
Proc Natl Acad Sci U S A ; 116(38): 18808-18814, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31484779

RESUMO

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 µM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.


Assuntos
Fluoretos/química , Elastase de Leucócito/antagonistas & inibidores , Inibidores de Serino Proteinase/química , Compostos de Enxofre/química , Química Click , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Elastase de Leucócito/química , Elastase de Leucócito/metabolismo , Estrutura Molecular , Ligação Proteica , Dobramento de Proteína , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/farmacologia , Ácidos Sulfínicos/síntese química , Ácidos Sulfínicos/química , Ácidos Sulfínicos/farmacologia
16.
Sci Rep ; 9(1): 11436, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391482

RESUMO

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.


Assuntos
Desenho de Fármacos , Glicoproteínas/farmacologia , Mutagênese , Engenharia de Proteínas/métodos , Inibidores de Serinopeptidase do Tipo Kazal/farmacologia , Inibidores de Serino Proteinase/farmacologia , Cristalografia por Raios X , Glicoproteínas/genética , Glicoproteínas/uso terapêutico , Glicoproteínas/ultraestrutura , Calicreínas/metabolismo , Calicreínas/ultraestrutura , Modelos Moleculares , Estrutura Terciária de Proteína , Inibidores de Serinopeptidase do Tipo Kazal/genética , Inibidores de Serinopeptidase do Tipo Kazal/uso terapêutico , Inibidores de Serinopeptidase do Tipo Kazal/ultraestrutura , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/uso terapêutico , Relação Estrutura-Atividade
17.
Protein J ; 38(4): 435-446, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31435809

RESUMO

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.


Assuntos
Quimotripsina/química , Inibidores de Serino Proteinase/química , Animais , Bovinos , Quimotripsina/metabolismo , Cinética , Modelos Moleculares , Phaseolus/metabolismo , Ligação Proteica , Inibidores de Serino Proteinase/metabolismo
18.
Bioorg Med Chem Lett ; 29(19): 126604, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445854

RESUMO

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/administração & dosagem , Compostos Macrocíclicos/química , Inibidores de Serino Proteinase/administração & dosagem , Inibidores de Serino Proteinase/química , Administração Oral , Disponibilidade Biológica , Humanos , Ligantes , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Estrutura Molecular , Inibidores de Serino Proteinase/farmacologia , Relação Estrutura-Atividade
19.
Bioorg Chem ; 92: 103199, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31446241

RESUMO

Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC50 values ranging from 8.05 µM to 82.08 µM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki values ranging from 4.12 µM to 11.01 µM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.


Assuntos
Flavonas/química , Ginkgo biloba/química , Hemostáticos/química , Extratos Vegetais/química , Folhas de Planta/química , Inibidores de Serino Proteinase/química , Trombina/antagonistas & inibidores , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Flavonas/metabolismo , Hemostáticos/farmacologia , Humanos , Cinética , Lisina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Extratos Vegetais/metabolismo , Ligação Proteica , Inibidores de Serino Proteinase/metabolismo , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem
20.
Arch Pharm (Weinheim) ; 352(8): e1900061, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31338866

RESUMO

Elastase is the only enzyme that has the capability to degrade elastin and collagen, the two proteins essential for skin and bones. The synthesis of some densely substituted piperidines functionalized with the trifluoromethyl group (4a-j) was carried out. The newly prepared compounds were subjected to elastase enzyme inhibitory potential and antioxidant activity assays. Among the series, 4i (IC50 = 0.341 ± 0.001 µM) exhibited the maximum inhibition against elastase. Binding analysis delineated that the fluorine atom of ligand 4i showed hydrogen and hydrophobic bonds with Thr41 and Thr96, with bond distances of 3.84 and 5.631 Å, respectively. The obtained results indicate that these trifluoromethyl functionalized piperidine derivatives could be considered as potential candidates to treat skin disorders.


Assuntos
Hidrocarbonetos Fluorados/farmacologia , Elastase Pancreática/antagonistas & inibidores , Piperidinas/farmacologia , Inibidores de Serino Proteinase/farmacologia , Animais , Relação Dose-Resposta a Droga , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Ligantes , Modelos Moleculares , Estrutura Molecular , Pâncreas/enzimologia , Elastase Pancreática/metabolismo , Piperidinas/síntese química , Piperidinas/química , Inibidores de Serino Proteinase/síntese química , Inibidores de Serino Proteinase/química , Relação Estrutura-Atividade , Suínos
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