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1.
Viruses ; 13(9)2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34578348

RESUMO

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzamidinas/farmacologia , COVID-19/metabolismo , COVID-19/virologia , Guanidinas/farmacologia , Interferon-alfa/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Benzamidinas/uso terapêutico , COVID-19/tratamento farmacológico , Cricetinae , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Guanidinas/uso terapêutico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon-alfa/uso terapêutico , Replicação Viral/efeitos dos fármacos
2.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443434

RESUMO

The aerial part of Biebersteinia heterostemon Maxim. (Geraniaceae Biebersteiniaceae) known as ming jian na bao in Chinese, has been traditionally used in Tibetan folk medicine for treatment of diabetes and hypertension. The aim of the present study was to evaluate the effects of galegine obtained from an ethanol extract of the entire Biebersteinia heterostemon plant on the rat's cardiovascular system in order to characterize its contributions as an antihypertensive agent. The antihypertensive effect of galegine was investigated in pentobarbital-anesthetized hypertensive rats at three dose levels based on the LD50 of galegine. Meanwhile a positive control group received dimaprit with the same procedure. Dimaprit infusion induced a significant hypotension which declined by an average margin of 20%. Simultaneously, single administration of galegine at the doses of 2.5, 5, and 10 mg/kg by intraperitoneal injection induced an immediate and dose-dependent decrease in mean arterial blood pressure (MABP) by an average margin of 40% with a rapid increase in heart rate (HR). We demonstrated that galegine is effective in reducing blood pressure in anesthetized hypertensive rats with rapid onset and a dose-related duration of the effects. The results indicate that galegine was the bioactive compound which can be used as a pharmacophore to design new hypertensive agents.


Assuntos
Anti-Hipertensivos/farmacologia , Guanidinas/farmacologia , Magnoliopsida/química , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dimaprit/farmacologia , Feminino , Guanidinas/química , Guanidinas/uso terapêutico , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Masculino , Camundongos , Ratos Sprague-Dawley
3.
mBio ; 12(4): e0097021, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34340553

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis. IMPORTANCE The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.


Assuntos
Benzamidinas/farmacologia , Ésteres/farmacologia , Guanidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/tratamento farmacológico , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Med Microbiol Immunol ; 210(4): 235-244, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34196781

RESUMO

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.


Assuntos
Anti-Infecciosos/farmacologia , COVID-19/prevenção & controle , COVID-19/virologia , Guanidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiocianatos/farmacologia , Inativação de Vírus , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Contenção de Riscos Biológicos , Humanos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/fisiologia , Manejo de Espécimes/métodos , Fatores de Tempo , Células Vero
5.
Cell Rep ; 36(3): 109415, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34270919

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants threatens efforts to contain the coronavirus disease 2019 (COVID-19) pandemic. The number of COVID-19 cases and deaths in India has risen steeply, and a SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the angiotensin converting enzyme 2 (ACE2) receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyze whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 enters two of eight cell lines tested with roughly 50% increased efficiency and is equally inhibited by two entry inhibitors. In contrast, B.1.617 is resistant against bamlanivimab, an antibody used for COVID-19 treatment. B.1.617 evades antibodies induced by infection or vaccination, although less so than the B.1.351 variant. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.


Assuntos
Enzima de Conversão de Angiotensina 2/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Antivirais/farmacologia , COVID-19/tratamento farmacológico , Ésteres/farmacologia , Guanidinas/farmacologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Linhagem Celular , Humanos , Inibidores de Proteases/farmacologia , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinação
6.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205045

RESUMO

SGLT-2i's exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i's empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i's (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i's improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i's might be partially mediated by inhibition of NHE1 and NOXs.


