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Antiretroviral drugs have made significant progress in treating HIV-1 and improving the quality of HIV-1-infected individuals. However, due to their limited permeability into the brain HIV-1 replication persists in brain reservoirs such as perivascular macrophages and microglia, which cause HIV-1-associated neurocognitive disorders. Therefore, it is highly desirable to find a novel therapy that can cross the blood-brain barrier (BBB) and target HIV-1 pathogenesis in brain reservoirs. A recently developed 2-amino-3-methylpentanoic acid [2-morpholin-4-yl-ethyl]-amide (LM11A-31), which is a p75 neutrotrophin receptor (p75 NTR ) modulator, can cross the BBB. In this study, we examined whether LM11A-31 treatment can suppress HIV-1 replication, oxidative stress, cytotoxicity, and inflammatory response in macrophages. Our results showed that LM11A-31 (100 nM) alone and/or in combination with positive control darunavir (5.5 µM) significantly suppresses viral replication and reduces cytotoxicity. Moreover, the HIV-1 suppression by LM11A-31 was comparable to the HIV-1 suppression by darunavir. Although p75 NTR was upregulated in HIV-1-infected macrophages compared to uninfected macrophages, LM11A-31 did not significantly reduce the p75 NTR expression in macrophages. Furthermore, our study illustrated that LM11A-31 alone and/or in combination with darunavir significantly suppress pro-inflammatory cytokines including IL-1β, IL-8, IL-18, and TNF-α and chemokines MCP-1 in HIV-induced macrophages. The suppression of these cytokines and chemokines by LM11A-31 was comparable to darunavir. In contrast, LM11A-31 did not significantly alter oxidative stress, expression of antioxidant enzymes, or autophagy marker proteins in U1 macrophages. The results suggest that LM11A-31, which can cross the BBB, has therapeutic potential in suppressing HIV-1 and inflammatory response in brain reservoirs, especially in macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mirzahosseini, Sinha, Zhou, Godse, Kodidela, Singh, Ishrat and Kumar.)

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents., Competing Interests: All authors were employed and compensated by Gilead Sciences and may be shareholders of the company.

The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs., (© 2024. The Author(s).)

There is an ongoing debate regarding whether low-level viremia (LLV), in particular persistent LLV, during HIV treatment with optimal adherence originates from low-level viral replication, viral production, or both. We performed an observational study in 30 individuals with LLV who switched to a boosted darunavir (DRV)-based therapy. In-depth virological analyses were used to characterize the viral population and the (activity) of the viral reservoir. Immune activation was examined using cell-bound and soluble markers. The primary outcome was defined as the effect on HIV-RNA and was categorized by responders (<50 cp/mL) or non-responders (>50 cp/mL). At week 24, 53% of the individuals were considered responders, 40% non-responders, and 7% could not be assigned. Sequencing showed no evolution or selection of drug resistance in the non-responders. Production of defective virus with mutations in either the protease (D25N) or RT active site contributed to persistent LLV in two individuals. We show that in about half of the study participants, the switch to a DRV-based regimen resulted in a viral response indicative of ongoing low-level viral replication as the cause of LLV before the switch. Our data confirm that in clinical management, high genetic barrier drugs like DRV are a safe choice, irrespective of the source of LLV.

Human Immunodeficiency Virus type 1 protease (HIV-1 PR) is one of the most challenging targets of antiretroviral therapy used in the treatment of AIDS-infected people. The performance of protease inhibitors (PIs) is limited by the development of protease mutations that can promote resistance to the treatment. The current study was carried out using statistics and bioinformatics tools. A series of thirty-three compounds with known enzymatic inhibitory activities against HIV-1 protease was used in this paper to build a mathematical model relating the structure to the biological activity. These compounds were designed by software; their descriptors were computed using various tools, such as Gaussian, Chem3D, ChemSketch and MarvinSketch. Computational methods generated the best model based on its statistical parameters. The model's applicability domain (AD) was elaborated. Furthermore, one compound has been proposed as efficient against HIV-1 protease with comparable biological activity to the existing ones; this drug candidate was evaluated using ADMET properties and Lipinski's rule. Molecular Docking performed on Wild Type, and Mutant Type HIV-1 proteases allowed the investigation of the interaction types displayed between the proteases and the ligands, Darunavir (DRV) and the new drug (ND). Molecular dynamics simulation was also used in order to investigate the complexes' stability allowing a comparative study on the performance of both ligands (DRV & ND). Our study suggested that the new molecule showed comparable results to that of darunavir and maybe used for further experimental studies. Our study may also be used as pipeline to search and design new potential inhibitors of HIV-1 proteases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Baassi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance., Competing Interests: ES, SL, SZ, GL, MH, AS, KK, EN, AA, NY, CS, RS No competing interests declared, (© 2023, Spielvogel et al.)

