Anti-human Immunodeficiency Virus Drug Research Articles

Comparison of a brand-name drug and its generic drugs - Impact of anti-human immunodeficiency virus drug exposure on the intestinal epithelial barrier

The use of unapproved anti-human immunodeficiency virus (HIV) drugs for its treatment and prevention has recently increased in Japan. Our research group investigated generic drugs of the Truvada combination tablet (TVD) that have not been approved in Japan, and reported differences in the pharmaceutical properties of their formulations. We also demonstrated that the pharmaceutical excipients in generic drugs may differ from those in TVD, and showed that some pharmaceutical excipients may affect epithelial cell barrier function. The present study investigated differences in the effects of TVD and its generic drugs (Generic A and Generic B) on epithelial cell barrier function. No significant differences were observed in epithelial cell barrier function following exposure to TVD, Generic A, and Generic B. However, significant increases in the mRNA levels of ABCB1 and Occludin, which regulate the epithelial cell barrier, were detected following exposure to Generic B. Therefore, the administration of Generic B for a longer duration or at a higher concentration may lead to changes in epithelial barrier function. Since the treatment and prevention of HIV requires the long-term (chronic) administration of drugs, the drugs used may change, such as from a brand-name drug to a generic drug and also from one generic drug to another. The present results suggest that the effects of brand-name and generic drugs, such as on intestinal epithelial barrier function, may differ with changes in the drugs being administered.

Drug-induced altered self-presentation increases tumor immunogenicity

Anti-human immunodeficiency virus (HIV) drug abacavir (ABC) binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activates CD8+ T cells by presenting altered abnormal peptides. Here, we examined the effect of ABC-induced altered self-presentation by HLA-B*57:01 on immunogenicity of cancer cells and CD8+ T-cell-dependent anti-tumor immunity. We established human-mouse chimeric HLA-B*57:01-expressing tumor cell lines (B16F10 and 3LL) and tested the anti-tumor effect of ABC in vivo. ABC treatment inhibited the growth of HLA-B*57:01-expressing tumors by a CD8+ T-cell-dependent mechanism. ABC treatment induced CXCR3-dependent infiltration of CD8+ T cells into HLA-B*57:01-expressing tumors, and activated those tumor-infiltrating CD8+ T cells to proliferate and secrete IFN-γ. The activation of CD8+ T cells using drug-induced altered self-presentation may be a new strategy to increase tumor immunogenicity and improve the efficacy of immunotherapy.

Molecular basis of RNA recognition by TBP of HIV-1 from multiple molecular dynamics simulations and energy predictions.

The frequent outbreaks of the AIDS (Acquired Immune Deficiency Syndrome) pandemic and the limited availability of anti-Human Immunodeficiency Virus (HIV) drugs highlight the urgent need to develop new antiviral drugs. A detailed understanding of the interactions between TAR-Binding Proteins (TBP) and RNA will facilitate the discovery of new anti-AIDS drugs. In order to characterize and explore the key interactions between RNA and TBP, we focused on the wild type (WT) and three mutant TBPs (TBP6.9, TBP6.7, and TBP6.3) with RNA, multiple molecular dynamics simulation and energy computation were performed. The results showed that 12 key residues played a major role in the interaction between TBP and RNA. The mutated residues of TBP changed the interaction between their surrounding residues and RNA, thus affecting the binding of TBP to RNA. In addition, structural and energy analyses showed that in contrast with WT TBP-RNA complex, the mutated residues had little effect on the backbone structure of TBP, but changes in the van der Waals interactions and electrostatic interaction associated with the side chains are responsible for the altered the binding between three mutant TBPs and RNA complexes. The discovery of TBP-RNA recognition mechanism in our work provides some useful insights and new opportunities for the development of anti-aids drugs.

