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1.
J Virol ; 96(18): e0064622, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36040176

RESUMO

Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. Furthermore, HAV causes acute liver failure or acute-on-chronic liver failure. However, no potent anti-HAV drugs are currently available in the clinical situations. There have been some reports that amantadine, a broad-spectrum antiviral, suppresses HAV replication in vitro. Therefore, we examined the effects of amantadine and rimantadine, derivates of adamantane, on HAV replication, and investigated the mechanisms of these drugs. In the present study, we evaluated the effects of amantadine and rimantadine on HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in cell culture infection systems. Amantadine and rimantadine significantly inhibited HAV replication at the post-entry stage in Huh7 cells. HAV infection inhibited autophagy by suppressing the autophagy marker light chain 3 and reducing number of lysosomes. Proteomic analysis on HAV-infected Huh7 cells treated by amantadine and rimantadine revealed the changes of the expression levels in 42 of 373 immune response-related proteins. Amantadine and rimantadine inhibited HAV replication, partially through the enhancement of autophagy. Taken together, our results suggest a novel mechanism by which HAV replicates along with the inhibition of autophagy and that amantadine and rimantadine inhibit HAV replication by enhancing autophagy. IMPORTANCE Amantadine, a nonspecific antiviral medication, also effectively inhibits HAV replication. Autophagy is an important cellular mechanism in several virus-host cell interactions. The results of this study provide evidence indicating that autophagy is involved in HAV replication and plays a role in the HAV life cycle. In addition, amantadine and its derivative rimantadine suppress HAV replication partly by enhancing autophagy at the post-entry phase of HAV infection in human hepatocytes. Amantadine may be useful for the control of acute HAV infection by inhibiting cellular autophagy pathways during HAV infection processes.


Assuntos
Amantadina , Autofagia , Vírus da Hepatite A , Hepatite A , Rimantadina , Replicação Viral , Amantadina/farmacologia , Amantadina/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Autofagia/efeitos dos fármacos , Linhagem Celular , Hepatite A/tratamento farmacológico , Anticorpos Anti-Hepatite A , Vírus da Hepatite A/efeitos dos fármacos , Humanos , Proteômica , Rimantadina/farmacologia , Rimantadina/uso terapêutico , Replicação Viral/efeitos dos fármacos
2.
Acta Trop ; 227: 106300, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34979144

RESUMO

Most of the patients infected with Chikungunya virus (CHIKV) develop chronic manifestations characterized by pain and deformity in joints, impacting their quality of life. The aminoadamantanes, in their turn, have been exploited due to their biological activities, with amantadine and memantine recently described with anti-CHIKV activities. Here we evaluated the antiviral activity of rimantadine hydrochloride (rtdH), a well-known antiviral agent against influenza A, its platinum complex (Pt-rtd), and the precursor cis-[PtCl2(dmso)2], against CHIKV infection in vitro. The rtdH demonstrated significant antiviral activity in all stages of CHIKV replication (29% in pre-treatment; 57% in early stages of infection; 60% in post-entry stages). The Pt-rtd complex protected the cells against infection in 92%, inhibited 100% of viral entry, mainly by a virucidal effect, and impaired 60% of post-entry stages. Alternatively, cis-[PtCl2(dmso)2] impaired viral entry in 100% and post-entry steps in 60%, but had no effect in protecting cells when administered prior to CHIKV infection. Collectively, the obtained data demonstrated that rtdH and Pt-rtd significantly interfered in the early stages of CHIKV life cycle, with the strongest effect observed to Pt-rtd complex, which reduced up to 100% of CHIKV infection. Moreover, molecular docking analysis and infrared spectroscopy data (ATR-FTIR) suggest an interaction of Pt-rtd with CHIKV glycoproteins, potentially related to the mechanism of inhibition of viral entry by Pt-rtd. Through a migration retardation assay, it was also shown that Pt-rtd and cis-[PtCl2(dmso)2] interacted with the dsRNA in 87% and 100%, respectively. The obtained results highlight the repurposing potential of rtdH as an anti-CHIKV drug, as well as the synthesis of promising platinum(II) metallodrugs with potential application for the treatment of CHIKV infections. Importance Chikungunya fever is a disease that can result in persistent symptoms due to the chronic infection process. Infected patients can develop physical disability, resulting and high costs to the health system and significant impacts on the quality of life of affected individuals. Additionally, there are no licensed vaccines or antivirals against the Chikungunya virus (CHIKV) and the virus is easily transmitted due to the abundance of viable vectors in epidemic regions. In this context, our study highlights the repurposing potential of the commercial drug rimantadine hydrochloride (rtdH) as an antiviral agent for the treatment of CHIKV infections. Moreover, our data demonstrated that a platinum(II)-rimantadine metallodrug (Pt-rtd) poses as a potent anti-CHIKV molecule with potential application for the treatment of Chikungunya fever. Altogether, rtdH and Pt-rtd significantly interfered in the early stages of CHIKV life cycle, reducing up to 100% of CHIKV infection in vitro.