Antiretroviral drugs have dramatically improved the prognosis of HIV-infected patients, with strikingly reduced morbidity and mortality. However, long-term use can be associated with signs of premature aging. Highly active antiretroviral therapy generally comprises two nucleoside reverse transcriptase inhibitors (NRTIs), with one of three additional antiretroviral drug classes, including protease inhibitors (PIs). One commonality between mitochondrial dysfunction (induced by NRTIs) and defects in lamin A (induced by PIs) is they can cause or accelerate cellular senescence, a state of essentially irreversible growth arrest, and the secretion of many bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP). We hypothesized that senescent cells increase following treatment with certain HIV therapies. We compared the effects of two distinct HIV PIs: ritonavir-boosted atazanavir (ATV/r) and ritonavir-boosted darunavir (DRN/r), used in combination treatments for HIV infection. Upon ATV/r, but not DRN/r, treatment, cells arrested growth, displayed multiple features of senescence, and expressed significantly upregulated levels of many SASP factors. Furthermore, mice receiving sustained ATV/r treatment showed an increase in senescent cells and age-related decline in physiological function. However, removing treatment reversed the features of senescence observed in vivo and cell culture. Given how these features disappeared with drug removal, certain features of senescence may not be prognostic as defined by an irreversible growth arrest. Importantly, for patients that are treated or have been treated with ATV/r, our data suggest that switching to another PI that does not promote premature aging conditions (DRN/r) may improve the associated age-related complications., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CL pro ) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CL pro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CL pro potency with EC50 values of 6.3 μM and 3.5 μM and were shown to bind to SARS-CoV-2 3CL pro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CL pro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CL pro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

Antiretroviral drug resistance is a therapeutic obstacle for people with HIV. HIV protease inhibitors darunavir and lopinavir are recommended for resistant infections. We characterized a protease mutant (PR10x) derived from a highly resistant clinical isolate including 10 mutations associated with resistance to lopinavir and darunavir. Compared to the wild-type protease, PR10x exhibits ∼3-fold decrease in catalytic efficiency and K i values of 2-3 orders of magnitude worse for darunavir, lopinavir, and potent investigational inhibitor GRL-519. Crystal structures of the mutant were solved in a ligand-free form and in complex with GRL-519. The structures show altered interactions in the active site, flap-core interface, hydrophobic core, hinge region, and 80s loop compared to the corresponding wild-type protease structures. The ligand-free crystal structure exhibits a highly curled flap conformation which may amplify drug resistance. Molecular dynamics simulations performed for 1 μs on ligand-free dimers showed extremely large fluctuations in the flaps for PR10x compared to equivalent simulations on PR with a single L76V mutation or wild-type protease. This analysis offers insight about the synergistic effects of mutations in highly resistant variants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 (IC 50 /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 IC 50 /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.