Effect of PEGylation on drug uptake, biodistribution, and tissue toxicity of efavirenz–ritonavir loaded PAMAM G4 dendrimers

The present investigations aimed to compare the efficiency of PAMAM G4 (PG4) and PEGylated PAMAM G4 (PPG4) dendrimers as novel nanocarriers for the treatment of HIV-1. Synthesized PG4 and PPG4 dendrimers were confirmed by electrospray ionization and particle size with its morphology. The anti-human immunodeficiency virus (HIV) drug efavirenz (EFV) with a booster dose of ritonavir (RTV) was encapsulated into PG4 and PPG4 formerly noted as PG4ER and PPG4ER, respectively. Further, evaluated for dendrimers mediated solubilization, drug release, cytotoxicity, drug uptake, plasma, and tissue pharmacokinetics, and histopathology. PG4ER and PPG4ER both promoted a prolonged release of EFV in weakly acidic pH 4 up to 84 h and 132 h, respectively. The results of the cytotoxicity assay and drug uptake study showed that PPG4ER was safe and biocompatible up to 12.5 µg/ml. The plasma pharmacokinetic profile of EFV and RTV was significantly increased by PPG4ER with prolonged t 1/2 up to three times as compared to free EFV–RTV and PG4ER. Histopathological analysis showed remarkably lower tissue toxicity in PPG4ER as compared to free EFV–RTV. Therefore, overall data suggested that PPG4 has a great potential for prolonged release of EFV and RTV with enhanced bioavailability and lower toxicity.

Anti-HIV drugs lopinavir/ritonavir activate bitter taste receptors.

Lopinavir and ritonavir (LPV/r) are the primary anti-human immunodeficiency virus (HIV) drugs recommended by the World Health Organization for treating children aged 3 years and above who are infected with the HIV. These drugs are typically available in liquid formulations to aid in dosing for children who cannot swallow tablets. However, the strong bitter taste associated with these medications can be a significant obstacle to adherence, particularly in young children, and can jeopardize the effectiveness of the treatment. Studies have shown that poor palatability can affect the survival rate of HIV-infected children. Therefore, developing more child-friendly protease inhibitor formulations, particularly those with improved taste, is critical for children with HIV. The molecular mechanism by which lopinavir and ritonavir activate bitter taste receptors, TAS2Rs, is not yet clear. In this study, we utilized a calcium mobilization assay to characterize the activation of bitter taste receptors by lopinavir and ritonavir. We discovered that lopinavir activates TAS2R1 and TAS2R13, while ritonavir activates TAS2R1, TAS2R8, TAS2R13, and TAS2R14. The development of bitter taste blockers that target these receptors with a safe profile would be highly desirable in eliminating the unpleasant bitter taste of these anti-HIV drugs.

Rapid simultaneous analysis of anti human immunodeficiency virus drugs in pharmaceutical formulation by smart spectrophotometry based on multivariate calibration and least squares support vector machine methods

In this study, two chemometrics methods, including partial least squares regression (PLS) and least squares support vector machine (LS-SVM) were applied for the simultaneous determination of zidovudine (ZDV) and lamivudine (LMV) in synthetic mixtures and anti-HIV pharmaceutical formulation. These approaches along with the spectrophotometric method were used to solve spectral overlapping problems between mentioned components. The results of PLS showed that the number of components for ZDV and LMV were 10 and 10 with mean square prediction error (MSPE) of 0.4045 and 2.1189, respectively. This method revealed recoveries ranging from 99.48% to 100.40% and 99.55% to 101.25% for ZDV and LMV, respectively. By applying leave-one-out cross-validation (LOO-CV), γ (regularization parameter) and σ2 (width of the function) values were found to be 50, 1500 and 210, 20 with root mean square error (RMSE) of 0.6156 and 0.3163 for ZDV and LMV, respectively. The mean recoveries obtained by the LS-SVM were 100.82% and 98.93% for ZDV and LMV, respectively. A comparison between the suggested methods and high-performance liquid chromatography (HPLC) as a reference technique was implemented, which did not show a significant difference. The results obtained in this research revealed that the chemometrics approaches can be efficient, simple, inexpensive, and precise for routine analysis and quality control of the drug.

PCSK9 Inhibitor: Safe Alternative to Fill the Treatment Gap in Statin-Limited Conditions?