Assuntos
Febre de Chikungunya , Rimantadina , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Platina/farmacologia , Platina/uso terapêutico , Qualidade de Vida , Rimantadina/farmacologia , Rimantadina/uso terapêutico , Replicação Viral
3.
Commun Biol ; 4(1): 1347, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853399

RESUMO

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.


Assuntos
Amantadina/farmacologia , Amilorida/análogos & derivados , Antivirais/farmacologia , Rimantadina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/fisiologia , Amilorida/farmacologia , Canais Iônicos/fisiologia
4.
Viruses ; 13(10)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34696509

RESUMO

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.


Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Memantina/farmacologia , Rimantadina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Dinamarca , Reposicionamento de Medicamentos , Humanos , Canais Iônicos/antagonistas & inibidores , Células Vero
5.
Artigo em Inglês | MEDLINE | ID: mdl-33989121

RESUMO

In this study, molecularly imprinted microspheres of a type capable of recognising amantadine and rimantadine were first synthesised, and three fluorescent tracers based on dansyl chloride, fluorescein isothiocyanate and 5-carboxytetramethylrhodamine were also synthesised. These reagents were used to develop and optimise a direct competitive fluorescence method on conventional 96-well microplate for detection of the two analytes. Results showed that this method achieved simple operation procedure, rapid assay process (30 min), high sensitivity (limits of detection 0.04-0.05 ng mL-1) and acceptable recycle performance (five times). After optimisation of several parameters, this method was used to detect amantadine and rimantadine in chicken muscle samples. Their recoveries from standards fortified blank samples were in the range of 62.3-93.7%. The analysis results for some real chicken samples were consistent with a confirmatory LC-MS/MS method. Therefore, this method could be used as a rapid, simple and accurate tool for routine screening the residues of amantadine and rimantadine in a large number of chicken muscle samples.


Assuntos
Amantadina/análise , Microesferas , Impressão Molecular , Rimantadina/análise , Animais , Galinhas , Estrutura Molecular , Músculos/química , Espectrometria de Fluorescência
6.
J Gen Virol ; 102(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33709903

RESUMO

The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments and thus helping virus assembly and release. This activity is essential for virus infectivity, making viroporins an attractive target for drug development. The protein sequence and drug sensitivity of p7 vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Here, we investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a-b, 2a-b, 3a and 4a using patch-clamp electrophysiology and cell-based assays. We established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity and its sensitivity to inhibitors. The results from the cell viability assay were consistent with control measurements using established assays of haemadsorption and intracellular pH, and agreed with data from patch-clamp electrophysiology. Hexamethylene amiloride (HMA) was the most potent inhibitor of p7 activity, but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. The alkyl-chain iminosugars NB-DNJ, NN-DNJ and NN-DGJ were tested and their activity was found to be genotype-specific. In the current study, we introduce cell viability assays as a rapid and cost-efficient technique to assess viroporin activity and identify channel inhibitors as potential novel antiviral drugs.


Assuntos
Hepacivirus/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Montagem de Vírus , Liberação de Vírus , Amantadina/farmacologia , Sequência de Aminoácidos , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Hepacivirus/efeitos dos fármacos , Humanos , Técnicas de Patch-Clamp , Rimantadina/farmacologia
7.
J Comput Aided Mol Des ; 35(1): 95-104, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33037548

RESUMO

We investigate the binding of native ß-cyclodextrin (ß-CD) and eight novel ß-CD derivatives with two different guest compounds, using isothermal calorimetry and 2D NOESY NMR. In all cases, the stoichiometry is 1:1 and binding is exothermic. Overall, modifications at the 3' position of ß-CD, which is at the secondary face, weaken binding by several kJ/mol relative to native ß-CD, while modifications at the 6' position (primary face) maintain or somewhat reduce the binding affinity. The variations in binding enthalpy are larger than the variations in binding free energy, so entropy-enthalpy compensation is observed. Characterization of the bound conformations with NOESY NMR shows that the polar groups of the guests may be situated at either face, depending on the host molecule, and, in some cases, both orientations are populated. The present results were used in the SAMPL7 blinded prediction challenge whose results are detailed in the same special issue of JCAMD.