PR S17 , a variant of human immunodeficiency virus type I protease (HIV-1 PR), has 17 mutated residues showing high levels of multidrug resistance. To describe the effects of these mutated residues on the dynamic properties and the binding mechanism of PR with substrate and inhibitor, focused on six systems (two complexes of WT PR and PR S17 with inhibitor Darunavir (DRV), two complexes of WT PR and PR S17 with substrate analogue CA-p2, two unligand WT PR and PR S17 ), we performed multiple molecular dynamics (MD) simulations combined with MM-PBSA and solvated interaction energy (SIE) methods. For both the unligand PRs and ligand-PR complexes, the results from simulations revealed 17 mutated residues alter the flap-flap distance, the distance from flap regions to catalytic sites, and the curling degree of the flap tips. These mutated residues changed the flexibility of the flap region in PR, and thus affected its binding energy with DRV and CA-p2, resulting in differences in sensitivity. Hydrophobic cavity makes an important contribution to the binding of PR and ligands. And most noticeable of all, the binding of the guanidine group in CA-p2 and Arg8' of PR S17 is useful for increasing their binding ability. These results have important guidance for the further design of drugs against multidrug resistant PR. IMPORTANCE Developing effective anti-HIV inhibitors is the current requirement to cope with the emergence of the resistance of mutants. Compared with the experiments, MD simulations along with energy calculations help reduce the time and cost of designing new inhibitors. Based on our simulation results, we propose two factors that may help design effective inhibitors against HIV-1 PR: (i) importance of hydrophobic cavity, and (ii) introduction of polar groups similar to the guanidine group.

Background: Several pre-clinical and clinical reports suggest that HIV-1 protease inhibitors, in addition to the antiretroviral properties, possess pleiotropic pharmacological effects including anticancer action. Therefore, we investigated the pro-apoptotic activity in tumor cells of two molecules, RDD-19 and RDD-142, which are hydroxyethylamine derivatives' precursors of darunavir and several HIV-1 protease inhibitors. Methods : Three hepatoma cell lines and one non-pathological cell line were treated with RDD-19 and RDD-142, and cell viability was assessed. The expression levels of several markers for ER stress, autophagy, cellular ubiquitination, and Akt activation were quantified in HepG2 cells treated with RDD-19 and RDD-142 to evaluate apoptotic and non-apoptotic cell death. Results: RDD-19 and RDD-142 showed a greater dose-dependent cytotoxicity towards the hepatic tumor cell line HepG2 compared to the non-pathological hepatic cell line IHH. Both molecules caused two types of cell death, a caspase-dependent apoptosis, which was ascertained by a series of biochemical and morphological assays, and a caspase-independent death that was characterized by the induction of ER stress and autophagy. The strong increase of ubiquitinated proteins inside the cells suggested that the target of these molecules could be the proteasome and in silico molecular docking analysis that was used to support the plausibility of this hypothesis. Furthermore, cells treated with the two compounds displayed decreased levels of p-AKT, which interferes with cell survival and proliferation. Conclusions: These findings demonstrate that two compounds, RDD-19 and RDD-142, have pleiotropic effects and that they may represent promising anticancer candidates.

The epidemic of the novel coronavirus disease (COVID-19) that started in 2019 has evoked an urgent demand for finding new potential therapeutic agents. In this study, we performed a molecular docking of anti-HIV drugs to refine HIV protease inhibitors and nucleotide analogues to target COVID-19. The evaluation was based on docking scores calculated by AutoDock Vina and top binding poses were analyzed. Our results suggested that lopinavir, darunavir, atazanavir, remdesivir, and tipranavir have the best binding affinity for the 3-chymotrypsin-like protease of COVID-19. The comparison of the binding sites of three drugs, namely, darunavir, atazanavir and remdesivir, showed an overlap region of the protein pocket. Our study showed a strong affinity between lopinavir, darunavir, atazanavir, tipranavir and COVID-19 protease. However, their efficacy should be confirmed by in vitro studies since there are concerns related to interference with their active sites., (© 2022 Linh Tran et al., published by Sciendo.)

