Reconstruction of the binding pathway of an anti-HIV drug, Indinavir, in complex with the HTLV-1 protease using unaggregated unbiased molecular dynamics simulation.

Retroviruses are a growing concern for the health of human beings, and one of the dangerous members of this family is the Human T-cell Leukemia Virus 1 (HTLV-1) virus. It has affected more than 20 million people so far, and since there are no registered treatments against it yet, urgent treatment solutions are needed. One of the most promising drug targets to fight this virus is the protease enzyme of the virus's protein machinery. In this study, by utilizing a computational method called Unaggregated Unbiased Molecular Dynamics (UUMD), we reconstructed the binding pathway of a HTLV-1 protease inhibitor, Indinavir, to find the details of the binding pathway, the influential residues, and also the stable states of the binding pathway. We achieved the native conformation of the inhibitor in 6 rounds, 360 replicas by performing over 4 micro-seconds of UMD simulations. We found 3 Intermediate states between the solvated state and the native conformation state in the binding pathway. We also discovered that aromatic residues such as Trp98 and Trp98', catalytic residues Asp32 and Asp32', and the flap region's residues have the most influential roles in the binding pathway and also have the most contribution to the total interaction energies. We believe that the details found in this study would be a great guide for developing new treatment solutions against the HTLV-1 virus by inhibiting the HTLV-1 protease.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

The mechanism for differential effect of nelfinavir and indinavir on collagen metabolism in human skin fibroblasts.

The mechanism for differential effects of human immune deficiency virus protease inhibitors (HIVPIs), nelfinavir (NEL) and indinavir (IND) on collagen metabolism disturbances was studied in human skin fibroblasts. It has been considered that HIVPIs-dependent deregulation of collagen biosynthesis involves prolidase (an enzyme providing proline for collagen biosynthesis), glutamine (Gln) (a substrate for proline biosynthesis), nuclear factor-κB (NF-κB) (a transcription factor that inhibit expression of type I collagen genes), ß1 integrin receptor and Akt signalling. It was found that NEL impaired collagen biosynthesis and the process was more pronounced in the presence of Gln, while IND stimulated collagen biosynthesis. NEL-dependent inhibition of collagen biosynthesis was accompanied by massive intracellular accumulation of type I collagen, while IND slightly induced this process. This effect of NEL was reversed by ascorbic acid but not N-acetylcysteine. The mechanism for the NEL-dependent defect in collagen metabolism was found at the level of prolidase activity, ß1 integrin signalling and NF-κB. NEL inhibited expression of ß1 integrin receptor, Akt and ERK1/2 and increased expression of p65 NF-κB. However, inhibitors of p65 NF-κB did not prevent NEL-dependent inhibition of collagen biosynthesis suggesting that this transcription factor is not involved in studied mechanism. Using PI3K inhibitor wortmannin that prevent phosphorylation of Akt revealed that NEL-dependent inhibition of Akt results in inhibition of collagen biosynthesis. The data suggest that differential effect of NEL and IND on collagen metabolism involves NEL-dependent down-regulation of Akt signalling and proline availability for collagen biosynthesis.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation.

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.

Hyperglycemia exacerbates antiretroviral drug combination induced blood-brain barrier endothelial toxicity.

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.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

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.

CD26/DPPIV inhibition alters the expression of immune response-related genes in the thymi of NOD mice.

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.

Herb-Drug Pharmacokinetic Interactions: Transport and Metabolism of Indinavir in the Presence of Selected Herbal Products.

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.

cAMP-dependent insulin modulation of synaptic inhibition in neurons of the dorsal motor nucleus of the vagus is altered in diabetic mice.

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.

In vivo assessment of antiretroviral therapy-associated side effects

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.

The HIV protease inhibitor indinavir down-regulates the expression of the pro-angiogenic MT1-MMP by human endothelial cells.

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.

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.

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: potential role of the NFkappa B pathway.

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO-IR) and 3T3-L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor ß-subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate-1 (IRS-1) on serine 307 (S307) in both CHO-IR cells and 3T3-L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin-stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling-1 and -3 (SOCS-1 and -3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO-IR cells were pre-treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR ß-subunit and phosphorylation of IRS-1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs.

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.

The antiretroviral protease inhibitors indinavir and nelfinavir stimulate Mrp1-mediated GSH export from cultured brain astrocytes.

