HIV protease inhibitors block adipocyte differentiation independently of lamin A/C

OBJECTIVES: To determine the importance of lamin A/C for fat cell differentiation in vitro and for the anti-adipogenic activity of HIV protease inhibitors such as indinavir. METHODS: Lipodystrophy-associated and processing-defective mutants of lamin A were stably expressed at high levels in 3T3-L1 pre-adipocytes. Additionally, 3T3-L1 pre-adipocytes with stable reduction of lamin A/C or emerin were derived. The cells were differentiated for 8 days into mature adipocytes in the presence or absence of indinavir or nelfinavir. RESULTS: 3T3-L1 cells stably expressing high levels of lipodystrophy-associated or processing-defective mutants of lamin A differentiated with comparable efficiencies to control cells. Similarly, cells with dramatically reduced lamin A levels differentiated as efficiently as controls. Although indinavir stimulated the accumulation of unprocessed lamin A, cells with dramatically reduced lamin A/C levels and no detectable prelamin A remained responsive to an indinavir-induced inhibition of adipogenesis. CONCLUSIONS: The ability of HIV protease inhibitor to stimulate the accumulation of unprocessed lamin A is neither necessary nor sufficient to explain their anti-adipogenic activity. Furthermore, lamin A/C plays a minimal role in the differentiation of 3T3-L1.     

A serine protease activity in C3H/10T1/2 cells that is inhibited by anticarcinogenic protease inhibitors.

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.     

MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells

OBJECTIVE: To evaluate the impact of the human multidrug resistance gene (MDR1) G1199A polymorphism (amino acid change Ser400Asn) on P-glycoprotein (P-gp)-dependent transepithelial permeability and uptake kinetics of HIV protease inhibitors (PI), by using recombinant epithelial cells expressing wild-type MDR1 (MDR1wt) or the G1199A variant (MDR1(1199A)). METHODS: Using a recombinant expression system developed previously, the transepithelial permeability and uptake kinetic parameters of five PI, amprenavir, indinavir, lopinavir, ritonavir, and saquinavir were estimated across polarized epithelial cells. RESULTS: For all PI, the transepithelial permeability ratio (basolateral-to-apical transport divided by apical-to-basolateral transport) was significantly greater in MDR1(1199A) cells than MDR1wt cells: amprenavir (1.7-fold), indinavir (1.8-fold), lopinavir (1.5-fold), ritonavir (2.8-fold), and saquinavir (2.1-fold). However, the impact of G1199A on P-gp activity appeared to primarily influence drug permeability in the apical-to-basolateral direction. Kinetic analysis of ritonavir and saquinavir uptake by MDR1wt- and MDR1(1199A)-expressing cells showed that Vmax was similar, while uptake Km was significantly higher in cells expressing the G1199A variant suggesting that alterations in P-gp-dependent efflux mediated by G1199A were due to changes in transporter affinity. CONCLUSIONS: Alterations in transepithelial permeability of HIV PI due to the G1199A polymorphism may impact oral bioavailability of PI and penetration into cells and tissues of the lymphoid and central nervous systems.     

The HIV type 1 protease L10I minor mutation decreases replication capacity and confers resistance to protease inhibitors

The effect of minor mutations in PR on treatment outcome has not been well established. We characterized the HIV protease minor mutations, L10I, compared to the minor mutation, L63P, and the major mutation D30N and their impact on viral fitness and resistance to protease inhibitors. Mutations were introduced individually and in combination by site-directed mutagenesis into the provirus pNL4.3ren and constructs used for replication capacity (RC) and resistance assays. A structure prediction of the protease carrying the L10I mutation was determined. The prevalence of the minor mutation L10I had a pattern similar to that found for major mutations D30N, with a low prevalence (4.9%) in naive patients and significantly higher prevalence in treated patients. Furthermore, viruses carrying the major mutation D30N or the minor mutation L10I showed a significant decrease in RC (p-value <0.05), whereas viruses carrying the minor mutation L63P had RC similar to wild-type virus. In addition, the L10I mutation conferred resistance to saquinavir, which was supported by the higher prevalence in the cohort of the L10I mutation among patients with SQV resistance. The molecular modeling suggests that L10I may affect the conformation of Leu-23, a critical residue in the substrate binding site. In conclusion, the L10I mutation impairs RC and confers resistance to SQV, similarly to other major mutations, which may be related with changes in the conformation in the protease binding site. The presence of this mutation in the genotype of HIV from patients should be taken into consideration when designing new optimize treatments.     

HIV type 1 protease inhibitors enhance bone marrow progenitor cell activity in normal subjects and in HIV type 1-infected patients

HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on bone marrow (BM) progenitor cells. In this study we investigated this hypothesis evaluating the effect of adding ritonavir (RTV) and indinavir (IND) on hematopoietic colony formation assays by colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, on apoptosis, on cytokine production and stromal cells, in subjects with HIV-1 infection, and in seronegative controls. After PI addition, CFC and LTC-IC assays in HIV-1-infected patients showed levels of colony growth significantly higher than those observed at baseline; the same PI activity on colony formation was observed in healthy subjects. No significant modifications on Fas, the membrane form of Fas (mFas) and Fas-ligand (FasL) expression, and on cytokine production were observed at BM level after the addition of PIs. At baseline, in HIV-1-infected patients, the majority of the stromal cells appeared as large and rounded, whereas after the addition of RTV or IND the stromal cells exhibited a "fibroblast-like" morphology and produced higher stem cell factor (SCF) and lower MIP-1alpha levels when compared with the stromal production without the addition of IND. RTV and IND increased colony growth of BM obtained either from HIV-1-infected patients or from normal individuals, in parallel with the normalization of functional and morphological characteristics of stromal cells.     