The intracellular penetration of darunavir, a second-generation HIV protease inhibitor, is limited by the activity of the efflux P-glycoprotein (ABCB1). ABCB1 expression and/or activity levels can vary between individuals due to genetic polymorphisms including the c.1199G>A, c.1236C>T, c.2677G>T and c.3435C>T variants, which could in part explain why the pharmacokinetics of darunavir are so variable from one individual to another. While a few clinical studies have failed to demonstrate an influence of these polymorphisms on darunavir pharmacokinetics, drug-drug interactions and methodological limitations may have prevented them from revealing the true influence of ABCB1 variants. In this work, we report on the intracellular accumulation of darunavir in recombinant HEK293 cell lines expressing wild-type ABCB1 or one of several variants: ABCB1 1199A, ABCB1 3435T, and ABCB1 1236T/2677T/3435T. We demonstrate that while ABCB1 expression limits intracellular accumulation of darunavir, there is no significant difference in efflux activity between cells expressing wild-type ABCB1 and those that express any of the studied variants.

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10-20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron-electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter K i and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (M pro ) 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 M pro . The frequently accessed sites on M pro 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.

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study., Competing Interests: The authors have declared that no competing interests exist.

HIV-1 protease inhibitors (PIs), such as darunavir (DRV), are the key component of antiretroviral therapy. However, HIV-1 often acquires resistance to PIs. Here, seven novel PIs were synthesized, by introducing single atom changes such as an exchange of a sulfur to an oxygen, scission of a single bond in P2'-cyclopropylaminobenzothiazole (or -oxazole), and/or P1-benzene ring with fluorine scan of mono- or bis-fluorine atoms around DRV's scaffold. X-ray structural analyses of the PIs complexed with wild-type Protease (PR WT ) and highly-multi-PI-resistance-associated PR DRV R P51 revealed that the PIs better adapt to structural plasticity in PR with resistance-associated amino acid substitutions by formation of optimal sulfur bond and adaptation of cyclopropyl ring in the S2'-subsite. Furthermore, these PIs displayed increased cell permeability and extreme anti-HIV-1 potency compared to DRV. Our work provides the basis for developing novel PIs with high potency against PI-resistant HIV-1 variants with a high genetic barrier.

Introduction. Cryptococcus species are pathogens commonly associated with cases of meningoencephalitis in individuals who are immunosuppressed due to AIDS. Aim. The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Cryptococcus species. Methodology. Susceptibility testing of darunavir and the common antifungals against 12 members of the Cryptococcus neoformans / Cryptococcus gattii species complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively. Results. Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. All Cryptococcus species strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation ( P <0.05). The combination of darunavir with antifungals caused a significant reduction in biofilm metabolic activity and biomass when compared to darunavir alone ( P <0.05). Conclusion. Darunavir presents antifungal activity against planktonic cells of Cryptococcus species and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms of Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.

HIV-associated nephropathy (HIVAN) is a rapidly progressive kidney disease that is caused by HIV infection of renal epithelial cells with subsequent expression of viral genes, including vpr. Antiretroviral therapy ameliorates HIVAN without eradicating HIV from the kidneys and the mechanism by which it protects kidneys is poorly understood. Since HIV protease inhibitors have "off target" cellular effects, we studied whether darunavir, the most commonly prescribed protease inhibitor, protects kidneys from HIV-induced injury via mechanisms independent of HIV protease and viral replication. Renal epithelial cells were transduced with lentiviruses encoding HIV (lacking protease and reverse transcriptase), Vpr, or vector control. Darunavir attenuated HIV and Vpr-induced activation of Stat3, Src, Erk, and cytokines, which are critical for HIVAN pathogenesis. We then studied HIV-transgenic mice, which develop HIVAN in the absence of HIV protease or reverse transcriptase. Mice were treated with darunavir, zidovudine, darunavir + zidovudine, or control. Darunavir and darunavir + zidovudine reduced albuminuria and histologic kidney injury and normalized expression of dysregulated proteins. RNA-seq analyses demonstrated that darunavir suppressed HIV-induced upregulation of immune response genes in human kidney cells. These data demonstrate that darunavir protects against HIV-induced renal injury via mechanisms that are independent of inhibition of HIV protease.

We generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2'-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2' ligands, respectively. GRL-001-15 and GRL-003-15 have meta -monofluorophenyl and para -monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (EC 50 s) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CC 50 s) of 38 and 11 μM, respectively. The activity of GRL-001-15 against multi-PI-resistant HIV-1 variants was generally greater than that of GRL-003-15. The EC 50 of GRL-001-15 against an HIV-1 variant that was highly resistant to multiple PIs, including darunavir (DRV) (HIV-1 DRV R P30 ), was 0.17 nM, and that of GRL-003-15 was 3.3 nM, while DRV was much less active, with an EC 50 of 216 nM. The emergence of HIV-1 variants resistant to GRL-001-15 and GRL-003-15 was significantly delayed compared to that of variants resistant to selected PIs, including DRV. Structural analyses of wild-type protease (PR WT ) complexed with the novel PIs revealed that GRL-001-15's meta -fluorine atom forms halogen bond interactions (2.9 and 3.0 Å) with Gly49 and Ile50, respectively, of the protease flap region and with Pro81' (2.7 and 3.2 Å), which is located close to the protease active site, and that two fluorine atoms of GRL-142-13 form multiple halogen bond interactions with Gly49, Ile50, Pro81', Ile82', and Arg8'. In contrast, GRL-003-15 forms halogen bond interactions with Pro81' alone, suggesting that the reduced antiviral activity of GRL-003-15 is due to the loss of the interactions with the flap region., (Copyright © 2019 American Society for Microbiology.)

The combination of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor elbasvir and the NS3/4A protease inhibitor grazoprevir is a potent, once-daily therapy indicated for the treatment of chronic HCV infection in individuals coinfected with human immunodeficiency virus (HIV). We explored the pharmacokinetic interactions of elbasvir and grazoprevir with ritonavir and ritonavir-boosted HIV protease inhibitors in three phase 1 trials. Drug-drug interaction trials with healthy participants were conducted to evaluate the effect of ritonavir on the pharmacokinetics of grazoprevir ( n  = 10) and the potential two-way pharmacokinetic interactions of elbasvir ( n  = 30) or grazoprevir ( n  = 39) when coadministered with ritonavir-boosted atazanavir, lopinavir, or darunavir. Coadministration of ritonavir with grazoprevir increased grazoprevir exposure; the geometric mean ratio (GMR) for grazoprevir plus ritonavir versus grazoprevir alone area under the concentration-time curve from 0 to 24 h (AUC 0-24 ) was 1.91 (90% confidence interval [CI]; 1.31 to 2.79). Grazoprevir exposure was markedly increased with coadministration of atazanavir-ritonavir, lopinavir-ritonavir, and darunavir-ritonavir, with GMRs for grazoprevir AUC 0-24 of 10.58 (90% CI, 7.78 to 14.39), 12.86 (90% CI, 10.25 to 16.13), and 7.50 (90% CI, 5.92 to 9.51), respectively. Elbasvir exposure was increased with coadministration of atazanavir-ritonavir, lopinavir-ritonavir, and darunavir-ritonavir, with GMRs for elbasvir AUC 0-24 of 4.76 (90% CI, 4.07 to 5.56), 3.71 (90% CI, 3.05 to 4.53), and 1.66 (90% CI, 1.35 to 2.05), respectively. Grazoprevir and elbasvir had little effect on atazanavir, lopinavir, and darunavir pharmacokinetics. Coadministration of elbasvir-grazoprevir with atazanavir-ritonavir, lopinavir-ritonavir, or darunavir-ritonavir is contraindicated, owing to an increase in grazoprevir exposure. Therefore, HIV treatment regimens without HIV protease inhibitors should be considered for HCV/HIV-coinfected individuals who are being treated with elbasvir-grazoprevir., (Copyright © 2019 American Society for Microbiology.)