Lipid-lowering therapy is of great importance in reducing the burden of atherosclerotic cardiovascular disease. Statins act as first-line therapy in the current lipid management guidelines. However, statin use is limited in (1) statin-induced adverse events, including statin-associated muscle symptoms, new-onset diabetes mellitus, drug-induced liver injuries, acute kidney injuries, cognitive effects, hemorrhagic strokes, and cataracts; (2) special populations, including pregnant and lactating patients, patients with decompensated cirrhosis, and patients on dialysis; (3) coadministration with statin-interactive drugs, such as anti-human immunodeficiency virus drugs, anti-hepatitis C virus drugs, and immunosuppressive drugs. These considerable statin-limited groups are in urgent need of safer alternative lipid-lowering options. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are attracting widespread attention for their documented safety in general populations and superior lipid-lowering properties. Therefore, questions have been raised whether PCSK9 inhibitors could be a safe alternative in patients who are intolerant to statin therapy. In this review, we discuss the safety of PCSK9 inhibitors in statin-limited conditions. We conclude that PCSK9 inhibitors are a safe alternative lipid-lowering therapy in various statin-limited conditions. Furthermore, we identify several limitations in the current literature and suggest future directions, for the refinement of lipid management regimens.

Frequency of CYP2B6 Alleles in Major Iranian Ethnicities, Affecting Response to Efavirenz.

Efavirenz is an antihuman immunodeficiency virus (HIV) drug metabolized by cytochrome P450 2B6 (CYP2B6) enzyme. Cytochrome P450 2B6 is an enzyme that in humans is encoded by the CYP2B6 gene. Polymorphisms of this gene play a crucial role in the metabolism of drugs such as Efavirenz. This study aims to evaluate the frequency of three clinically significant CYP2B6 polymorphisms (CYP2B6 ∗ 6 (516G > T), CYP2B6 ∗ 4 (785A > G), and CYP2B6 ∗ 5 (1459C > T)) in three major Iranian ethnicities. One hundred forty-seven participants from three main Iranian ethnicities were included in this study. After DNA extraction, CYP2B6 ∗ 6 (516G > T), CYP2B6 ∗ 4 (785A > G), and CYP2B6 ∗ 5 (1459C > T) were genotyped using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR). The frequency of the mutated allele in the Iranian population for CYP2B6 ∗ 6 (516G > T) was 41.50 (95% CI: 35.81, 47.36), which was significantly lower than in Kurds (59.62, 95% CI: 45.10, 72.99). Similarly, Kurds had a higher frequency of mutated allele of CYP2B6 ∗ 5 (1459C > T) (46.15%, 95% CI: 32.23, 60.53) than in Iranians (24.49%, 95% CI: 19.68, 29.82). The frequency of A and G alleles of CYP2B6 ∗ 4 (785A > G) was 62.59% (95% CI: 56.78, 68.13) and 37.41 (95% CI: 31.87, 43.22), respectively. Kurds are at higher risk of adverse drug reactions (ADRs) and insufficient anti-HIV response compared to other Iranians.

Probing the interaction of anti-HIV drug Darunavir with dsDNA and HSA using electrochemical and spectroscopic measurements

Investigation of electrochemical and spectroscopic characteristics of anti-human immunodeficiency virus (HIV) drug provides important information related to the efficacy of the drug in relation with its interaction with several important biomolecules. In the present investigation we have developed an electrochemical and spectroscopic method for the detection of anti-HIV drug Darunavir (DRV) using the carbon paste as the working electrode. The analytical method has generated the detection limit of 1.86 µM (S/N = 3). The electrochemical investigations have also been carried out for the exploration of the interaction of DRV with double stranded deoxyribose nucleic acid DNA (dsDNA) and human serum albumin (HSA). Electrochemical investigations were supported from the spectroscopic measurements in evaluating the interaction. The results obtained from voltammetric and spectroscopic experiments shows strong interaction between the drug and the macromolecules. It has been observed that DRV forms strong complexes with HSA and dsDNA with the formation constants of 2.7 × 104 and 4.2 × 104 M−1 respectively. The formation constants are varied with the pH of the solution, which leads to the assertion of the mechanism of the interaction between DRV and dsDNA.

