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1.
Int J Mol Sci ; 25(13)2024 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-39000315

RESUMO

Aprotinin is a broad-spectrum inhibitor of human proteases that has been approved for the treatment of bleeding in single coronary artery bypass surgery because of its potent antifibrinolytic actions. Following the outbreak of the COVID-19 pandemic, there was an urgent need to find new antiviral drugs. Aprotinin is a good candidate for therapeutic repositioning as a broad-spectrum antiviral drug and for treating the symptomatic processes that characterise viral respiratory diseases, including COVID-19. This is due to its strong pharmacological ability to inhibit a plethora of host proteases used by respiratory viruses in their infective mechanisms. The proteases allow the cleavage and conformational change of proteins that make up their viral capsid, and thus enable them to anchor themselves by recognition of their target in the epithelial cell. In addition, the activation of these proteases initiates the inflammatory process that triggers the infection. The attraction of the drug is not only its pharmacodynamic characteristics but also the possibility of administration by the inhalation route, avoiding unwanted systemic effects. This, together with the low cost of treatment (≈2 Euro/dose), makes it a good candidate to reach countries with lower economic means. In this article, we will discuss the pharmacodynamic, pharmacokinetic, and toxicological characteristics of aprotinin administered by the inhalation route; analyse the main advances in our knowledge of this medication; and the future directions that should be taken in research in order to reposition this medication in therapeutics.


Assuntos
Antivirais , Aprotinina , Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Aprotinina/uso terapêutico , Aprotinina/farmacologia , Aprotinina/química , Humanos , Antivirais/uso terapêutico , Antivirais/farmacologia , Antivirais/administração & dosagem , Administração por Inalação , SARS-CoV-2/efeitos dos fármacos , COVID-19/virologia , Animais , Reposicionamento de Medicamentos/métodos , Inibidores de Serina Proteinase/uso terapêutico , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/administração & dosagem
2.
Sci Rep ; 14(1): 16197, 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39003338

RESUMO

Type-II transmembrane serine proteases are effective pharmacological targets for host defence against viral entry and in certain cancer cell progressions. These serine proteases cleave viral spike proteins to expose the fusion peptide for cell entry, which is essential to the life cycle of the virus. TMPRSS2 inhibitors can also fight against respiratory viruses that employ them for cell entry. Our study combining virtual screening, all-atom molecular dynamics, and well-tempered metadynamics simulation identifies vicenin-2, neohesperidin, naringin, and rhoifolin as promising TMPRSS2 antagonists. The binding energies obtained are - 16.3, - 15.4, - 13.6, and - 13.8 kcal/mol for vicenin-2, neohesperidin, naringin, and rhoifolin respectively. The RMSD, RMSF, PCA, DCCM, and binding free energy profiles also correlate with the stable binding of these ligands at the active site of TMPRSS2. The study reveals that these molecules could be promising lead molecules for combating future outbreaks of coronavirus and other respiratory viruses.


Assuntos
Simulação de Dinâmica Molecular , Serina Endopeptidases , Serina Endopeptidases/metabolismo , Serina Endopeptidases/química , Humanos , Antivirais/farmacologia , Antivirais/química , Simulação de Acoplamento Molecular , Ligação Proteica , Termodinâmica , SARS-CoV-2/efeitos dos fármacos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia
3.
Vet Res ; 55(1): 78, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877574

RESUMO

Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.


Assuntos
Estresse do Retículo Endoplasmático , Células Epiteliais , Estresse Oxidativo , Serpinas , Trichinella spiralis , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Trichinella spiralis/fisiologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Suínos , Serpinas/metabolismo , Serpinas/genética , Inibidores de Serina Proteinase/farmacologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Jejuno/efeitos dos fármacos
4.
J Cardiothorac Surg ; 19(1): 399, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38937755