Combinations of antiretroviral drugs are successfully used for the treatment of acquired immune deficiency syndrome and reduce the incidence of severe human immunodeficiency virus (HIV)-associated dementia. To test whether such drugs affect the GSH metabolism of brain cells, we have exposed astrocyte-rich primary cultures to various antiretroviral compounds. Treatment of the cultures with the protease inhibitors indinavir or nelfinavir in low micromolar concentrations resulted in a time- and concentration-dependent depletion of cellular GSH from viable cells which was accompanied by a matching increase in the extracellular GSH content. In contrast, the reverse transcriptase inhibitors zidovudine, lamivudine, efavirenz or nevirapine did not alter cellular or extracellular GSH levels. Removal of indinavir from the medium by washing the cells terminated the stimulated GSH export immediately, while the nelfinavir-induced accelerated GSH export was maintained even after removal of nelfinavir. The stimulation of the GSH export from viable astrocytes by indinavir or nelfinavir was completely prevented by the application of MK571, an inhibitor of the multidrug resistance protein 1. These data demonstrate that indinavir and nelfinavir stimulate multidrug resistance protein 1-mediated GSH export from viable astrocytes and suggest that treatment of patients with such inhibitors may affect the GSH homeostasis in brain.

Exposure to HIV-protease inhibitors selects for increased expression of P-glycoprotein (ABCB1) in Kaposi's sarcoma cells.

BACKGROUND: Given that HIV-protease inhibitors (HIV-PIs) are substrates/inhibitors of the multidrug transporter ABCB1, can induce ABCB1 expression, and are used in combination with doxorubicin for AIDS-Kaposi's Sarcoma (KS) treatment, the role that ABCB1 plays in mediating multidrug resistance (MDR) in a fully transformed KS cell line (SLK) was explored. METHODS: The KS cells were exposed to both acute and chronic treatments of physiological concentrations of different HIV-PIs (indinavir, nelfinavir, atazanavir, ritonavir, or lopinavir), alone or together with doxorubicin. The ABCB1 mRNA and protein expression levels were then assessed by qRT-PCR and western blotting, flow cytometry, and immunofluorescence. RESULTS: Chronic treatment of SLK cells with one of the five HIV-PIs alone or together resulted in increased resistance to doxorubicin. Co-treatment with one of the HIV-PIs in combination with doxorubicin resulted in a synergistic increase in resistance to doxorubicin, and the degree of resistance was found to correlate with the expression of ABCB1. The SLK cells were also revealed to be cross-resistant to the structurally unrelated drug paclitaxel. CONCLUSION: These studies suggest that ABCB1 is primarily responsible for mediating MDR in SLK cells selected with either HIV-PIs alone or in combination with doxorubicin. Therefore, the roles that ABCB1 and drug cocktails play in mediating MDR in KS in vivo should be evaluated.

Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells.

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs.

Analyses of nanoformulated antiretroviral drug charge, size, shape and content for uptake, drug release and antiviral activities in human monocyte-derived macrophages.

Long-term antiretroviral therapy (ART) for human immunodeficiency virus type one (HIV-1) infection shows limitations in pharmacokinetics and biodistribution while inducing metabolic and cytotoxic aberrations. In turn, ART commonly requires complex dosing schedules and leads to the emergence of viral resistance and treatment failures. We posit that the development of nanoformulated ART could preclude such limitations and affect improved clinical outcomes. To this end, we wet-milled 20 nanoparticle formulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz, collectively referred to as "nanoART," then assessed their performance using a range of physicochemical and biological tests. These tests were based on cell-nanoparticle interactions using monocyte-derived macrophages and their abilities to uptake and release nanoformulated drugs and affect viral replication. We demonstrate that physical characteristics such as particle size, surfactant coating, surface charge, and most importantly shape are predictors of cell uptake and antiretroviral efficacy. These studies bring this line of research a step closer to developing nanoART that can be used in the clinic to affect the course of HIV-1 infection.

Human immunodeficiency virus protease inhibitors reduce the growth of human tumors via a proteasome-independent block of angiogenesis and matrix metalloproteinases.

Human immunodeficiency virus protease inhibitors (HIV-PIs), such as indinavir and saquinavir, have been shown to block angiogenesis and tumor cell invasion and to induce tumor cell apoptosis and growth arrest, respectively, both in vitro and in vivo. These findings have suggested that HIV-PIs or their analogues can be used as antitumor drugs. To this regard, indinavir and saquinavir were assessed for their ability to inhibit in vivo the growth of highly prevalent human tumors, such as lung, breast, colon and hepatic adenocarcinomas. We show here that both HIV-PIs significantly inhibited the growth of all adenocarcinomas tested in the mice model. This was not mediated by effects on proteasome-dependent cell growth arrest or on apoptosis but by the block of angiogenesis and matrix metalloproteinase activity. Accordingly, therapeutic steadystate concentrations of indinavir or saquinavir were highly effective in inhibiting invasion of tumor cells in vitro. In contrast, growth arrest was induced only by high concentrations of saquinavir that are not reached or are only transiently present in plasma of treated patients, likely through a proteasome-mediated mechanism. These data suggest that HIV-PIs or their analogues, characterized by a better biodistribution and lower toxicity, may represent a new class of antitumor drugs capable of targeting both matrix metalloproteinases and the proteasome for a most effective antitumor therapy.