Assuntos
Células Endoteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Transporte de Sódio-Glucose/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Canagliflozina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucosídeos/farmacologia , Guanidinas/farmacologia , Humanos , Inflamação/genética , Inflamação/patologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Estresse Oxidativo/genética , Pirazóis/farmacologia , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Transporte de Sódio-Glucose/genética , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Trocador 1 de Sódio-Hidrogênio/genética , Estresse Mecânico , Sulfonas/farmacologia
7.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200418

RESUMO

This paper reports the synthesis of branched alkylene guanidines using microfluidic technologies. We describe the preparation of guanidine derivatives at lower temperatures, and with significantly less time than that required in the previously applicable method. Furthermore, the use of microfluidics allows the attainment of high-purity products with a low residual monomer content, which can expand the range of applications of this class of compounds. For all the samples obtained, the molecular-weight characteristics are calculated, based on which the optimal condensation conditions are established. Additionally, in this work, the antiviral activity of the alkylene guanidine salt against the SARS-CoV-2 virus is confirmed.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Guanidinas/síntese química , Guanidinas/farmacologia , Microfluídica/métodos , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19 , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Chlorocebus aethiops , Concentração Inibidora 50 , Espectrometria de Massas por Ionização por Electrospray , Células Vero
8.
Am J Physiol Cell Physiol ; 321(3): C471-C488, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288721

RESUMO

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control.


Assuntos
Colo/metabolismo , Enterócitos/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colo/citologia , Colo/efeitos dos fármacos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Guanidinas/farmacologia , Células HT29 , Homeostase/genética , Humanos , Concentração de Íons de Hidrogênio , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia
9.
Clin Biochem ; 96: 56-62, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252447

RESUMO

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Ésteres/sangue , Guanidinas/sangue , Espectrometria de Massas em Tandem/métodos , COVID-19/sangue , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Esterases/antagonistas & inibidores , Esterases/metabolismo , Ésteres/metabolismo , Ésteres/farmacologia , Guanidinas/farmacologia , Humanos , Hidrólise/efeitos dos fármacos , Isoflurofato/química , Isoflurofato/farmacologia , Paraoxon/sangue , Paraoxon/química , Paraoxon/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação
10.
J Med Chem ; 64(12): 8684-8709, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34110814

RESUMO

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H2 receptor (H2R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H2R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D2-like receptors (especially to the D3R). To improve the selectivity, a series of novel carbamoylguanidine-type ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H1-4 and D2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H2- and D2long/3-receptors. These results provide a solid base for the exploration of the H2R functions in the brain in further studies.


Assuntos
Guanidinas/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos H2/metabolismo , Tiazóis/farmacologia , Animais , Sítios de Ligação , Guanidinas/síntese química , Guanidinas/metabolismo , Cobaias , Células HEK293 , Agonistas dos Receptores Histamínicos/síntese química , Agonistas dos Receptores Histamínicos/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ratos , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/química , Receptores de Dopamina D3/metabolismo , Receptores Histamínicos H2/química , Células Sf9 , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/metabolismo
11.
ACS Chem Neurosci ; 12(13): 2503-2519, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34100603

RESUMO

This article describes the discovery of novel potent muscarinic receptor antagonists identified during a search for more active histamine H3 receptor (H3R) ligands. The idea was to replace the flexible seven methylene linker with a semirigid 1,4-cyclohexylene or p-phenylene substituted group of the previously described histamine H3R antagonists ADS1017 and ADS1020. These simple structural modifications of the histamine H3R antagonist led to the emergence of additional pharmacological effects, some of which unexpectedly showed strong antagonist potency at muscarinic receptors. This paper reports the routes of synthesis and pharmacological characterization of guanidine derivatives, a novel chemotype of muscarinic receptor antagonists binding to the human muscarinic M2 and M4 receptors (hM2R and hM4R, respectively) in nanomolar concentration ranges. The affinities of the newly synthesized ADS10227 (1-{4-{4-{[4-(phenoxymethyl)cyclohexyl]methyl}piperazin-1-yl}but-1-yl}-1-(benzyl)guanidine) at hM2R and hM4R were 2.8 nM and 5.1 nM, respectively.


Assuntos
Antagonistas dos Receptores Histamínicos H3 , Receptores Histamínicos H3 , Colinérgicos , Guanidinas/farmacologia , Histamina , Antagonistas dos Receptores Histamínicos , Antagonistas dos Receptores Histamínicos H3/farmacologia , Humanos , Antagonistas Muscarínicos , Relação Estrutura-Atividade
12.
J Virol ; 95(19): e0086121, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34160253

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.