RESUMO

BACKGROUND: We aimed to assess the efficacy of the neutrophil elastase inhibitor, sivelestat, in the treatment of sepsis-induced acute respiratory distress syndrome (ARDS) and septic cardiomyopathy (SCM). METHODS: Between January 2019 and December 2021, we conducted a randomized trial on patients who had been diagnosed with sepsis-induced acute respiratory distress syndrome (ARDS) and septic cardiomyopathy (SCM) at Wuhan Union Hospital. The patients were divided into two groups by random envelop method, the Sivelestat group and the Control group. We measured the serum concentrations of Interleukin (IL)-6, IL-8, Tumor necrosis factor-α (TNF-α), and High-mobility group box 1 (HMGB1) at five time points, which were the baseline, 12 h, 24 h, 48 h, and 72 h after admission to the ICU. We evaluated the cardiac function by sonography and the heart rate variability (HRV) with 24-hour Holter recording between the time of admission to the intensive care unit (ICU) and 72 h after Sivelestat treatment. RESULTS: From January 2019 to December 2021, a total of 70 patients were included in this study. The levels of IL-6, IL-8, and TNF-α were significantly lower in the Sivelestat group at different time points (12 h, 24 h, 48 h, and 72 h). HMGB1 levels were significantly lower at 72 h after Sivelestat treatment (19.46 ± 2.63pg/mL vs. 21.20 ± 2.03pg/mL, P = 0.003). The stroke volume (SV), tricuspid annular plane systolic excursion (TAPSE), early to late diastolic transmitral flow velocity (E/A), early (e') and late (a') diastoles were significantly low in the Control group compared with the Sivelestat group. Tei index was high in the Control group compared with the Sivelestat group (0.60 ± 0.08 vs. 0.56 ± 0.07, P = 0.029). The result of HRV showed significant differences in standard deviation of normal-to-normal intervals (SDNN), low frequency (LF), and LF/HF (high frequency) between the two groups. CONCLUSIONS: Sivelestat can significantly reduce the levels of serum inflammatory factors, improve cardiac function, and reduce heart rate variability in patients with Sepsis-induced ARDS and SCM.


Assuntos
Cardiomiopatias , Glicina , Síndrome do Desconforto Respiratório , Sepse , Sulfonamidas , Humanos , Masculino , Feminino , Glicina/análogos & derivados , Glicina/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/sangue , Sepse/tratamento farmacológico , Sepse/complicações , Sepse/sangue , Pessoa de Meia-Idade , Síndrome do Desconforto Respiratório/tratamento farmacológico , Sulfonamidas/uso terapêutico , Resultado do Tratamento , Idoso , Inibidores de Serina Proteinase/uso terapêutico
5.
Int J Mol Sci ; 25(10)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38791221

RESUMO

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.


Assuntos
Anticorpos Monoclonais , Bothrops , Peptídeos , Serina Proteases , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Serina Proteases/química , Serina Proteases/metabolismo , Antivenenos/química , Antivenenos/imunologia , Antivenenos/farmacologia , Regiões Determinantes de Complementaridade/química , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/química , Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia
7.
Bioorg Med Chem Lett ; 109: 129814, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38815872

RESUMO

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Serina Endopeptidases , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Relação Estrutura-Atividade , Humanos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/síntese química , Estrutura Molecular , Relação Dose-Resposta a Droga , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química
8.
Eur Respir Rev ; 33(172)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38657996

RESUMO

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.


Assuntos
Alérgenos , Imunidade Inata , Serina Proteases , Humanos , Alérgenos/imunologia , Serina Proteases/metabolismo , Serina Proteases/imunologia , Animais , Poluição do Ar em Ambientes Fechados/efeitos adversos , Inibidores de Serina Proteinase/uso terapêutico , Exposição por Inalação/efeitos adversos , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/enzimologia
9.
Int J Mol Sci ; 25(8)2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38674153

RESUMO

Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with other drugs to treat fever and viral diseases caused by various factors. In our previous study, a novel serine protease inhibitor-encoding gene (MDSPI16) with improved anti-inflammatory activity was selected from a constructed suppressive subducted hybridization library of housefly larvae. This protein could easily induce an immune response in animals and had a short half-life, which limited its wide application in the clinic. Thus, in this study, mPEG-succinimidyl propionate (mPEG-SPA, Mw = 5 kDa) was used to molecularly modify the MDSPI16 protein, and the modified product mPEG-SPA-MDSPI16, which strongly inhibited elastase production, was purified. It had good stability and safety, low immunogenicity, and a long half-life, and the IC50 for elastase was 86 nM. mPEG-SPA-MDSPI16 effectively inhibited the expression of neutrophil elastase and decreased ROS levels. Moreover, mPEG-SPA-MDSPI16 exerted anti-inflammatory effects by inhibiting activation of the NF-κB signaling pathway and the MAPK signaling pathway in neutrophils. It also exerted therapeutic effects on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. In summary, mPEG-SPA-MDSPI16 is a novel anti-inflammatory protein modified with PEG that has the advantages of safety, nontoxicity, improved stability, and strong anti-inflammatory activity in vivo and in vitro and is expected to become an effective anti-inflammatory drug.