Effects of Genetic Polymorphisms of Cathepsin A on Metabolism of Tenofovir Alafenamide

Cathepsin A (CatA) is important as a drug-metabolizing enzyme responsible for the activation of prodrugs, such as the anti-human immunodeficiency virus drug Tenofovir Alafenamide (TAF). The present study was undertaken to clarify the presence of polymorphisms of the CatA gene in healthy Japanese subjects and the influence of gene polymorphism on the expression level of CatA protein and the drug-metabolizing activity. Single-strand conformation polymorphism method was used to analyze genetic polymorphisms in healthy Japanese subjects. Nine genetic polymorphisms were identified in the CatA gene. The polymorphism (85_87CTG>-) in exon 2 was a mutation causing a deletion of leucine, resulting in the change of the leucine 9-repeat (Leu9) to 8-repeat (Leu8) in the signal peptide region of CatA protein. The effect of Leu8 on the expression level of CatA protein was evaluated in Flp-In-293 cells with a stably expressed CatA, resulting in the expression of CatA protein being significantly elevated in variant 2 with Leu8 compared with Leu9. Higher concentrations of tenofovir alanine (TFV-Ala), a metabolite of TAF, were observed in the Leu8-expressing cells than in the Leu9-expressing cells using LC/MS/MS. Our findings suggest that the drug metabolic activity of CatA is altered by the genetic polymorphism.

Variable short duration treatment versus standard treatment, with and without adjunctive ribavirin, for chronic hepatitis C: the STOP-HCV-1 non-inferiority, factorial RCT

Background High cure rates with licensed durations of therapy for chronic hepatitis C virus suggest that many patients are overtreated. New strategies in individuals who find it challenging to adhere to standard treatment courses could significantly contribute to the elimination agenda. Objectives To compare cure rates using variable ultrashort first-line treatment stratified by baseline viral load followed by retreatment, with a fixed 8-week first-line treatment with retreatment with or without adjunctive ribavirin. Design An open-label, multicentre, factorial randomised controlled trial. Randomisation Randomisation was computer generated, with patients allocated in a 1 : 1 ratio using a factorial design to each of biomarker-stratified variable ultrashort strategy or fixed duration and adjunctive ribavirin (or not), using a minimisation algorithm with a probabilistic element. Setting NHS. Participants A total of 202 adults (aged ≥ 18 years) infected with chronic hepatitis C virus genotype 1a/1b or 4 for ≥ 6 months, with a detectable plasma hepatitis C viral load and no significant fibrosis [FibroScan® (Echosens, Paris, France) score F0–F1 or biopsy-proven minimal fibrosis], a hepatitis C virus viral load < 10,000,000 IU/ml, no previous exposure to direct-acting antiviral therapy for this infection and not pregnant. Patients co-infected with human immunodeficiency virus were eligible if human immunodeficiency virus viral load had been < 50 copies/ml for > 24 weeks on anti-human immunodeficiency virus drugs. Interventions Fixed-duration 8-week first-line therapy compared with variable ultrashort first-line therapy, initially for 4–6 weeks (continuous scale) stratified by screening viral load (variable ultrashort strategy 1, mean 32 days of treatment) and then, subsequently, for 4–7 weeks (variable ultrashort strategy 2 mean 39 days of duration), predominantly with ombitasvir, paritaprevir, ritonavir (Viekirax®; AbbVie, Chicago, IL, USA), and dasabuvir (Exviera®; AbbVie, Chicago, IL, USA) or ritonavir. All patients in whom first-line treatment was unsuccessful were immediately retreated with 12 weeks’ sofosbuvir, ledipasvir (Harvoni®, Gilead Sciences, Inc., Foster City, CA, USA) and ribavirin. Main outcome measure The primary outcome was overall sustained virological response (persistently undetectable) 12 weeks after the end of therapy (SVR12). Results A total of 202 patients were analysed. All patients in whom the primary outcome was evaluable achieved SVR12 overall [100% (197/197), 95% confidence interval 86% to 100%], demonstrating non-inferiority between fixed- and variable-duration strategies (difference 0%, 95% confidence interval –3.8% to 3.7%, prespecified non-inferiority margin 4%). A SVR12 following first-line treatment was achieved in 91% (92/101; 95% confidence interval 86% to 97%) of participants randomised to the fixed-duration strategy and by 48% (47/98; 95% confidence interval 39% to 57%) allocated to the variable-duration strategy. However, the proportion achieving SVR12 was significantly higher among those allocated to variable ultrashort strategy 2 [72% (23/32), 95% confidence interval 56% to 87%] than among those allocated to variable ultrashort strategy 1 [36% (24/66), 95% confidence interval 25% to 48%]. Overall, a SVR12 following first-line treatment was achieved by 72% (70/101) (95% confidence interval 65% to 78%) of patients treated with ribavirin and by 68% (69/98) (95% confidence interval 61% to 76%) of those not treated with ribavirin. A SVR12 with variable ultrashort strategies 1 and 2 was 52% (25/48) (95% confidence interval 38% to 65%) with ribavirin, compared with 44% (22/50) (95% confidence interval 31% to 56) without. However, at treatment failure, the emergence of viral resistance was lower with ribavirin [12% (3/26), 95% confidence interval 2% to 30%] than without [38% (11/29), 95% confidence interval 21% to 58%; p = 0.01]. All 10 individuals who became undetectable at day 3 of treatment achieved first-line SVR12 regardless of treatment duration. Five participants in the variable-duration arm and five in the fixed-duration arm experienced serious adverse events (p = 0.69), as did five participants receiving ribavirin and five participants receiving no ribavirin. Conclusions SVR12 rates were significantly higher when ultrashort treatment varied between 4 and 7 weeks, rather than between 4 and 6 weeks. We found no evidence of ribavirin significantly affecting first-line SVR12, with unsuccessful first-line short-course therapy also not compromising subsequent retreatment with sofosbuvir, ledipasvir and ribavirin. Future work A priority for future work needs to be the development and evaluation of robust predictive measures to identify those patients who can be cured with ultrashort courses of therapy. Trial registration Current Controlled Trials ISRCTN37915093, EudraCT 2015-005004-28 and CTA 19174/0370/001-0001. Funding This project was funded by the Efficacy and Mechanism Evaluation programme, a MRC and National Institute for Health Research (NIHR) partnership. This will be published in full in Efficacy and Mechanism Evaluation; Vol. 8, No. 17. See the NIHR Journals Library website for further project information.