Assuntos
Ciclodextrinas/metabolismo , Cicloexanóis/metabolismo , Rimantadina/metabolismo , Termodinâmica , beta-Ciclodextrinas/metabolismo , Ciclodextrinas/química , Cicloexanóis/química , Entropia , Estrutura Molecular , Rimantadina/química , beta-Ciclodextrinas/química
8.
Biophys J ; 119(9): 1811-1820, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33080223

RESUMO

The ubiquitous mutation from serine (WT) to asparagine at residue 31 (S31N) in the influenza A M2 channel renders it insensitive to amantadine (AMT) and rimantadine (RMT) block, but it is unknown whether the inhibition results from weak binding or incomplete block. Two-electrode voltage clamp (TEVC) of transfected Xenopus oocytes revealed that the M2 S31N channel is essentially fully blocked by AMT at 10 mM, demonstrating that, albeit weak, AMT binding in a channel results in complete block of its proton current. In contrast, RMT achieves only a modest degree of block in the M2 S31N channel at 1 mM, with very little increase in block at 10 mM, indicating that the RMT binding site in the channel saturates with only modest block. From exponential curve fits to families of proton current wash-in and wash-out traces, the association rate constant (k1) is somewhat decreased for both AMT and RMT in the S31N, but the dissociation rate constant (k2) is dramatically increased compared with WT. The potentials of mean force (PMF) from adaptive biasing force (ABF) molecular dynamics simulations predict that rate constants should be exquisitely sensitive to the charge state of the His37 selectivity filter of M2. With one exception out of eight cases, predictions from the simulations with one and three charged side chains bracket the experimental rate constants, as expected for the acidic bath used in the TEVC assay. From simulations, the weak binding can be accounted for by changes in the potentials of mean force, but the partial block by RMT remains unexplained.


Assuntos
Influenza Humana , Rimantadina , Amantadina/farmacologia , Antivirais/farmacologia , Transtornos Dissociativos , Humanos , Proteínas da Matriz Viral/genética
9.
Molecules ; 25(17)2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32883012

RESUMO

A series of nineteen amino acid analogues of amantadine (Amt) and rimantadine (Rim) were synthesized and their antiviral activity was evaluated against influenza virus A (H3N2). Among these analogues, the conjugation of rimantadine with glycine illustrated high antiviral activity combined with low cytotoxicity. Moreover, this compound presented a profoundly high stability after in vitro incubation in human plasma for 24 h. Its thermal stability was established using differential and gravimetric thermal analysis. The crystal structure of glycyl-rimantadine revealed that it crystallizes in the orthorhombic Pbca space group. The structure-activity relationship for this class of compounds was established, with CoMFA (Comparative Molecular Field Analysis) 3D-Quantitative Structure Activity Relationships (3D-QSAR) studies predicting the activities of synthetic molecules. In addition, molecular docking studies were conducted, revealing the structural requirements for the activity of the synthetic molecules.


Assuntos
Adamantano/análogos & derivados , Adamantano/farmacologia , Antivirais/farmacologia , Simulação por Computador , Orthomyxoviridae/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Adamantano/síntese química , Adamantano/química , Animais , Antivirais/síntese química , Antivirais/química , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Cristalografia por Raios X , Análise Diferencial Térmica , Cães , Estabilidade de Medicamentos , Humanos , Ligação de Hidrogênio , Análise dos Mínimos Quadrados , Células Madin Darby de Rim Canino , Conformação Molecular , Simulação de Acoplamento Molecular , Domínios Proteicos , Rimantadina/sangue , Rimantadina/química , Temperatura , Proteínas da Matriz Viral/química
10.
PLoS Pathog ; 16(8): e1008716, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32780760

RESUMO

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.


Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/virologia , Rimantadina/farmacologia , Proteínas da Matriz Viral/antagonistas & inibidores , Zanamivir/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
11.
Comput Biol Med ; 122: 103848, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658735

RESUMO

The recent outbreak of coronavirus disease-19 (COVID-19) continues to drastically affect healthcare throughout the world. To date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection. Therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against SARS-CoV-2 for lead identification and repurposing prospects. This study addresses the identification of small molecules that specifically bind to any of the three essential proteins (RdRp, 3CL-protease and helicase) of SARS-CoV-2. By applying computational approaches we screened a library of 4574 compounds also containing FDA-approved drugs against these viral proteins. Shortlisted hits from initial screening were subjected to iterative docking with the respective proteins. Ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds. Finally, to minimize chances of false positives, we performed docking of the identified molecules with 100 irrelevant proteins of diverse classes thereby ruling out the non-specific binding. Three FDA-approved drugs showed binding to 3CL-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation. A drug-like molecule showed binding to RdRp in its catalytic pocket blocking the key catalytic residues. Two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis. This study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Amidas , COVID-19 , Carbamatos , Domínio Catalítico , Simulação por Computador , Ciclopropanos , Dimerização , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Inibidores de Proteases/química , Quinoxalinas/farmacologia , Rimantadina/farmacologia , SARS-CoV-2 , Sulfonamidas , Proteínas Virais/química
12.
Vopr Virusol ; 65(1): 16-20, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32496716

RESUMO

INTRODUCTION: Adamantanthane-type drugs such as rimantadine and amantadine have long been used to treat diseases caused by influenza A virus. However, as a result of the mutations, influenza viruses have become resistant to aminoadamantans. The target for these drugs was the protein channel M2. Influenza A virus M2 viroporin in the protein shell forms fairly specific ion channels with a diameter of about 11 Å, specializing in transporting protons inside the viral particle (virion). Restoration of the antiviral properties of adamantanthane-type drugs consists in the selection of advanced functional groups bound by the carbocycle to find new sites of binding to the protein target M2. The purpose of the study is to identify the antiviral properties of new adamantanum derivatives to the pandemic strain of influenza A virus in vitro. MATERIAL AND METHODS: Compounds of aminoadamantans with amino acids and other organic molecules were obtained by classical peptide synthesis methods. The structure of the compound was tested by means of physical and chemical methods. Antiviral properties of synthetic compounds were studied in vitro on monolayer MDCK cells infected with pandemic strain of influenza A/California/07/2009 virus in two schemes of administration of investigated compounds and virus. RESULTS: The reference strain of the influenza virus A/California/07/2009(H1N1) was sensitive to the compounds under test in varying degrees. The antiviral activity of the compounds was expressed in a 50% inhibitory concentration (IС50) ranging from 0.5 to 2.5 мкM, which is generally a good indicator for the Rimantadine/Amantadine resistant strain. DISCUSSION: The values of the IС50 for compounds introduced two hours before contact with the virus were slightly higher than those for single-moment introduction of the substance and virus. The effect of increasing the inhibitory concentration in the prophylactic scheme of compounds was valid for all compounds of the experiment. CONCLUSION: The presented synthetic compounds are active against the variant of influenza A virus resistant to Rimantadine and Amantadine preparations. The obtained compounds can be used as model structures for creation of a new drug of direct action against advanced strains of influenza A virus.


Assuntos
Adamantano/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Adamantano/análogos & derivados , Animais , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/genética , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mutação , Rimantadina/efeitos adversos , Rimantadina/farmacologia
13.
Mol Biol (Mosk) ; 54(2): 321-332, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392203

RESUMO

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H^(+) proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.


Assuntos
Antivirais/farmacologia , Compostos Aza/farmacologia , Ciclo-Octanos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Proteínas da Matriz Viral/antagonistas & inibidores , Amantadina , Rimantadina
14.
J Sep Sci ; 43(3): 631-638, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31680400

RESUMO

The first dispersive liquid liquid microextraction scheme followed by liquid chromatography-post column derivatization for the determination of the antiviral drug rimantadine in urine samples is demonstrated. The effect of the type and volume of organic extraction solvent, type and volume of disperser solvent, sample pH, ionic strength, extraction time, and centrifugation speed on the extraction efficiency were studied. Rimantadine and the internal standard (amantadine) were chromatographed using a reversed phase monolithic stationary phase with a mixture of equal volumes of methanol and phosphate buffer (pH = 3) as mobile phase. On-line post-column derivatization of the analyte was performed using a "two-stream" manifold with o-phthalaldehyde and N-acetyl-cysteine at alkaline medium. Under the optimized extraction conditions, the enrichment factor of rimantadine was 58. The linear range was 5-100 µg/L with correlation coefficient r of 0.9984 while the limit of detection achieved was 0.5 µg/L. The within-day and between-day precision for the tested concentration levels were less than 14.3% and the mean recoveries obtained from the spiked samples were ranged between 87.5 and 113.9%. The main advantages of the proposed method are the simplicity of operation, rapidity, low cost, and low limit of detection of the analyte.