Assuntos
Lesão Pulmonar Aguda , Lipopolissacarídeos , Inibidores de Serina Proteinase , Animais , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/induzido quimicamente , Camundongos , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/uso terapêutico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , NF-kappa B/metabolismo , Masculino , Elastase de Leucócito/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Modelos Animais de Doenças
10.
J Virol ; 98(5): e0190323, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38593045

RESUMO

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses. IMPORTANCE: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.


Assuntos
Antivirais , Benzotiazóis , Tratamento Farmacológico da COVID-19 , Oligopeptídeos , SARS-CoV-2 , Inibidores de Serina Proteinase , Replicação Viral , Animais , Feminino , Humanos , Camundongos , Antivirais/farmacologia , Chlorocebus aethiops , COVID-19/virologia , Modelos Animais de Doenças , Pulmão/virologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Peptidomiméticos/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/uso terapêutico , Células Vero , Replicação Viral/efeitos dos fármacos , Oligopeptídeos/farmacologia , Benzotiazóis/farmacologia
11.
Sci Rep ; 14(1): 7703, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38565937

RESUMO

Bioactive molecules in tick saliva are considered to be key to successful feeding and further the transmission of tick-borne pathogens. Problems such as pathogen transmission and animal weight loss result in tick infestation can cause tremendous economic losses to the livestock industry. Therefore, the development of a universal tick vaccine is urgently needed. In this paper, three serine protease inhibitor (serpin) proteins RMS-3, L7LRK7 and L7LTU1 were analyzed with bioinformatics methods. Subsequently the proteins were expressed and purified, and inoculated into Kunming mice for immune protection analysis. The amino acid sequence similarities between RMS-3, L7LRK7 and L7LTU1 were up to 90% in Rhipicephalus sanguineus. The recombinant RMS-3 + L7LRK7 + L7LTU1 showed anticoagulant reaction function and could inhibit the activity of CD4+ lymphocytes, when inoculated into Kunming mice. Additionally, After the immunized mice were challenged with Rhipicephalus sanguineus, the percentage of larvae and nymphs that were fully engorged dropped to 40.87% (P < 0.05) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, and 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, in comparison with the control group. These data indicate that RmS-3 + L7LRK7 + L7LTU1 has good immune protection and has the potential to be developed into a vaccine against the larvae and nymphs of R. sanguineus.


Assuntos
Animais não Endogâmicos , Rhipicephalus sanguineus , Rhipicephalus , Vacinas , Camundongos , Animais , Inibidores de Serina Proteinase/metabolismo , Rhipicephalus/metabolismo , Ninfa , Larva
12.
Eur Heart J Cardiovasc Pharmacother ; 10(4): 342-352, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38658193

RESUMO

The ODYSSEY OUTCOMES trial, comprising over 47 000 patient-years of placebo-controlled observation, demonstrated important reductions in the risk of recurrent ischaemic cardiovascular events with the monoclonal antibody to proprotein convertase subtilisin/kexin type 9 alirocumab, as well as lower all-cause death. These benefits were observed in the context of substantial and persistent lowering of low-density lipoprotein cholesterol with alirocumab compared with that achieved with placebo. The safety profile of alirocumab was indistinguishable from matching placebo except for a ∼1.7% absolute increase in local injection site reactions. Further, the safety of alirocumab compared with placebo was evident in vulnerable groups identified before randomization, such as the elderly and those with diabetes mellitus, previous ischaemic stroke, or chronic kidney disease. The frequency of adverse events and laboratory-based abnormalities was generally similar to that in placebo-treated patients. Thus, alirocumab appears to be a safe and effective lipid-modifying treatment over a duration of at least 5 years.


Assuntos
Anticorpos Monoclonais Humanizados , LDL-Colesterol , Inibidores de PCSK9 , Humanos , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticolesterolemiantes/efeitos adversos , Anticolesterolemiantes/uso terapêutico , Biomarcadores/sangue , Doenças Cardiovasculares/prevenção & controle , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/sangue , LDL-Colesterol/sangue , Dislipidemias/tratamento farmacológico , Dislipidemias/sangue , Dislipidemias/diagnóstico , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/imunologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Inibidores de Serina Proteinase/efeitos adversos , Inibidores de Serina Proteinase/uso terapêutico , Fatores de Tempo , Resultado do Tratamento
13.
FASEB J ; 38(6): e23566, 2024 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-38526868

RESUMO

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Camundongos , Humanos , Receptor 4 Toll-Like/genética , Receptor PAR-2/genética , Doença de Chagas/genética , Doença de Chagas/parasitologia , Antivirais/farmacologia , Inibidores de Serina Proteinase/farmacologia , Inflamação , Serina , Serina Endopeptidases/genética
14.
Fish Shellfish Immunol ; 148: 109525, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38537926

