Involvement of CYP3A4 and MDR1 in altered metabolism and transport of indinavir in 1,25(OH)2D3-treated Caco-2 cells.

Altered drug concentrations may induce unexpected toxicity or treatment failure; thus, understanding the factors that alter the pharmacokinetic profiles of drugs is crucial for optimal disease treatment. Vitamin D receptor (VDR), a nuclear receptor, regulates the expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), which are crucial determinants of drug pharmacokinetics. In this study, we investigated the effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a VDR ligand, on the metabolism, transport, and pharmacokinetics of indinavir, a dual substrate of CYP3A4 and MDR1. 1,25(OH)2D3 treatment for three days upregulated the expression levels of CYP3A4 and MDR1 in Caco-2 cells and consequently led to an increase in the level of a metabolite formed via CYP3A4 (indinavir M6) and the efflux ratio of indinavir in transport study. The increase in the metabolic reaction was also confirmed through a metabolism assay performed using the lysate of 1,25(OH)2D3-treated Caco-2 cells. In the Ussing chamber study conducted with the rat intestine, 1,25(OH)2D3 treatment did not alter the transport of indinavir into the basolateral side but increased indinavir M6 formation. Similarly, plasma levels of the metabolite increased in 1,25(OH)2D3-treated rats; however, systemic exposure to indinavir led to insignificant alterations. Considering the overlapping substrate specificities for CYP3A4 and MDR1 and their significant roles in drug pharmacokinetics, VDR may play an important role in drug interactions of CYP3A4 and MDR1 substrates for accessing more effective and safe disease treatments.

Discovery of Novel HIV Protease Inhibitors Using Modern Computational Techniques.

The human immunodeficiency virus type 1 (HIV-1) has continued to be a global concern. With the new HIV incidence, the emergence of multi-drug resistance and the untoward side effects of currently used anti-HIV drugs, there is an urgent need to discover more efficient anti-HIV drugs. Modern computational tools have played vital roles in facilitating the drug discovery process. This research focuses on a pharmacophore-based similarity search to screen 111,566,735 unique compounds in the PubChem database to discover novel HIV-1 protease inhibitors (PIs). We used an in silico approach involving a 3D-similarity search, physicochemical and ADMET evaluations, HIV protease-inhibitor prediction (IC50/percent inhibition), rigid receptor-molecular docking studies, binding free energy calculations and molecular dynamics (MD) simulations. The 10 FDA-approved HIV PIs (saquinavir, lopinavir, ritonavir, amprenavir, fosamprenavir, atazanavir, nelfinavir, darunavir, tipranavir and indinavir) were used as reference. The in silico analysis revealed that fourteen out of the twenty-eight selected optimized hit molecules were within the acceptable range of all the parameters investigated. The hit molecules demonstrated significant binding affinity to the HIV protease (PR) when compared to the reference drugs. The important amino acid residues involved in hydrogen bonding and п-п stacked interactions include ASP25, GLY27, ASP29, ASP30 and ILE50. These interactions help to stabilize the optimized hit molecules in the active binding site of the HIV-1 PR (PDB ID: 2Q5K). HPS/002 and HPS/004 have been found to be most promising in terms of IC50/percent inhibition (90.15%) of HIV-1 PR, in addition to their drug metabolism and safety profile. These hit candidates should be investigated further as possible HIV-1 PIs with improved efficacy and low toxicity through in vitro experiments and clinical trial investigations.

Hybrid QM/MM Free-Energy Evaluation of Drug-Resistant Mutational Effect on the Binding of an Inhibitor Indinavir to HIV-1 Protease.

A human immunodeficiency virus-1 (HIV-1) protease is a homodimeric aspartic protease essential for the replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analogues have been developed. However, serious drug-resistant mutants have emerged. For understanding the molecular mechanism of the drug resistance, an accurate examination of the impacts of the mutations on ligand binding and enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of indinavir, a potent transition state analogue inhibitor, to the wild-type protein and a V82T/I84V drug-resistant mutant of the HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free-energy optimization technique which combines a highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of the MM protein environment by long-time molecular dynamics simulations. Through the free-energy calculations of protonation states of catalytic groups at the binding pocket and of the ligand-binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug resistance through the direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

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.

Toxoplasma gondii UBL-UBA shuttle proteins regulate several important cellular processes.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.

Screening potential FDA-approved inhibitors of the SARS-CoV-2 major protease 3CLpro through high-throughput virtual screening and molecular dynamics simulation.

It has been confirmed that the new coronavirus SARS-CoV-2 caused the global pandemic of coronavirus disease 2019 (COVID-19). Studies have found that 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for virus replication, and could be used as a potential target to inhibit SARS-CoV-2. In this work, 3CLpro was used as the target to complete the high-throughput virtual screening of the FDA-approved drugs, and Indinavir and other 10 drugs with high docking scores for 3CLpro were obtained. Studies on the binding pattern of 3CLpro and Indinavir found that Indinavir could form the stable hydrogen bond (H-bond) interactions with the catalytic dyad residues His41-Cys145. Binding free energy study found that Indinavir had high binding affinity with 3CLpro. Subsequently, molecular dynamics simulations were performed on the 3CLpro and 3CLpro-Indinavir systems, respectively. The post-dynamic analyses showed that the conformational state of the 3CLpro-Indinavir system transformed significantly and the system tended to be more stable. Moreover, analyses of the residue interaction network (RIN) and H-bond occupancy revealed that the residue-residue interaction at the catalytic site of 3CLpro was significantly enhanced after binding with Indinavir, which in turn inactivated the protein. In short, through this research, we hope to provide more valuable clues against COVID-19.