Assuntos
Microextração em Fase Líquida , Rimantadina/urina , Cromatografia Líquida de Alta Pressão/instrumentação , Voluntários Saudáveis , Humanos , Microextração em Fase Líquida/instrumentação
15.
Cancer Res ; 80(4): 732-746, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31848196

RESUMO

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias de Cabeça e Pescoço/terapia , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/terapia , Rimantadina/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Adolescente , Adulto , Idoso , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral/transplante , Quimiorradioterapia/métodos , Estudos de Coortes , Modelos Animais de Doenças , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/virologia , Voluntários Saudáveis , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/antagonistas & inibidores , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Células RAW 264.7 , RNA-Seq , Rimantadina/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Adulto Jovem
17.
Molecules ; 24(21)2019 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-31684129

RESUMO

A zone-fluidics (ZF) based automated fluorimetric sensor for the determination of pharmaceutically active adamantine derivatives, i.e., amantadine (AMA), memantine (MEM) and rimantadine (RIM) is reported. Discrete zones of the analytes and reagents (o-phthalaldehyde and N-acetylcysteine) mix and react under stopped-flow conditions to yield fluorescent iso-indole derivatives (λex/ λem = 340/455 nm). The proposed ZF sensor was developed and validated to prove suitable for quality control tests (assay and content uniformity) of commercially available formulations purchased from the Greek market (EU licensed) and from non-EU web-pharmacies at a sampling rate of 16 h-1. Interestingly, a formulation obtained through the internet and produced in a third-non-EU-country (AMA capsules, 100 mg per cap), was found to be out of specifications (mean assay of 85.3%); a validated HPLC method was also applied for confirmatory purposes.


Assuntos
Amantadina/isolamento & purificação , Fluorometria/métodos , Memantina/isolamento & purificação , Rimantadina/isolamento & purificação , Amantadina/química , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes/química , Indóis/química , Memantina/química , Microfluídica , Rimantadina/química
18.
Molecules ; 24(9)2019 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-31083636

RESUMO

This work discloses a new procedure for the resolution of commercially available racemic rimantadine hydrochloride to enantiomerically pure (S)-rimantadine using (R)-phenoxypropionic acid as a recyclable resolving reagent. Good chemical yields, operational ease, and low-cost structure underscore the preparative value of this method for the production of enantiomerically pure rimantadine for medicinal or synthetic studies.


Assuntos
Rimantadina/química , Aminas/química , Estrutura Molecular , Estereoisomerismo
20.
Interdiscip Sci ; 11(2): 292-299, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30194627

RESUMO

HCV p7 protein is a cation-selective ion channel, playing an essential role during the life cycle of HCV viruses. To understand the cation-selective mechanism, we constructed a hexameric model in lipid bilayers of HCV p7 protein for HCB JFH-1 strain, genotype 2a. In this structural model, His9 and Val6 were key factors for the HCV cation-selective ion channel. The histidine residues at position 9 in the hexameric model formed a first gate for HCV p7 channel, acting as a selectivity filter for cations. The valines mentioned above formed a second gate for HCV p7 channel, serving as a hydrophobic filter for the dehydrated cations. The binding pocket for the channel blockers, e.g., amantadine and rimantadine, was composed of residues 20-26 in H2 helix and 52-60 in H3 helix in i + 2 monomer. However, the molecular volumes for both amantadine and rimantadine were too small for the binding pocket of HCV p7 channel. Thus, designing a compound similar with rimantadine and having much larger volume would be an effective strategy for discovering inhibitors against HCV p7 channel. To achieve this point, we used rimantadine as a structural template to search ChEMBL database for the candidates employing favorable binding affinities to HCV p7 channel. As a result, six candidates were identified to have potential to be novel inhibitors against HCV p7 channel.


Assuntos
Antivirais/farmacologia , Biologia Computacional/métodos , Desenho de Fármacos , Hepacivirus/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Hepacivirus/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Rimantadina/química , Rimantadina/farmacologia , Relação Estrutura-Atividade , Proteínas Virais/química
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