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 μM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 μM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 μM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

COVID-19 Treatments and Vaccines: A year in Review

COVID-19 Treatments and Vaccines: A year in Review

Inhibition of Bitter Taste from Oral Tenofovir Alafenamide.

Children have difficulty swallowing capsules. Yet, when presented with liquid formulations, children often reject oral medications due to their intense bitterness. Presently, effective strategies to identify methods, reagents, and tools to block bitterness remain elusive. For a specific bitter-tasting drug, identification of the responsible bitter receptors and discovery of antagonists for those receptors can provide a method to block perceived bitterness. We have identified a compound (6-methylflavone) that can block responses to an intensely bitter-tasting anti-human immunodeficiency virus (HIV) drug, tenofovir alafenamide (TAF), using a primary human taste bud epithelial cell culture as a screening platform. Specifically, TAS2R39 and TAS2R1 are the main type 2 taste receptors responding to TAF observed via heterologously expressing specific TAS2R receptors into HEK293 cells. In this assay, 6-methylflavone blocked the responses of TAS2R39 to TAF. In human sensory testing, 8 of 16 subjects showed reduction in perceived bitterness of TAF after pretreating (or "prerinsing") with 6-methylflavone and mixing 6-methylflavone with TAF. Bitterness was completely and reliably blocked in two of these subjects. These data demonstrate that a combined approach of human taste cell culture-based screening, receptor-specific assays, and human psychophysical testing can successfully discover molecules for blocking perceived bitterness of pharmaceuticals, such as the HIV therapeutic TAF. Our hope is to use bitter taste blockers to increase medical compliance with these vital medicines. SIGNIFICANCE STATEMENT: Identification of a small molecule that inhibits bitter taste from tenofovir alafenamide may increase the compliance in treating children with human immunodeficiency virus infections.