Assuntos
Antivirais/farmacologia , Ésteres/farmacologia , Guanidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/química , Serina Endopeptidases/farmacologia , Serina Proteases/farmacologia , Antivirais/química , COVID-19/tratamento farmacológico , COVID-19/prevenção & controle , Carcinoma de Células Escamosas , Ésteres/química , Ésteres/metabolismo , Guanidinas/química , Guanidinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Neoplasias Bucais , Domínios Proteicos , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Internalização do Vírus/efeitos dos fármacos
13.
Am J Physiol Gastrointest Liver Physiol ; 321(2): G185-G199, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34132108

RESUMO

Recent studies in our lab have shown that the KV7 channel activator, flupirtine, inhibits colonic epithelial Cl- secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na+ absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22Na+ fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na+ transport in the rat colon. Flupirtine stimulated electroneutral Na+ absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (ISC; i.e., Cl- secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na+/H+ exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na+ absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na+ channel (ENaC)-mediated Na+ absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na+ absorption in the proximal colon. Finally, the parallel effects of flupirtine on ISC (Cl- secretion) and Na+ absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na+ absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons.NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal KV7 K+ channels markedly stimulates Na+ absorption and inhibits Cl- secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).


Assuntos
Aminopiridinas/farmacologia , Colo/metabolismo , Sistema Nervoso Entérico/metabolismo , Absorção Intestinal , Sódio/metabolismo , Animais , Colo/efeitos dos fármacos , Canais Epiteliais de Sódio/metabolismo , Guanidinas/farmacologia , Masculino , Moduladores de Transporte de Membrana/farmacologia , Metacrilatos/farmacologia , Ratos , Ratos Sprague-Dawley , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia
14.
Food Microbiol ; 99: 103797, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119092

RESUMO

Postharvest treatments with sanitizers and fungicides are applied to increase the quality, safety and shelf life of fresh produce including cantaloupes (also known as rockmelons). The primary role of sanitizers during cantaloupe washing is to prevent cross contamination of potentially pathogenic bacteria in washwater. Postharvest fungicide sprays or dips are employed to inhibit spoilage-causing fungi. While assessing the compatibility of these antimicrobials based on the measurement of active ingredients levels provides some indication of antimicrobial capacity, there is limited data on whether the interaction between these chemicals in wash water modifies their overall efficacy against relevant microorganisms. The aim of this research was to determine how chlorine- and peroxyacetic acid-based sanitizers interact with commercial guazatine- and imazalil-based fungicide formulations used on cantaloupes, and whether mixing these augments or suppresses anti-microbial activity against relevant human pathogens and spoilage fungi in wash water. The results were unpredictable: while most combinations were antimicrobial, the chlorine-based sanitizer when mixed with the guazatine-based fungicide had significantly reduced efficacy against pathogenic Salmonella spp. (~2.7 log) and the fungal spoilage organisms, Trichothecium roseum and Rhizopus stolonifera. Mixing the chlorine-based sanitizer with an imazalil-based fungicide produced a range of outcomes with antagonistic, indifferent and synergistic interactions observed for the fungal species tested. The peroxyacetic acid-based sanitizer led to indifferent interactions with the guazatine-based fungicide, while antagonism and synergy were observed when mixed with the imazalil-based fungicide. This study demonstrates that mixing postharvest agrichemicals used in the cantaloupe industry may increase the risk of microbial contamination and thereby potentially compromise food safety and quality.


Assuntos
Cucumis melo/microbiologia , Desinfetantes/farmacologia , Conservação de Alimentos/métodos , Fungicidas Industriais/farmacologia , Cloro/química , Cloro/farmacologia , Desinfetantes/química , Interações Medicamentosas , Contaminação de Alimentos/prevenção & controle , Conservação de Alimentos/instrumentação , Frutas/microbiologia , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Fungicidas Industriais/química , Guanidinas/química , Guanidinas/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Ácido Peracético/química , Ácido Peracético/farmacologia , Salmonella/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento
15.
Commun Biol ; 4(1): 682, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083759