Pretreatment resistance to hepatitis C virus protease inhibitors boceprevir/telaprevir in hepatitis C subgenotype 1a-infected patients from Manitoba

BACKGROUND:

Traditional therapy with pegylated interferon and ribavirin combined with the new protease inhibitors boceprevir or telaprevir has demonstrated improved outcomes in hepatitis C virus (HCV)-infected patients. Prevalence data regarding pre-existing drug-resistant variants to these two new virus inhibitors in the Canadian population are not available.

OBJECTIVE:

To detect pre-existing mutations conferring resistance to boceprevir and/or telaprevir in Canadian patients infected with HCV genotype 1a.

METHODS:

Resistance-associated mutations (RAMs) were evaluated in 85 patients infected with HCV genotype 1a who had not yet received antiviral therapy. The NS3 protease gene was sequenced and common RAMs were identified based on a recently published list.

RESULTS:

The overall prevalence of pre-existing RAMs to boceprevir and telaprevir was higher compared with other similar studies. All of the observed RAMs were associated with a low level of resistance. A surprisingly high proportion of patients had the V55A RAM (10.6%). None of the mutations associated with a high level of resistance were observed. The simultaneous presence of two low-level resistance mutations (V36L and V55A) was observed in only one patient. Three other patients had both T54S RAM and V55I mutations, which may require a higher concentration of the protease drugs. The prevalence of various mutations in Aboriginal Canadian patients was higher (37.5%) compared with Caucasians (16.39%) (P=0.038).

CONCLUSIONS:

The present study was the first to investigate pre-existing drug resistance to boceprevir/telaprevir in Canadian HCV-infected patients. A relatively high proportion of untreated HCV genotype 1a patients in Manitoba harbour low-level RAMs, especially patients of Aboriginal descent, which may contribute to an increased risk of treatment failure.     

Hepatitis C virus (HCV) protease variability and anti-HCV protease inhibitor resistance in HIV/HCV-coinfected patients

Objectives

Data on the natural selection of isolates harbouring mutations within the NS3 protease, conferring resistance to hepatitis C virus (HCV) protease inhibitors (PIs), are limited for HIV/HCV‐coinfected patients. The aim of this study was to describe the natural prevalence of mutations conferring resistance to HCV PIs in HIV/HCV‐coinfected patients compared with HCV‐monoinfected patients.

Methods

The natural prevalences of HCV PI resistance mutations in 120 sequences from HIV/HCV‐coinfected patients (58 genotype 1a, 18 genotype 1b and 44 genotype 4) and 501 sequences from HCV‐monoinfected patients (476 genotype 1 and 25 genotype 4), retrieved from GenBank as a control group, were compared.

Results

Of 76 sequences from HIV/HCV genotype 1‐coinfected patients, six (7.9%) showed amino acid substitutions associated with HCV PI resistance (V36L, n=1; V36M, n=2; T54S, n=2; R155K, n=1). In 31 of 476 (6.5%) HCV genotype 1 sequences retrieved from the GenBank database, HCV PI resistance mutations were found. The difference was not statistically significant (P=0.6). All of the sequences from HIV/HCV genotype 4‐coinfected patients and those retrieved from the GenBank database had amino acid changes at position 36 (V36L).

Conclusion

Our study suggests that the natural prevalence of strains resistant to HCV PIs does not differ between HCV‐monoinfected and HIV/HCV‐coinfected patients. Further studies on larger cohorts are needed to confirm these findings and to evaluate the impact of these mutations in clinical practice.     

石杉碱甲对D-半乳糖致衰老大鼠海马及下丘Lamin A／C、prelamin A及FACE1表达的影响

目的研究D-半乳糖致衰老大鼠海马及下丘laminA／C、prelaminA、FACEl的水平变化,并进-步明确石杉碱甲对D-半乳糖诱导的这些蛋白变化的影响及其意义。方法36只健康雄性SD大鼠随机分成对照组、D-半乳糖治疗组和D-半乳糖联合石杉碱甲组。D-半乳糖组每天皮下注射D-半乳糖300mg／Kg,共8W;D-半乳糖联合石杉碱甲组每天D-半乳糖300mg／Kg联合石杉碱甲0．1mg／Kg共同皮下注射,共8W。对照组皮下注射等量生理盐水。应用蛋白免疫印迹杂交（Western blotting）方法检测3组大鼠海马、下丘laminA／C、prelaminA和FACEl的表达。结果海马中,laminA在对照组、D半乳糖＋石杉碱甲组、D-半乳糖组中逐渐降低,laminC水平逐渐升高,prelaminA在对照组中最高,D-半乳糖组与D-半乳糖＋石杉碱甲组无明显差异;FACEl在D-半乳糖＋石杉碱甲组水平最高,对照组中水平最低。下丘中,laminA水平在D-半乳糖＋石杉碱甲组最高,D-半乳糖组中最低;D-半乳糖组和D-半乳糖＋石杉碱甲组laminC水平较对照组升高,D-半乳糖组与D-半乳糖＋石杉碱甲组之间的差异无统计学意义;prelaminA水平在对照组中最低,D-半乳糖＋石杉碱甲组中最高;FACE1水平在对照组、D-半乳糖＋石杉碱甲组、D-半乳糖组中逐渐升高。结论LaminA降低、laminC及FACE1升高预示着D-半乳糖诱导的海马及下丘衰老。石杉碱甲可以在-定程度上改善部分指标的变化,说明它有-定的延缓海马及下丘衰老的作用。