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1.
Chem Res Toxicol ; 31(2): 68-80, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29355304

RESUMO

Cytochromes P450 (CYPs) oxidize alkylated amines commonly found in drugs and other biologically active molecules, cleaving them into an amine and an aldehyde. Metabolic studies usually neglect to report or investigate aldehydes, even though they can be toxic. It is assumed that they are efficiently detoxified into carboxylic acids and alcohols. Nevertheless, some aldehydes are reactive and escape detoxification pathways to cause adverse events by forming DNA and protein adducts. Herein, we modeled N-dealkylations that produce both amine and aldehyde metabolites and then predicted the reactivity of the aldehyde. This model used a deep learning approach previously developed by our group to predict other types of drug metabolism. In this study, we trained the model to predict N-dealkylation by human liver microsomes (HLM), finding that including isozyme-specific metabolism data alongside HLM data significantly improved results. The final HLM model accurately predicted the site of N-dealkylation within metabolized substrates (97% top-two and 94% area under the ROC curve). Next, we combined the metabolism, metabolite structure prediction, and previously published reactivity models into a bioactivation model. This combined model predicted the structure of the most likely reactive metabolite of a small validation set of drug-like molecules known to be bioactivated by N-dealkylation. Applying this model to approved and withdrawn medicines, we found that aldehyde metabolites produced from N-dealkylation may explain the hepatotoxicity of several drugs: indinavir, piperacillin, verapamil, and ziprasidone. Our results suggest that N-dealkylation may be an under-appreciated bioactivation pathway, especially in clinical contexts where aldehyde detoxification pathways are inhibited. Moreover, this is the first report of a bioactivation model constructed by combining a metabolism and reactivity model. These results raise hope that more comprehensive models of bioactivation are possible. The model developed in this study is available at http://swami.wustl.edu/xenosite/ .


Assuntos
Indinavir/metabolismo , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Piperacilina/metabolismo , Piperazinas/metabolismo , Tiazóis/metabolismo , Verapamil/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Aminas/química , Aminas/metabolismo , Remoção de Radical Alquila , Humanos , Indinavir/farmacologia , Fígado/efeitos dos fármacos , Microssomos Hepáticos/química , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Piperacilina/farmacologia , Piperazinas/farmacologia , Tiazóis/farmacologia , Verapamil/farmacologia
2.
Behav Brain Res ; 338: 32-39, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28943428

RESUMO

The insulin-regulated glucose transporter, GluT4, is a key molecule in peripheral insulin signaling. Although GluT4 is abundantly expressed in neurons of specific brain regions such as the hippocampus, the functional role of neuronal GluT4 is unclear. Here, we used pharmacological inhibition of GluT4-mediated glucose uptake to determine whether GluT4 mediates insulin-mediated glucose uptake in the hippocampus. Consistent with previous reports, we found that glucose utilization increased in the dorsal hippocampus of male rats during spontaneous alternation (SA), a hippocampally-mediated spatial working memory task. We previously showed that insulin signaling within the hippocampus is required for processing this task, and that administration of exogenous insulin enhances performance. At baseline levels of hippocampal insulin, inhibition of GluT4-mediated glucose uptake did not affect SA performance. However, inhibition of an upstream regulator of GluT4, Akt, did impair SA performance. Conversely, when a memory-enhancing dose of insulin was delivered to the hippocampus prior to SA-testing, inhibition of GluT4-mediated glucose transport prevented cognitive enhancement. These data suggest that baseline hippocampal cognitive processing does not require functional hippocampal GluT4, but that cognitive enhancement by supra-baseline insulin does. Consistent with these findings, we found that in neuronal cell culture, insulin increases glucose utilization in a GluT4-dependent manner. Collectively, these data demonstrate a key role for GluT4 in transducing the procognitive effects of elevated hippocampal insulin.


Assuntos
Transportador de Glucose Tipo 4/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Insulina/farmacologia , Memória de Curto Prazo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Animais , Sulfato de Atazanavir/farmacologia , Glucose/metabolismo , Hipocampo/metabolismo , Indinavir/farmacologia , Masculino , Microinjeções , Nelfinavir/farmacologia , Ratos , Ratos Sprague-Dawley
3.
Diabetes ; 66(3): 587-597, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27797912

RESUMO

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.