The New NNRTI ACC007 Combined with Lamivudine and Tenofovir Disoproxil Fumarate Show Synergy Anti-HIV Activity In Vitro

Acquired immunodeficiency syndrome can hardly be cured currently and people with human immunodeficiency virus (HIV) need lifelong treatment that may result in the emergence of drug resistance which leads to failed treatment. Thus, the development of new anti- HIV drugs and new treatment regimens are necessary. The aim of this study is to analyze the combined anti-HIV activity of tenofovir disoproxil fumarate, lamivudine and ACC007, a new non-nucleoside reverse transcriptase inhibitor. The antiviral activity of tenofovir disoproxil fumarate, lamivudine and ACC007 alone or in combination against different HIV-1 strains was determined by the detection of HIV-1 p24 level through enzyme-linked immunosorbent assay. ACC007 showed EC50 of nanomolar range (from 3.03 nM to 252.59 nM) against all HIV-1 strains used in this study except the HIV-1A17, with EC50 of 1.57 μM. The combined antiviral activity of ACC007, lamivudine and tenofovir disoproxil fumarate showed synergy antiviral activity against all HIV-1 strains used in this study. The three-drug combination showed moderate synergism against HIV-1A17, HIV-14755-5, HIV-1K103N and HIV-1V106M, with a combination index value ranging from 0.71 to 0.87, and showed synergism against the other HIV-1 strains with combination index value from 0.35 to 0.67. The combination with ACC007 significantly increases the dose reduction index value of lamivudine and tenofovir disoproxil fumarate, compared with two-drug combination. ACC007 exhibits potent antiviral activity alone or with 3TC and TDF, and exerts synergistic effect against all HIV strains used in our investigation in vitro.

Arrestin Recruitment to C-C Chemokine Receptor 5: Potent C-C Chemokine Ligand 5 Analogs Reveal Differences in Dependence on Receptor Phosphorylation and Isoform-Specific Recruitment Bias.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.

Detection of Abacavir-Induced Structural Alterations in Human Leukocyte Antigen-B*57 : 01 Using Phage Display.

The interaction of human leukocyte antigen (HLA) with specific drugs is associated with delayed-type hypersensitivity reactions, which cause severe cutaneous toxicity. Such interactions induce structural alterations in HLA complexes via several different mechanisms such as the hapten theory, p-i concept, and altered peptide repertoire model, leading to the activation of cytotoxic T cells. To date, comprehensive detection of such structural alterations in preclinical studies has been difficult. Here, we evaluated structural alterations in HLA complexes focusing on the interaction between the HLA-B*57 : 01 allele and abacavir (an anti-human immunodeficiency virus drug), representing a model of abacavir hypersensitivity syndrome induced by changes in the peptide repertoire on the HLA molecule. We employed a phage display method using a commercially available antibody library to screen specific phage antibodies able to recognize HLA-B*57 : 01. The affinity of selected phage antibodies increased because of structural alterations in HLA-B*57 : 01 following exposure to abacavir, indicating that specific phage antibodies can identify drug-mediated structural changes in HLA complexes. We also identified an unreported structural change in HLA-B*57 : 01 using the phage display method, whereby abacavir increased the expression of peptide-deficient HLA-B*57 : 01 on the cell surface. These results suggest that phage display technology is a useful method for detecting structural changes in HLA complexes. This technology represents a potential novel strategy for predicting HLA-associated hypersensitivity reactions by drugs in pre-clinical studies.

The Anti-Angiogenic Effects of Anti-Human Immunodeficiency Virus Drugs.

