Ritonavir increases CD36, ABCA1 and CYP27 expression in THP-1 macrophages

Ritonavir, a protease inhibitor used in combination antiretroviral therapy for HIV-1 infection, is associated with an increased risk of premature atherosclerosis. The aim of the present study was to assess the effects of ritonavir, in the absence of added lipoproteins, on the expression of genes that control cholesterol trafficking in human monocytes/macrophages. Design: THP-1 cells were used to study the effects of ritonavir on the expression of CD36, ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1), scavenger receptor B class I (SR-BI), caveolin-1 and sterol 27-hydroxylase (CYP27). Exposure to ritonavir (2.5 mug/ml) increased CD36 protein (28%, P < 0.05) and mRNA (38%, P < 0.05) in differentiated THP-1 macrophages, but not in undifferentiated monocytes. This effect was not related to the increase in PPARgamma expression (51%, P < 0.05) caused by ritonavir. Ritonavir also reduced SR-BI protein levels (46%, P < 0.05) and increased CYP27 (43%, P < 0.05) and ABCA1 (49%, P < 0.05) mRNA expression. Liver X receptor alpha (LXRalpha) mRNA, protein and binding activity were also increased by ritonavir treatment. Conclusions: We propose that ritonavir induces ABCA1 expression in THP-1 macrophages through LXRalpha. The increase in ABCA1 and other cholesterol efflux mediators, such as CYP27, may compensate CD36 induction. Therefore, we suggest that the net effect of ritonavir on macrophages in the absence of lipoproteins is not clearly proatherogenic.     

Studies on flexibility and binding affinity of Asp25 of HIV-1 protease mutants

We have investigated and highlighted the behavior of binding residue, Asp25 by computational analysis, which play an important role in understanding docking process with drug molecule, Ritonavir (Norvir^®) and the flexibility nature of the Human Immunodeficiency Virus-1 (HIV-1) protease enzyme. It is well known that Ritonavir is a potent and a selective HIV-1 protease inhibitor. Molecular dockings were performed in order to gain insights regarding the binding mode of this inhibitor. In our analysis, we observed Ritonavir had different rank orders of scores against different mutant of this enzyme. Asp25 of the enzyme was found to be the active site for all the mutants. The results clearly suggest that Ritonavir is not able to appropriately bind at the active site of each HIV-1 protease mutant due to RMSD difference of the amino acid (Asp) at the position 25 of all mutants. These findings support the concept that 3D space of active site is a qualitative assessment for binding affinity of inhibitor with an enzyme. The investigation on the flexibility nature of Asp25 by normal mode analysis, show that binding residue posses less flexibility due to its solvation potential. The overall analysis of our study brings clarity to the binding behavior with respect to the different mutants with Ritonavir on the basis RMSD and also on the flexible nature of HIV-1 protease enzyme with respect to Asp25 position.     

The Antiretroviral Protease Inhibitor Ritonavir Accelerates Glutathione Export from Cultured Primary Astrocytes

Antiretroviral protease inhibitors are a class of important drugs that are used for the treatment of human immunodeficiency virus infections. Among those compounds, ritonavir is applied frequently in combination with other antiretroviral protease inhibitors, as it has been reported to boost their therapeutic efficiency. To test whether ritonavir affects the viability and the glutathione (GSH) metabolism of brain cells, we have exposed primary astrocyte cultures to this protease inhibitor. Application of ritonavir in low micromolar concentrations did not compromise cell viability, but caused a time- and concentration-dependent loss of GSH from the cells which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for ritonavir in a concentration of 3 μM. The ritonavir-induced stimulated GSH export from astrocytes was completely prevented by MK571, an inhibitor of the multidrug resistance protein 1. In addition, continuous presence of ritonavir was essential to maintain the stimulated GSH export, since removal of ritonavir terminated the stimulated GSH export. Ritonavir was more potent to stimulate GSH export from astrocytes than the antiretroviral protease inhibitors indinavir and nelfinavir, but combinations of ritonavir with indinavir or nelfinavir did not further stimulate astrocytic GSH export compared to a treatment with ritonavir alone. The strong effects of ritonavir and other antiretroviral protease inhibitors on the GSH metabolism of astrocytes suggest that a chronic treatment of patients with such compounds may affect their brain GSH metabolism.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

HIV protease inhibitor induces fatty acid and sterol biosynthesis in liver and adipose tissues due to the accumulation of activated sterol regulatory element-binding proteins in the nucleus

The mechanism by which human immunodeficiency virus (HIV) protease inhibitor therapy adversely induces lipodystrophy and hyperlipidemia has not been defined. This study explored the mechanism associated with the adverse effects of the prototype protease inhibitor ritonavir in mice. Ritonavir treatment increased plasma triglyceride and cholesterol levels through increased fatty acid and cholesterol biosynthesis in adipose and liver. Ritonavir treatment also resulted in hepatic steatosis and hepatomegaly. These abnormalities, which were especially pronounced after feeding a Western type high fat diet, were due to ritonavir-induced accumulation of the activated forms of sterol regulatory binding protein (SREBP)-1 and -2 in the nucleus of liver and adipose, resulting in elevated expression of lipid metabolism genes. Interestingly, protease inhibitor treatment did not alter SREBP mRNA levels in these tissues. Thus, the adverse lipid abnormalities associated with protease inhibitor therapy are caused by the constitutive induction of lipid biosynthesis in liver and adipose tissues due to the accumulation of activated SREBP in the nucleus.     