Assuntos
Glicemia/metabolismo , Encéfalo/metabolismo , Intolerância à Glucose/genética , Hipoglicemia/genética , Resistência à Insulina/genética , Animais , Western Blotting , Dieta Hiperlipídica , Epinefrina/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4 , Homeostase/genética , Hipotálamo/citologia , Hipotálamo/metabolismo , Técnicas In Vitro , Indinavir/farmacologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley
4.
Neurotoxicology ; 56: 1-6, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27345270

RESUMO

In this study, we sought to investigate how concomitant hyperglycemia influences the impact of combination antiretroviral therapy on blood-brain barrier (BBB) endothelial function. Immortalized human brain microvascular endothelial cell line (hCMEC/D3) was exposed to azidothymidine (AZT; a nucleoside reverse transcriptase inhibitor) and/or indinavir (IND; protease inhibitor) in normal glycemic (5.5mM) or hyperglycemic (HG; 25mM) media containing D-glucose for 24-72h. Cellular reactive oxygen species (ROS) and mitochondria-specific superoxide levels were assayed in addition to membrane potential to determine the extent of mitochondrial dysfunction. Nrf2 expression was analyzed by immunofluorescence. Our results indicated a significant increase in BBB endothelial toxicity (decreased ATP) by HG and AZT+IND with progression of time (24-72h). Concurrent HG and antiviral drug combination synergistically elevated BBB endothelial ROS induced by either condition alone. Further, HG and AZT+IND mutually interact to elicit a pronounced increase in mitochondrial superoxide levels post 24h (vs. either condition alone or controls). In addition, HG and AZT+IND complemented each other to induce potential loss of mitochondrial membrane potential. While HG or AZT+IND alone for 24h increased Nrf2 nuclear distribution, co-exposure conditions induced a potential loss of Nrf2 expression/nuclear translocation in BBB endothelium. In summary, our data strongly suggest that antiretroviral drug combination potentially interacts with concomitant HG and triggers exacerbated mitochondrial dysfunction and BBB endothelial toxicity, possibly through dysregulation of Nrf2 signaling. Thus, this study warrants the critical need for safety evaluation and monitoring of neurovascular complications of HAART regimens in HIV-infected diabetic patient cohort.


Assuntos
Antivirais/farmacocinética , Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Indinavir/farmacologia , Zidovudina/farmacologia , Trifosfato de Adenosina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Fatores de Tempo
5.
PLoS One ; 11(3): e0151286, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26982200

RESUMO

BACKGROUND: HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. RESULTS: A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. CONCLUSIONS: This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.


Assuntos
Protease de HIV/metabolismo , Schizosaccharomyces/enzimologia , Inibidores da Protease de HIV/farmacologia , Indinavir/farmacologia , Estresse Oxidativo , Schizosaccharomyces/efeitos dos fármacos
6.
Mol Cell Endocrinol ; 426: 101-12, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-26911933

RESUMO

The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of ß-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and ß-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Indinavir/farmacologia , Animais , Apresentação do Antígeno/genética , Feminino , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Imunomodulação/genética , Camundongos Endogâmicos NOD , Timo/efeitos dos fármacos , Timo/metabolismo , Transcriptoma
7.
Molecules ; 20(12): 22113-27, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26690396

RESUMO

Patients receiving anti-retroviral drug treatment are sometimes simultaneously taking herbal remedies, which may result in pharmacokinetic herb-drug interactions. This study aimed to determine if pharmacokinetic interactions exist between selected commercially available herbal products (i.e., Linctagon Forte(®), Viral Choice(®) and Canova(®)) and indinavir in terms of in vitro transport and metabolism. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in the presence and absence of the selected herbal products and verapamil (positive control). Metabolism of indinavir was determined in LS180 cells in the presence and absence of the selected herbal products as well as ketoconazole (positive control). The secretory transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte(®) and Viral Choice(®) when compared to that of indinavir alone. Canova(®) only slightly affected the efflux of indinavir compared to that of the control group. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir's bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made.


Assuntos
Fármacos Anti-HIV/farmacologia , Venenos de Crotalídeos/farmacologia , Interações Ervas-Drogas , Indinavir/farmacologia , Extratos Vegetais/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Células CACO-2 , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Humanos , Cetoconazol/farmacologia , Verapamil/farmacologia
8.
J Comput Chem ; 36(25): 1885-92, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26198456