The growth and metastasis of malignant tumors benefit from the formation of blood vessels within the tumor area. There, new vessels originate from angiogenesis (the sprouting of pre-existing neighboring vessels) and/or vasculogenesis (the mobilization of bone marrow-derived endothelial cell precursors which incorporate in tumor vasculature and then differentiate into mature endothelial cells). These events are induced by soluble molecules (the angiogenic factors) and modulated by endothelial cell interactions with the perivascular matrix. Given angiogenesis/vasculogenesis relevance to tumor progression, anti-angiogenic drugs are often employed to buttress surgery, chemotherapy or radiation therapy in the treatment of a wide variety of cancers. Most of the anti-angiogenic drugs have been developed to functionally impair the angiogenic vascular endothelial growth factor: however, this leaves other angiogenic factors unaffected, hence leading to drug resistance and escape. Other anti-angiogenic strategies have exploited classical inhibitors of enzymes remodeling the perivascular matrix. Disappointingly, these inhibitors have been found toxic and/or ineffective in clinical trials, even though they block angiogenesis in pre-clinical models. These findings are stimulating the identification of other anti-angiogenic compounds. In this regard, it is noteworthy that drugs utilized for a long time to counteract human immune deficiency virus (HIV) can directly and effectively hamper molecular pathways leading to blood vessel formation. In this review the mechanisms leading to angiogenesis and vasculogenesis, and their susceptibility to anti-HIV drugs will be discussed.

Design, synthesis and DNA interaction studies of new fluorescent platinum complex containing anti-HIV drug didanosine

Forming coordination complexes with nucleoside analogues may be helpful in studying anti-tumour activity of them. Therefore, to improve the clinical efficacy of nucleoside analogue and design new ones, a new fluorescent platinum (Pt) complex with anti-human immunodeficiency virus drug didanosine (ddI); K[PtCl(OCH3)2(ddI)]; was synthesized and characterized. The ultraviolet–visible (UV-vis) spectroscopy, infrared, thermogravimetric analysis, mass assignments and elemental analysis confirmed the preparation of the complex. The molecular ion peaks seen at the positive mass spectrum of Pt complex confirm coordination of the drug to metal centre. The interaction of this complex with calf thymus DNA (ct-DNA) was studied using several spectroscopic techniques such as UV absorption, fluorescence spectroscopy and dynamic viscosity measurements. Hyperchromism of the band in the UV-vis spectra and the intrinsic binding constant (0.56 ± 0.25) × 104 M−1, decreasing in Hoechst-DNA fluorescence by adding Pt complex concentration and also relatively small changes in DNA viscosity indicated that this complex could interact as a groove-binder. According to the UV spectra and the fluorescence quenching of the complex in our case seems to be primarily caused by complex formation between the Pt complex and DNA. The thermodynamic parameters showed that hydrogen bond and van der Waals interactions play main roles in the binding of Pt complex to ct-DNA. The free energy values are negative, showing the spontaneity of the Pt complex–DNA binding. The docking simulation was performed and the results confirm a preference of groove site of synthesized complex on DNA helix. The knowledge gained from this study will be helpful to further understand the DNA binding mechanism and can also provide much fruitful information for designing a new type of anti-cancer drugs.Communicated by Ramaswamy H. Sarma

Synthesis and anti-human immunodeficiency virus activity of substituted ( o,o-difluorophenyl)-linked-pyrimidines as potent non-nucleoside reverse transcriptase inhibitors.

With the worldwide number of human immunodeficiency virus positive patients stagnant and the increasing emergence of viral strains resistant to current treatment, the development of novel anti-human immunodeficiency virus drug candidates is a perpetual quest of medicinal chemists. Herein, we report a novel group of diarylpyrimidines, non-nucleoside reverse transcriptase inhibitors, which represents an important class of current anti-human immunodeficiency virus therapy. Series of diarylpyrimidines containing o,o-difluorophenyl (A-arm), 4-cyanophenylamino (B-arm), and a small substituent (e.g. NH2, OMe) at positions 2, 4, and 6 of the pyrimidine ring were prepared. The A-arm was modified in the para position (F or OMe) and linked to the central pyrimidine core with a variable spacer (CO, O, NH). Antiviral activities of 20 compounds were measured against wild type human immunodeficiency virus-1 and mutant reverse transcriptase strains (K103N, Y181C) using a cytoprotection assay. To the most promising structural motives belong the o,o-difluoro-p-methoxy A-arm in position 4, and the amino group in position 6 of pyrimidine. Single digit nanomolar activities with no significant toxicity (CC50 > 17,000 nM) were found for compounds 35 (EC50 = 2 nM), 37 (EC50 = 3 nM), and 13 (EC50 = 4 nM) having O, NH, and CO linkers, respectively.