RESUMO

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of Coronavirus Disease-2019 (COVID-19), a respiratory disease, has infected almost one hundred million people since the end of 2019, killed over two million, and caused worldwide social and economic disruption. Because the mechanisms of SARS-CoV-2 infection of host cells and its pathogenesis remain largely unclear, there are currently no antiviral drugs with proven efficacy. Besides severe respiratory and systematic symptoms, several comorbidities increase risk of fatal disease outcome. Therefore, it is required to investigate the impacts of COVID-19 on pre-existing diseases of patients, such as cancer and other infectious diseases. In the current study, we report that SARS-CoV-2 encoded proteins and some currently used anti-COVID-19 drugs are able to induce lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV), one of major human oncogenic viruses, through manipulation of intracellular signaling pathways. Our data indicate that those KSHV + patients especially in endemic areas exposure to COVID-19 or undergoing the treatment may have increased risks to develop virus-associated cancers, even after they have fully recovered from COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/complicações , Herpesvirus Humano 8/fisiologia , SARS-CoV-2/fisiologia , Sarcoma de Kaposi/etiologia , Ativação Viral , Azitromicina/farmacologia , Benzamidinas/farmacologia , COVID-19/tratamento farmacológico , Linhagem Celular , Guanidinas/farmacologia , Infecções por Herpesviridae/induzido quimicamente , Infecções por Herpesviridae/etiologia , Herpesvirus Humano 8/efeitos dos fármacos , Humanos , Vírus Oncogênicos/efeitos dos fármacos , Vírus Oncogênicos/fisiologia , SARS-CoV-2/efeitos dos fármacos , Sarcoma de Kaposi/induzido quimicamente , Proteínas Virais/metabolismo , Ativação Viral/efeitos dos fármacos
16.
Biomed Res Int ; 2021: 5591851, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055984

RESUMO

Proteins undergo glycation resulting in the generation of advanced glycation end products (AGEs) that play a central role in the onset and advancement of diabetes-associated secondary complications. Aminoguanidine (AG) acts as an antiglycating agent by inhibiting AGE generation by blocking reactive carbonyl species (RCS) like, methylglyoxal (MGO). Previous studies on antiglycating behavior of AG gave promising results in the treatment of diabetes-associated microvascular complications, but it was discontinued as it was found to be toxic at high concentrations (>10 mmol/L). The current article aims at glycation inhibition by conjugating gold nanoparticles (Gnp) with less concentration of AG (0.5-1.0 mmol/L). The HPLC results showed that AG-Gnp fairly hampers the formation of glycation adducts. Moreover, the in vivo studies revealed AG-Gnp mediated inhibition in the production of total-AGEs and -N ε -(carboxymethyl)lysine (CML) in the diabetic rat model. This inhibition was found to be directly correlated with the antioxidant parameters, blood glucose, insulin, and glycosylated hemoglobin levels. Furthermore, the histopathology of AG-Gnp-treated rats showed good recovery in the damaged pancreatic tissue as compared to diabetic rats. We propose that this approach might increase the efficacy of AG at relatively low concentrations to avoid toxicity and might facilitate to overcome the hazardous actions of antiglycating drugs.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Ouro/química , Guanidinas/farmacologia , Nanopartículas Metálicas/química , Animais , Glicemia , Complicações do Diabetes/metabolismo , Modelos Animais de Doenças , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação/efeitos dos fármacos , Guanidinas/química , Lisina/análogos & derivados , Masculino , Proteínas/metabolismo , Aldeído Pirúvico , Ratos , Ratos Wistar
17.
Geroscience ; 43(3): 1497-1511, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33890206

RESUMO

AMP-activated protein kinase (AMPK) is a central regulator of both lifespan and health across multiple model organisms. ß-Guanidinopropionic acid (GPA) is an endogenous AMPK activator previously shown to improve metabolic function in young and obese mice. In this study, we tested whether age of administration significantly affects the physiological outcomes of GPA administration in mice. We report that intervention starting at 7-8 months (young) results in activation of AMPK signaling and a phenotype consisting of lower body mass, improved glucose control, enhanced exercise tolerance, and altered mitochondrial electron transport chain flux similar to previous reports. When GPA treatment is started at 18-19 months (old), the effect of GPA on AMPK signaling is blunted compared to younger mice despite similar accumulation of GPA in skeletal muscle. Even so, GPA administration in older animals delayed age-related declines in lean mass, improved measures of gait performance and circadian rhythm, and increased fat metabolism as measured by respiratory exchange ratio. These results are likely partially driven by the relative difference in basal function and metabolic plasticity between young and old mice. Our results suggest that age-related declines in AMPK sensitivity may limit potential strategies targeting AMPK signaling in older subjects and suggest that further research and development is required for AMPK activators to realize their full potential.