RESUMO

Human immunodeficiency virus (HIV)-1 protease is one of the most promising drug target commonly utilized to combat Acquired Immune Deficiency Syndrome (AIDS). However, with the emergence of drug resistance arising from mutations, the efficiency of protease inhibitors (PIs) as a viable treatment for AIDS has been greatly reduced. I50V mutation as one of the most significant mutations occurring in HIV-1 protease will be investigated in this study. Molecular dynamics (MD) simulation was utilized to examine the effect of I50V mutation on the binding of two PIs namely indinavir and amprenavir to HIV-1 protease. Prior to the simulations conducted, the electron density distributions of the PI and each residue in HIV-1 protease are derived by combining quantum fragmentation approach molecular fractionation with conjugate caps and Poisson-Boltzmann solvation model based on polarized protein-specific charge scheme. The atomic charges of the binding complex are subsequently fitted using delta restrained electrostatic potential (delta-RESP) method to overcome the poor charge determination of buried atom. This way, both intraprotease polarization and the polarization between protease and the PI are incorporated into partial atomic charges. Through this study, the mutation-induced affinity variations were calculated and significant agreement between experiments and MD simulations conducted was observed for both HIV-1 protease-drug complexes. In addition, the mechanism governing the decrease in the binding affinity of PI in the presence of I50V mutation was also explored to provide insights pertaining to the design of the next generation of anti-HIV drugs.


Assuntos
Carbamatos/farmacologia , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/enzimologia , Indinavir/farmacologia , Mutação Puntual , Sulfonamidas/farmacologia , Infecções por HIV/tratamento farmacológico , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/química , HIV-1/genética , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica
9.
Biofactors ; 41(3): 198-208, 2015 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-26040542

RESUMO

This work presents results concerning influence of indinavir (protease inhibitor, PI(1)) and zidovudine (nucleoside and nucleotide inhibitor of reverse transcriptase, NRTI) administered to pregnant Wistar rat females on organic and mineral constituents of bones and teeth (mandibles, skulls, tibiae, femurs, and incisors) of their offspring at the age of: 7, 14, and 28 days studied by means of induced laser and X-ray fluorescence spectroscopy supported by digital radiography. Influence of indinavir administered to pregnant female rats on bone of their offspring revealed mainly in changes of mineral concentration: lowered Ca concentration and disturbances of trace elements. Zidovudine influenced organic matter more than inorganic matrix which was seen in enhancement of LIF fluorescence. However, there was also an unexpected increase of bone density for rats from zidovudine group, unlike indinavir group, observed. Our studies suggest that studied antiretroviral agents given to pregnant women, may have different destructive impact on bone state of their offspring in the first period of life. Maternal administration of zidovudine may delay development of organic matrix, while indinavir may have adverse effects on inorganic structure.


Assuntos
Antirretrovirais/farmacologia , Cálcio/metabolismo , Indinavir/farmacologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Oligoelementos/metabolismo , Zidovudina/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/crescimento & desenvolvimento , Fêmur/metabolismo , Humanos , Incisivo/efeitos dos fármacos , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Mandíbula/efeitos dos fármacos , Mandíbula/crescimento & desenvolvimento , Mandíbula/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Wistar , Crânio/efeitos dos fármacos , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo
10.
Sci Rep ; 5: 10323, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25988960

RESUMO

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.


Assuntos
Farmacorresistência Viral/genética , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Fármacos Anti-HIV/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Fluorometria , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Indinavir/farmacologia , Lopinavir/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologia
11.
Bioorg Med Chem Lett ; 25(7): 1538-40, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25704890

RESUMO

The inhibitive activities of the human immunodeficiency virus protease inhibitors ritonavir (RTV) boosted indinavir (IDV) and RTV boosted lopinavir (LPV) for erythrocytic stage malaria were evaluated in rhesus macaques. The IDV/RTV regimen effectively inhibits the replication of Plasmodium knowlesi with clinically relevant doses, whereas the LPV/RTV regimen did not show activity against plasmodium infection.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/efeitos dos fármacos , Indinavir/farmacologia , Malária/tratamento farmacológico , Plasmodium knowlesi/efeitos dos fármacos , Ritonavir/farmacologia , Animais , Relação Dose-Resposta a Droga , Eritrócitos/parasitologia , Macaca mulatta , Malária/parasitologia , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
12.
Biomaterials ; 37: 383-94, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25453966

RESUMO

With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million people bear the infection worldwide. One of the main causes of therapeutic failure is the frequent administration of several antiretrovirals that results in low patient compliance and treatment cessation. In this work, we have developed an innovative Nanoparticle-in-Microparticle Delivery System (NiMDS) comprised of pure drug nanocrystals of the potent protease inhibitor indinavir free base (used as poorly water-soluble model protease inhibitor) produced by nanoprecipitation that were encapsulated within mucoadhesive polymeric microparticles. Pure drug nanoparticles and microparticles were thoroughly characterized by diverse complementary techniques. NiMDSs displayed an encapsulation efficiency of approximately 100% and a drug loading capacity of up to 43% w/w. In addition, mucoadhesiveness assays ex vivo conducted with bovine gut showed that film-coated microparticles were retained for more than 6 h. Finally, pharmacokinetics studies in mongrel dogs showed a dramatic 47- and 95-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the free unprocessed drug. These results support the outstanding performance of this platform to reduce the dose and the frequency of administration of protease inhibitors, a crucial step to overcome the current patient-incompliant therapy.