RESUMO

Serine protease inhibitors Kazal type (SPINKs) function in physiological and immunological processes across multicellular organisms. In the present study, we identified a SPINK gene, designated as CqSPINK, in the red claw crayfish Cherax quadricarinatus, which is the ortholog of human SPINK5. The deduced CqSPINK contains two Kazal domains consisting of 45 amino acid residues with a typical signature motif C-X3-C-X5-PVCG-X5-Y-X3-C-X6-C-X12-14-C. Each Kazal domain contains six conserved cysteine residues forming three pairs of disulfide bonds, segmenting the structure into three rings. Phylogenetic analysis revealed CqSPINK as a homolog of human SPINK5. CqSPINK expression was detected exclusively in hepatopancreas and epithelium, with rapid up-regulation in hepatopancreas upon Vibrio parahaemolyticus E1 challenge. Recombinant CqSPINK protein (rCqSPINK) was heterologously expressed in Escherichia coli and purified for further study. Proteinase inhibition assays demonstrated that rCqSPINK could potently inhibit proteinase K and subtilisin A, weakly inhibit α-chymotrypsin and elastase, but extremely weak inhibit trypsin. Furthermore, CqSPINK inhibited bacterial secretory proteinase activity from Bacillus subtilis, E. coli, and Staphylococcus aureus, and inhibited B. subtilis growth. These findings suggest CqSPINK's involvement in antibacterial immunity through direct inhibition of bacterial proteases, contributing to resistance against pathogen invasion.


Assuntos
Astacoidea , Inibidores de Serina Proteinase , Humanos , Animais , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/química , Filogenia , Escherichia coli , Proteínas Recombinantes/genética , Bactérias/metabolismo
15.
Food Funct ; 15(7): 3600-3614, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38469889

RESUMO

Food ingredients have critical effects on the maturation and development of the immune system, which innate - lymphoid (ILCs) and myeloid - cells play key roles as important regulators of energy storage and hepatic fat accumulation. Therefore, the objective of this study is to define potential links between a dietary immunonutritional induction of the selective functional differentiation of monocytes-derived macrophages, ILCs and lipid homeostasis in hepatocarcinoma (HCC)-developing mice. Hepatic chemically injured (diethylnitrosamine/thiacetamide) Rag2-/- and Rag2-/-Il2-/- mice were administered with serine-type protease inhibitors (SETIs) obtained from Chenopodium quinoa. Early HCC-driven immunometabolic imbalances (infiltrated macrophages, glucose homeostasis, hepatic lipid profile, ILCs expansion, inflammatory conditions, microbiota) in animals put under a high-fat diet for 2 weeks were assessed. It was also approached the potential of SETIs to cause functional adaptations of the bioenergetics of human macrophage-like cells (hMLCs) in vitro conditioning their capacity to accumulate fat. It is showed that Rag2-/-Il2-/- mice, lacking ILCs, are resistant to the SETIs-induced hepatic macrophages (CD68+F4/80+) activation. Feeding SETIs to Rag2-/- mice, carrying ILCs, promoted the expansion towards ILC3s (CD117+Nkp46+CD56+) and reduced that of ILC2s (CD117+KLRG1+) into livers. In vitro studies demonstrate that hMLCs, challenged to SETIs, develop a similar phenotype of that found in mice and bioenergetic adaptations leading to increased lipolysis. It is concluded that SETIs promote liver macrophage activation and ILCs adaptations to ameliorate HCC-driven immunometabolic imbalances.


Assuntos
Carcinoma Hepatocelular , Chenopodium quinoa , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Imunidade Inata , Linfócitos , Interleucina-2 , Inibidores de Serina Proteinase , Neoplasias Hepáticas/tratamento farmacológico , Lipídeos , Serina
16.
Int J Biol Macromol ; 265(Pt 1): 130852, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38508547

RESUMO

In the intricate realm of animal biology, a multitude of vital processes heavily rely on precisely orchestrated proteinase cascades, but the potential for havoc makes proteinase inhibitors indispensable, with serine proteinase inhibitors (serpins) at the forefront, serving as custodians of homeostasis and participating in various critical biological processes. Importantly, there are still many unexplored facets of serpin functionality. In this study, we focused on the serpin family proteins from Marsupenaeus japonicus, utilizing a fine-tuned pretrained protein language model. This approach led to the identification and evolutionary validation of 28 serpins, one of which, referred to as Mjserpin-1, was both computationally and experimentally demonstrated to show potential as an antiviral and apoptosis inhibitor. Our research unveils exciting prospects for the fusion of state-of-the-art artificial intelligence and rich bioinformatics, holding the promise of significant discoveries that could pave the way for future therapeutic advancements.