Kinetics of cytochrome P450 3A4 inhibition by heterocyclic drugs defines a general sequential multistep binding process.

Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug-drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O-debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6ß-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron-nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450-inhibitor complexes exist en route to the final enzyme-inhibitor complex with full inhibitory activity.

Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine against main protease and RNA-dependent RNA polymerase of SARS-CoV-2: A molecular docking and drug repurposing approach.

BACKGROUND: Outbreak of COVID-19 has been recognized as a global health concern since it causes high rates of morbidity and mortality. No specific antiviral drugs are available for the treatment of COVID-19 till date. Drug repurposing strategy helps to find out the drugs for COVID-19 treatment from existing FDA approved antiviral drugs. In this study, FDA approved small molecule antiviral drugs were repurposed against the major viral proteins of SARS-CoV-2. METHODS: The 3D structures of FDA approved small molecule antiviral drugs were retrieved from PubChem. Virtual screening was performed to find out the lead antiviral drug molecules against main protease (Mpro) and RNA-dependent RNA polymerase (RdRp) using COVID-19 Docking Server. Furthermore, lead molecules were individually docked against protein targets using AutoDock 4.0.1 software and their drug-likeness and ADMET properties were evaluated. RESULTS: Out of 65 FDA approved small molecule antiviral drugs screened, Raltegravir showed highest interaction energy value of -9 kcal/mol against Mpro of SARS-CoV-2 and Indinavir, Tipranavir, and Pibrentasvir exhibited a binding energy value of ≥-8 kcal/mol. Similarly Indinavir showed the highest binding energy of -11.5 kcal/mol against the target protein RdRp and Dolutegravir, Elbasvir, Tipranavir, Taltegravir, Grazoprevir, Daclatasvir, Glecaprevir, Ledipasvir, Pibrentasvir and Velpatasvir showed a binding energy value in range from -8 to -11.2 kcal/mol. The antiviral drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine also exhibited good bioavailability and drug-likeness properties. CONCLUSION: This study suggests that the screened small molecule antiviral drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine could serve as potential drugs for the treatment of COVID-19 with further validation studies.

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.

Indinavir Increases Midazolam N-Glucuronidation in Humans: Identification of an Alternate CYP3A Inhibitor Using an In Vitro to In Vivo Approach.

Midazolam is a widely used index substrate for assessing effects of xenobiotics on CYP3A activity. A previous study involving human hepatocytes showed the primary route of midazolam metabolism, 1'-hydroxylation, shifted to N-glucuronidation in the presence of the CYP3A inhibitor ketoconazole, which may lead to an overprediction of the magnitude of a xenobiotic-midazolam interaction. Because ketoconazole is no longer recommended as a clinical CYP3A inhibitor, indinavir was selected as an alternate CYP3A inhibitor to evaluate the contribution of the N-glucuronidation pathway to midazolam metabolism. The effects of indinavir on midazolam 1'-hydroxylation and N-glucuronidation were first characterized in human-derived in vitro systems. Compared with vehicle, indinavir (10 µM) inhibited midazolam 1'-hydroxylation by recombinant CYP3A4, human liver microsomes, and high-CYP3A activity cryopreserved human hepatocytes by ≥70%; the IC50 obtained with hepatocytes (2.7 µM) was within reported human unbound indinavir Cmax (≤5 µM). Midazolam N-glucuronidation in hepatocytes increased in the presence of indinavir in both a concentration-dependent (1-33 µM) and time-dependent (0-4 hours) manner (by up to 2.5-fold), prompting assessment in human volunteers (n = 8). As predicted by these in vitro data, indinavir was a strong inhibitor of the 1'-hydroxylation pathway, decreasing the 1'-hydroxymidazolam/midazolam area under the plasma concentration versus time curve (AUC)0-12h ratio by 80%. Although not statistically significant, the midazolam N-glucuronide/midazolam AUC0-12h ratio increased by 40%, suggesting a shift to the N-glucuronidation pathway. The amount of midazolam N-glucuronide recovered in urine increased 4-fold but remained <10% of the oral midazolam dose (2.5 mg). A powered clinical study would clarify whether N-glucuronidation should be considered when assessing the magnitude of a xenobiotic-midazolam interaction.

Computationally Assessing the Bioactivation of Drugs by N-Dealkylation.

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/ .

Insulin modulates hippocampally-mediated spatial working memory via glucose transporter-4.

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.

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.

Understanding the basis of I50V-induced affinity decrease in HIV-1 protease via molecular dynamics simulations using polarized force field.

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.

The changes in bone organic and inorganic matrix in newborn rats after maternal application of antiretroviral agents: Indinavir and zidovudine.

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.