Assuntos
Sistemas de Liberação de Medicamentos , Indinavir/administração & dosagem , Indinavir/farmacocinética , Nanopartículas/química , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacocinética , Adesividade/efeitos dos fármacos , Administração Oral , Alginatos/química , Animais , Bovinos , Quitosana/química , Cães , Relação Dose-Resposta a Droga , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Indinavir/sangue , Indinavir/farmacologia , Nanopartículas/ultraestrutura , Inibidores de Proteases/sangue , Inibidores de Proteases/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo , Difração de Raios X
13.
PLoS One ; 9(12): e113957, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25464510

RESUMO

BACKGROUND: Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas' disease. METHODOLOGY AND PRINCIPAL FINDINGS: HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages. CONCLUSIONS AND SIGNIFICANCE: The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.


Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores de Proteases/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Carbamatos/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Indinavir/farmacologia , Insetos Vetores/parasitologia , Lopinavir/farmacologia , Microscopia Eletrônica de Transmissão , Nelfinavir/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologia , Sulfonamidas/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
14.
PLoS Comput Biol ; 10(11): e1003886, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25375675

RESUMO

Despite the success of highly active antiretroviral therapy (HAART) in the management of human immunodeficiency virus (HIV)-1 infection, virological failure due to drug resistance development remains a major challenge. Resistant mutants display reduced drug susceptibilities, but in the absence of drug, they generally have a lower fitness than the wild type, owing to a mutation-incurred cost. The interaction between these fitness costs and drug resistance dictates the appearance of mutants and influences viral suppression and therapeutic success. Assessing in vivo viral fitness is a challenging task and yet one that has significant clinical relevance. Here, we present a new computational modelling approach for estimating viral fitness that relies on common sparse cross-sectional clinical data by combining statistical approaches to learn drug-specific mutational pathways and resistance factors with viral dynamics models to represent the host-virus interaction and actions of drug mechanistically. We estimate in vivo fitness characteristics of mutant genotypes for two antiretroviral drugs, the reverse transcriptase inhibitor zidovudine (ZDV) and the protease inhibitor indinavir (IDV). Well-known features of HIV-1 fitness landscapes are recovered, both in the absence and presence of drugs. We quantify the complex interplay between fitness costs and resistance by computing selective advantages for different mutants. Our approach extends naturally to multiple drugs and we illustrate this by simulating a dual therapy with ZDV and IDV to assess therapy failure. The combined statistical and dynamical modelling approach may help in dissecting the effects of fitness costs and resistance with the ultimate aim of assisting the choice of salvage therapies after treatment failure.


Assuntos
Aptidão Genética , Infecções por HIV/virologia , HIV-1/genética , Modelos Biológicos , Fármacos Anti-HIV/farmacologia , Estudos Transversais , Farmacorresistência Viral/genética , Genótipo , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Indinavir/farmacologia , Mutação , Resultado do Tratamento , Zidovudina/farmacologia
15.
Mem Inst Oswaldo Cruz ; 109(4): 484-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25075786

RESUMO

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.


Assuntos
Gorduras na Dieta/sangue , Inibidores da Protease de HIV/farmacologia , Indinavir/farmacologia , Animais , Autoanticorpos/sangue , Biomarcadores/sangue , Glicemia/análise , Colesterol/sangue , Cricetinae , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Lipoproteínas LDL/efeitos dos fármacos , Modelos Animais , Triglicerídeos/sangue
16.
J Med Chem ; 57(14): 6266-72, 2014 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-25006983

RESUMO

HTLV-1 protease (HTLV-1 PR) is an aspartic protease which represents a promising drug target for the discovery of novel anti-HTLV-1 drugs. The X-ray structure of HTLV-1 PR in complex with the well-known and approved HIV-1 PR inhibitor Indinavir was determined at 2.40 Å resolution. In this contribution, we describe the first crystal structure in complex with a nonpeptidic inhibitor that accounts for rationalizing the rather moderate affinity of Indinavir against HTLV-1 PR and provides the basis for further structure-guided optimization strategies.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Indinavir/química , Indinavir/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
17.
Am J Physiol Regul Integr Comp Physiol ; 307(6): R711-20, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24990858