Assuntos
Serpinas , Animais , Serpinas/genética , Serpinas/metabolismo , Inibidores de Serina Proteinase/farmacologia , Inteligência Artificial , Peptídeo Hidrolases , Aprendizado de Máquina
17.
Arterioscler Thromb Vasc Biol ; 44(5): 1156-1164, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38545781

RESUMO

BACKGROUND: Pediatric patients with homozygous familial hypercholesterolemia (HoFH) have an increased risk of atherosclerotic cardiovascular disease and difficulty meeting low-density lipoprotein cholesterol (LDL-C) goals. In this post hoc analysis, we evaluated pooled safety and efficacy data from 3 studies in pediatric patients with HoFH treated with the PCSK9 (proprotein convertase subtilisin/kexin type 9) monoclonal antibody inhibitor evolocumab. METHODS: Patients with HoFH aged 10 to 17 years received treatment with open-label evolocumab 420 mg subcutaneously monthly or biweekly in the TAUSSIG, RAMAN, or HAUSER-OLE clinical studies. All patients received background statins with or without ezetimibe. Study duration ranged from 12 to 260 weeks. The primary end point was treatment-emergent adverse events per 100 patient-years. Efficacy end points were changes from baseline to week 12 in lipids and PCSK9. RESULTS: Of the 39 patients in the pooled analysis, 69.2% were males, median age was 13.0 years, and 79.5% (31/39) had genotyped HoFH with LDLR pathogenic variants. Overall, median exposure to evolocumab was 18.2 (Q1, Q3: 3.0, 18.5) months. Treatment-emergent adverse events with an exposure-adjusted patient incidence rate of ≥5% were upper respiratory tract infection (6.6%), influenza (5.2%), and acne (5.0%) per 100 patient-years. Exposure-adjusted patient incidence of serious treatment-emergent adverse events was 13.3% per 100 patient-years. Excluding 4 patients receiving lipoprotein apheresis, week 12 median percentage change from baseline in LDL-C was -2.9% (Q1, Q3: -21.7, 1.5); however, 42.9% (15/35) of patients achieved ≥15% reduction in LDL-C from baseline. Residual LDLR (LDL receptor) activity was not associated with a reduction in LDL-C. CONCLUSIONS: In this pooled data analysis from 3 studies in pediatric patients with HoFH, evolocumab was well tolerated, with no new safety signals reported. These safety findings are consistent with findings from previous studies of evolocumab. Patients showed marked variability in LDL-C reduction. Results from this pooled analysis support guidelines suggesting a trial of PCSK9 inhibitor therapy regardless of estimated residual LDLR function. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01624142, NCT03403374, and NCT02624869.


Assuntos
Anticorpos Monoclonais Humanizados , Anticolesterolemiantes , LDL-Colesterol , Homozigoto , Hiperlipoproteinemia Tipo II , Inibidores de PCSK9 , Adolescente , Criança , Feminino , Humanos , Masculino , Fatores Etários , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Anticolesterolemiantes/efeitos adversos , Biomarcadores/sangue , LDL-Colesterol/sangue , Quimioterapia Combinada , Ezetimiba/uso terapêutico , Ezetimiba/efeitos adversos , Predisposição Genética para Doença , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/diagnóstico , Fenótipo , Pró-Proteína Convertase 9/genética , Inibidores de Serina Proteinase/efeitos adversos , Inibidores de Serina Proteinase/uso terapêutico , Fatores de Tempo , Resultado do Tratamento , Estudos Clínicos como Assunto
18.
J Med Chem ; 67(6): 4833-4854, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38477709

RESUMO

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.


Assuntos
Serina Proteases , Inibidores de Serina Proteinase , Animais , Camundongos , Serina Proteases/metabolismo , Relação Estrutura-Atividade , Inibidores de Serina Proteinase/química , Conformação Molecular , Peptídeos
20.
Molecules ; 29(5)2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38474448

RESUMO

Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.


Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Sulfonas , Humanos , Prótese Vascular , Antibacterianos/farmacologia , Biofilmes , Inibidores de Serina Proteinase/farmacologia , Staphylococcus aureus , Materiais Biocompatíveis , Puromicina , Peptídeo Hidrolases
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