The effect of the HIV protease inhibitor ritonavir on proliferation, differentiation, lipogenesis, gene expression and apoptosis of human preadipocytes and adipocytes.

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 microM) on expression of mRNA of PPARgamma2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15% of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.     

The HIV protease inhibitor ritonavir increases lipoprotein production and has no effect on lipoprotein clearance in mice

This study examined the effect of human immunodeficiency virus (HIV) protease inhibitor therapy on lipoprotein production and catabolism in vivo. The HIV protease inhibitor ritonavir was given to C57BL/6 mice fed either a basal low-fat diet or a Western type high-fat diet. Fasted mice were injected with Triton WR1339 followed by hourly blood collection to monitor lipoprotein production. Results showed that ritonavir increased VLDL triglyceride production by 30% over a 4 h period when mice were fed the low-fat basal diet. The ritonavir effect was more pronounced under high-fat feeding conditions, with a 2-fold increase in VLDL triglyceride production rate. Ritonavir did not alter hepatic expression levels of diacylglycerol acyltransferase or microsomal triglyceride transfer protein, but increased hepatic apolipoprotein B (apoB) secretion rates under both low- and high-fat dietary conditions. In contrast to its effect on lipoprotein production, ritonavir did not alter triglyceride-rich lipoprotein clearance from circulation under either dietary condition. Taken together, these results indicate that the hyperlipidemic effect of HIV protease inhibitors is a direct result of increased hepatic lipoprotein production. The mechanism appears to be related to their role in preventing proteasome-mediated degradation of apoB and activated sterol regulatory element binding proteins in the liver.     

X-ray crystallographic structure of ABT-378 (lopinavir) bound to HIV-1 protease

The crystal structure of ABT-378 (lopinavir), bound to the active site of HIV-1 protease is described. A comparison with crystal structures of ritonavir, A-78791, and BILA-2450 shows some analogous features with previous reported compounds. A cyclic urea unit in the P(2) position of ABT-378 is novel and makes two bidentate hydrogen bonds with Asp 29 of HIV-1 protease. In addition, a previously unreported shift in the Gly 48 carbonyl position is observed. A discussion of the structural features responsible for its high potency against wild-type HIV protease is given along with an analysis of the effect of active site mutations on potency in in vitro assays.     

Calpain regulates enterocyte brush border actin assembly and pathogenic Escherichia coli-mediated effacement

This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN2, MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.     

Construction of a human immunodeficiency virus type 1 (HIV-1) library containing random combinations of amino acid substitutions in the HIV-1 protease due to resistance by protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.     

Structure–activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers. Part II: P2/P3 region and discovery of cobicistat (GS-9350)

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.     

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.     

Resistance-Associated Loss of Viral Fitness in Human Immunodeficiency Virus Type 1: Phenotypic Analysis of Protease and gag Coevolution in Protease Inhibitor-Treated Patients

We have studied the phenotypic impact of adaptative Gag cleavage site mutations in patient-derived human immunodeficiency virus type 1 (HIV-1) variants having developed resistance to the protease inhibitor ritonavir or saquinavir. We found that Gag mutations occurred in a minority of resistant viruses, regardless of the duration of the treatment and of the protease mutation profile. Gag mutations exerted only a partial corrective effect on resistance-associated loss of viral fitness. Reconstructed viruses with resistant proteases displayed multiple Gag cleavage defects, and in spite of Gag adaptation, several of these defects remained, explaining the limited corrective effect of cleavage site mutations on fitness. Our data provide clear evidence of the interplay between resistance and fitness in HIV-1 evolution in patients treated with protease inhibitors.     

X-ray structure of HIV-1 protease tethered dimer complexed to ritonavir

We report here the 2.5A structure of HIV-1 protease tethered-dimer ritonavir complex. The inhibitor bound in the active site has different conformations in the two orientations. There is only one hydrogen bond between the inhibitor and the enzyme. The conserved flap-water is not found in the present complex.     

Analysis of HIV-1 CRF_01 A/E protease inhibitor resistance: structural determinants for maintaining sensitivity and developing resistance to atazanavir

A series of HIV-1 protease mutants has been designed in an effort to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to that of the subtype B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pretherapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to it aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pretherapy B protease showed that the ability of atazanavir to maintain its binding affinity for variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.     

New water-soluble prodrugs of HIV protease inhibitors based on O-->N intramolecular acyl migration

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns–Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O→N intramolecular acyl migration reaction at the -hydroxy-β-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300 mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37 °C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t_1/2 value of 32 h that may not be suitable for practical use.     

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.