Assuntos
Proteínas Quinases Ativadas por AMP , Propionatos , Fatores Etários , Animais , Guanidinas/farmacologia , Camundongos
18.
Acc Chem Res ; 54(8): 1866-1877, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33733746

RESUMO

Antimicrobial resistance to existing antibiotics represents one of the greatest threats to human health and is growing at an alarming rate. To further complicate treatment of bacterial infections, many chronic infections are the result of bacterial biofilms that are tolerant to treatment with antibiotics because of the presence of metabolically dormant persister cell populations. Together these threats are creating an increasing burden on the healthcare system, and a "preantibiotic" age is on the horizon if significant action is not taken by the scientific and medical communities. While the golden era of antibiotic discovery (1940s-1960s) produced most of the antibiotic classes in clinical use today, followed by several decades of limited development, there has been a resurgence in antibiotic drug discovery in recent years fueled by the academic and biotech sectors. Historically, great success has been achieved by developing next-generation variants of existing classes of antibiotics, but there remains a dire need for the identification of novel scaffolds and/or antimicrobial targets to drive future efforts to overcome resistance and tolerance. In this regard, there has been no more valuable source for the identification of antibiotics than natural products, with 69-77% of approved antibiotics either being such compounds or being derived from them.Our group has developed a program centered on the chemical synthesis and chemical microbiology of marine natural products with unusual structures and promising levels of activity against multidrug-resistant (MDR) bacterial pathogens. As we are motivated by preparing and studying the biological effects of these molecules, we are not initially pursuing a biological question but instead are allowing the observed phenotypes and activities to guide the ultimate project direction. In this Account, our recent efforts on the synoxazolidinone, lipoxazolidinone, and batzelladine natural products will be discussed and placed in the context of the field's greatest challenges and opportunities. Specifically, the synoxazolidinone family of 4-oxazolidinone-containing natural products has led to the development of several chemical methods to prepare antimicrobial scaffolds and has revealed compounds with potent activity as adjuvants to treat bacterial biofilms. Bearing the same 4-oxazolidinone core, the lipoxazolidinones have proven to be potent single-agent antibiotics. Finally, our synthetic efforts toward the batzelladines revealed analogues with activity against a number of MDR pathogens, highlighted by non-natural stereochemical isomers with superior activity and simplified synthetic access. Taken together, these studies provide several distinct platforms for the development of novel therapeutics that can add to our arsenal of scaffolds for preclinical development and can provide insight into the biochemical processes and pathways that can be targeted by small molecules in the fight against antimicrobial-resistant and -tolerant infections. We hope that this work will serve as inspiration for increased efforts by the scientific community to leverage synthetic chemistry and chemical microbiology toward novel antibiotics that can combat the growing crisis of MDR and tolerant bacterial infections.


Assuntos
Antibacterianos/síntese química , Produtos Biológicos/síntese química , Alcaloides/síntese química , Alcaloides/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Guanidina/análogos & derivados , Guanidina/síntese química , Guanidina/farmacologia , Guanidinas/síntese química , Guanidinas/farmacologia , Testes de Sensibilidade Microbiana , Oxazolidinonas/síntese química , Oxazolidinonas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Estereoisomerismo , Relação Estrutura-Atividade
19.
Bioorg Med Chem Lett ; 40: 127939, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33713780

RESUMO

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.


Assuntos
Benzoatos/farmacologia , Enteropeptidase/antagonistas & inibidores , Guanidinas/farmacologia , Inibidores da Tripsina/farmacologia , Animais , Benzoatos/síntese química , Benzoatos/farmacocinética , Células CHO , Bovinos , Cricetulus , Dieta Hiperlipídica , Fezes/química , Guanidinas/síntese química , Guanidinas/farmacocinética , Humanos , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/enzimologia , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Proteínas/metabolismo , Relação Estrutura-Atividade , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/farmacocinética
20.
EBioMedicine ; 65: 103255, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33676899

RESUMO

BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Ésteres/farmacologia , Guanidinas/farmacologia , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células HEK293 , Humanos , Pulmão/patologia , Pulmão/virologia , Proteínas de Membrana/biossíntese , Simulação de Dinâmica Molecular , Serina Endopeptidases/biossíntese , Serina Proteases/biossíntese , Células Vero , Ativação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
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