Antiretroviral Protease Inhibitors Accelerate Glutathione Export from Viable Cultured Rat Neurons

Antiretroviral protease inhibitors are crucial components of the antiretroviral combination therapy that is successfully used for the treatment of patients with HIV infection. To test whether such protease inhibitors affect the glutathione (GSH) metabolism of neurons, cultured cerebellar granule neurons were exposed to indinavir, nelfinavir, lopinavir or ritonavir. In low micromolar concentrations these antiretroviral protease inhibitors did not acutely compromise the cell viability, but caused a time- and concentration-dependent increase in the accumulation of extracellular GSH which was accompanied by a matching loss in cellular GSH. The stimulating effect by indinavir, lopinavir and ritonavir on GSH export was immediately terminated upon removal of the protease inhibitors, while the nelfinavir-induced stimulated GSH export persisted after washing the cells. The stimulation of neuronal GSH export by protease inhibitors was completely prevented by MK571, an inhibitor of the multidrug resistance protein 1, suggesting that this transporter mediates the accelerated GSH export during exposure of neurons to protease inhibitors. These data suggest that alterations in brain GSH metabolism should be considered as potential side-effects of a treatment with antiretroviral protease inhibitors.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K_i and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2 h with arsenate in concentrations of up to 10 mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3 mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.     

The effects of protease inhibitors on basal and insulin-stimulated lipid metabolism,insulin binding,and signaling

The objective of our research was to investigate the effects of the protease inhibitors ritonavir, saquinavir, and indinavir on triglyceride synthesis, lipolysis, insulin binding, and signaling in differentiating 3T3 L1 pre-adipocytes. Saquinavir, ritonavir, and indinavir all stimulated triglyceride (TG) synthesis. Additionally, all concentrations of protease inhibitors employed (i.e., 0.1 micro M to 10 micro M) significantly decreased insulin-stimulated TG synthesis. No effects of any of the protease inhibitors were observed either on basal lipolysis or after stimulation of lipolysis with 100 nM noradrenaline. Specific (125)I-insulin binding was observed to be decreased by exposure to all the protease inhibitors throughout the period of adipocyte phenotype development. This was mediated by indinavir through a receptor decrease and had no effect on receptor affinity. During differentiation with ritonavir (i.e., 1-11 days post addition of differentiating cocktail), insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation was ascertained (day 11) and found to be decreased in the ritonavir exposed cells when compared with control cells. The results reported herein demonstrate protease inhibitor effects on basal TG synthesis while exhibiting decreased insulin-stimulated TG synthesis at physiological concentrations of protease inhibitors. These effects may be subsequent to decreased insulin binding and/or IRS-1 tyrosine phosphorylation.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Comparison of protease genotype and phenotype in HIV-1 infected patients exposed to more than one protease inhibitor

Many patterns of mutations selected by HIV-1 protease inhibitors have been described, but in most cases isolates with these patterns have been obtained from pre-clinical studies or after failures of monotherapies. We compared genotype and phenotype in HIV-1 infected patients who have failed more than one PI-including regimen. Phenotypic resistance could arise also in the absence of specific primary mutations and in the presence of different substitutions among those known to confer resistance to ritonavir, indinavir or nelfinavir. The number of secondary mutations was significantly associated with phenotypic resistance for each protease inhibitor. Thus, more study of mutational patterns in heavily pretreated patients is warranted; in the mean time treatment choices might be optimized if phenotyping could integrate genotyping within this setting.     

Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.     

Simple and rapid high-performance liquid chromatographic method for nelfinavir,M8 nelfinavir metabolite,ritonavir and saquinavir assay in plasma

A simple reversed-phase liquid chromatographic method has been developed to determine protease inhibitors concentrations in plasma. Plasma samples (250 micro l) containing protease inhibitors were prepared by a simple deproteinization (recovery: 92, 91, 91 and 90.5% for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite, respectively). Chromatography was accomplished using a Hypersil octadecylsilyl column (100 x 4.6 mm I.D.) and a mobile phase composed of acetonitrile, tetrahydrofuran and dihydrogenophosphate buffer (pH 4) (32:10:58, v/v). Ultraviolet detection at 210 nm was used. The limit of detection was 200 ng/ml for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite. Calibration curves were linear up to 20000 ng/ml, with correlation coefficients better than 0.997 for all compounds. Intra- and inter-day coefficients of variation of the assay were 相似文献   

Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry

A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC-MS-MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.     

Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes

Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH–bimane-conjugate (GS–B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS–B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.

     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Indinavir reduces Cryptosporidium parvum infection in both in vitro and in vivo models

The use of highly active antiretroviral therapy in persons with acquired immunodeficiency syndrome has reduced the prevalence of infection with Cryptosporidium parvum and the length and severity of its clinical course. This effect has in most cases been attributed to the recovery of the host immunity; however, some works suggest that human immunodeficiency virus protease inhibitors, indinavir in particular, which is one of the human immunodeficiency virus protease inhibitors used in highly active antiretroviral therapy, may be capable of controlling Microsporidia and Cryptosporidium infections, which are refractory to other treatments. The objective of the present study was to investigate the effect of human immunodeficiency virus protease inhibitors on C. parvum infections. Since preliminary experiments using ritonavir, saquinavir, and indinavir showed a drastic reduction of C. parvum infection both in vivo (neonatal Balb/c mice) and in vitro (human ileocecal adenocarcinoma tumour cell line) models, indinavir alone was tested in successive experiments. In vitro, the treatment of the sporulated oocysts with different concentrations of indinavir reduced the percentage of human ileocecal adenocarcinoma tumour cell line infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data show that indinavir directly interferes with the cycle of C. parvum, resulting in a marked reduction in oocyst shedding and in the number of intracellular parasites. Protease inhibitors could be considered as good candidates for the treatment of cyptosporidiosis in immunosuppressed persons.