RESUMO

Pathologies in which insulin is dysregulated, including diabetes, can disrupt central vagal circuitry, leading to gastrointestinal and other autonomic dysfunction. Insulin affects whole body metabolism through central mechanisms and is transported into the brain stem dorsal motor nucleus of the vagus (DMV) and nucleus tractus solitarius (NTS), which mediate parasympathetic visceral regulation. The NTS receives viscerosensory vagal input and projects heavily to the DMV, which supplies parasympathetic vagal motor output. Normally, insulin inhibits synaptic excitation of DMV neurons, with no effect on synaptic inhibition. Modulation of synaptic inhibition in DMV, however, is often sensitive to cAMP-dependent mechanisms. We hypothesized that an effect of insulin on GABAergic synaptic transmission may be uncovered by elevating resting cAMP levels in GABAergic terminals. We used whole cell patch-clamp recordings in brain stem slices from control and diabetic mice to identify insulin effects on inhibitory neurotransmission in the DMV in the presence of forskolin to elevate cAMP levels. In the presence of forskolin, insulin decreased the frequency of inhibitory postsynaptic currents (IPSCs) and the paired-pulse ratio of evoked IPSCs in DMV neurons from control mice. This effect was blocked by brefeldin-A, a Golgi-disrupting agent, or indinavir, a GLUT4 blocker, indicating that protein trafficking and glucose transport were involved. In streptozotocin-treated, diabetic mice, insulin did not affect IPSCs in DMV neurons in the presence of forskolin. Results suggest an impairment of cAMP-induced insulin effects on GABA release in the DMV, which likely involves disrupted protein trafficking in diabetic mice. These findings provide insight into mechanisms underlying vagal dysregulation associated with diabetes.


Assuntos
Tronco Encefálico/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Inibição Neural , Transmissão Sináptica , Nervo Vago/metabolismo , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiopatologia , Brefeldina A/farmacologia , Colforsina/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/fisiopatologia , Estimulação Elétrica , Feminino , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Indinavir/farmacologia , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos , Inibição Neural/efeitos dos fármacos , Transporte Proteico , Transmissão Sináptica/efeitos dos fármacos , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiopatologia , Ácido gama-Aminobutírico/metabolismo
18.
Mem. Inst. Oswaldo Cruz ; 109(4): 484-487, 03/07/2014. tab, graf
Artigo em Inglês | LILACS | ID: lil-716314

RESUMO

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.


Assuntos
Animais , Cricetinae , Gorduras na Dieta/sangue , Inibidores da Protease de HIV/farmacologia , Indinavir/farmacologia , Autoanticorpos/sangue , Biomarcadores/sangue , Glicemia/análise , Colesterol/sangue , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Lipoproteínas LDL/efeitos dos fármacos , Modelos Animais , Triglicerídeos/sangue
19.
Angiogenesis ; 17(4): 831-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24719186

RESUMO

In addition to contrast human immunodeficiency virus (HIV) replication, the HIV protease inhibitors (HIV-PI) have reduced tumour incidence or clinical progression in infected patients. In this regard, we have previously shown that, independently of its anti-viral activity, the HIV-PI indinavir (IDV) directly blocks matrix metalloproteinase (MMP)-2 proteolytic activation, thus efficiently inhibiting tumour angiogenesis in vitro, in animal models, and in humans. Herein we investigated the molecular mechanism for IDV anti-angiogenic effect. We found that treatment of human primary endothelial cells with therapeutic IDV concentrations decreases the expression of membrane type (MT)1-MMP, which is the major activator of MMP-2. This occurs for both the constitutive expression of MT1-MMP and that up-regulated by angiogenic factors. In either cases, reduction of MT1-MMP levels by IDV is preceded by the inhibition of the binding of the specificity protein (Sp)1 transcription factor to the promoter region of the MT1-MMP gene in endothelial cell nuclei. As MT1-MMP is key for tumour angiogenesis, these results support the use of IDV or its derivatives in anti-cancer therapy. This is recommended by the low toxicity of the drug, and the large body of data on its pharmacokinetic.


Assuntos
Células Endoteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Inibidores da Protease de HIV/química , Indinavir/farmacologia , Metaloproteinase 14 da Matriz/metabolismo , Animais , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição Sp1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
J Biol Chem ; 289(23): 16100-13, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24706759

RESUMO

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.


Assuntos
Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Inibidores da Protease de HIV/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Inibidores da Protease de HIV/metabolismo , Humanos , Indinavir/metabolismo , Indinavir/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos
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