A fixed-dose combination consisting of darunavir (Drv), cobicistat (Cobi), emtricitabine (2',3'-dideoxy-5-fluoro-3'-thiacytidine [FTC]), and tenofovir alafenamide (Taf) has been recently approved by the European Medicines Agency for the treatment of HIV infection, and is the first ever protease-inhibitor-based single-tablet regimen. This article provides a detailed description of its pharmacokinetic, efficacy, and safety profile. The pharmacokinetics of single compounds were analyzed, with a special focus on contrasts between Drv/Cobi and Drv/ritonavir (Rtv). When comparing Cobi and Rtv, multiple interactions must be taken into account: in comparison to Rtv, Cobi is a more selective CYP3A4 inhibitor and has no clinical effect on other isoenzymes inhibited by Rtv (eg, 2C8 and 2C9). Moreover, unlike Cobi, Rtv shows in vivo induction activity on some CYP isoenzymes (eg, 1A2, 2C19, 2C8, 2C9, and 2B6), glucuronyltransferases (eg, UGT1A4), and Pgp. Drv-Cobi-FTC-Taf has recently been demonstrated to be of equal efficacy to Drv-Rtv and other protease inhibitors in both experienced (EMERALD study) and naïve (AMBER study) patients. Moreover, kidney and bone safety profiles have been shown to be good, as has central nervous system tolerance. Total cholesterol:low-density-lipoprotein cholesterol and total cholesterol:high-density-lipoprotein cholesterol ratios are generally high in Drv-Cobi-FTC-Taf vs Rtv-Drv-FTC + tenofovir disoproxil fumarate. An unlikely role of Drv in influencing cardiovascular risk in HIV infection has also been reported. Kidney safety profile is influenced by Cobi, with an increase in creatinine plasma concentration of 0.05-0.1 mg/dL and a parallel glomerular filtration-rate reduction of 10 mL/min within the first 4 weeks after Cobi introduction, which remains stable during treatment. Bone and central nervous system safety profiles were found to be good in randomized clinical trials of both experienced and naïve patients. The efficacy and safety of Drv/Cobi/FTC/Taf are comparable to other drug regimens recommended for HIV treatment., Competing Interests: Disclosure NS has received grants for board membership by Janssen and ViiV, payment for lectures, including service on speakers’ bureaux from MSD, and travel grant/supports from Gilead. AG has received grants/research support from AbbVie, Astellas, BMS, Boehringer, Gilead, Janssen, MSD, Novartis, Pfizer, Roche, ViiV, and ANLAIDS Sezione Lombarda, receipt of honoraria or consultation fees from Bristol-Myers Squibb, Gilead, Janssen, MSD, Novartis, and ViiV, fees for participation in a company-sponsored speakers’ bureau from Gilead, and travel grant/supports from Bristol-Myers Squibb, Gilead, Jansen, and ViiV. AB has received grants for board membership from Janssen, grants/research support from Gilead and ANLAIDS Sezione Lombarda, and travel grants/support from ViiV, Gilead, AbbVie, MSD, and BMS. The authors report no other conflicts of interest in this work.

HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor's ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.

Nanonizationhas been extensively investigated to increase theoral bioavailability of hydrophobicdrugsin general andantiretrovirals(ARVs)used inthe therapy of the human immunodeficiency virus (HIV) infection in particular. Weanticipatedthatin the caseofprotease inhibitors, a family of pH-dependent ARVsthatdisplay high aqueous solubility undertheacidconditionsof thestomach andextremely low solubilityunder the neutral ones ofthe small intestine, this strategy might failowing to an uncontrolled dissolution-re-precipitation process that will take place along the gastrointestinal tract.To tackle thisbiopharmaceutical challenge, in this work, wedesigned, produced and fully characterized a novelNanoparticle-in-MicroparticleDelivery System(NiMDS)comprised of pure nanoparticlesofthefirst-line protease inhibitor darunavir(DRV) and itsboosting agentritonavir (RIT) encapsulated within film-coated microparticles.For this, a clinically relevant combination of pure DRV and RIT nanoparticles wassynthesized by a sequential nanoprecipitation/solvent diffusion and evaporation method employing sodium alginateas viscosity stabilizer. Then, pure nanoparticles were encapsulated within calcium alginate/chitosanmicroparticlesthat were film-coated with a series ofpoly(methacrylate) copolymers with differential solubility in the gastrointestinal tract. This coating ensured full stability under gastric-like pH and sustained drug release under intestinal one. PharmacokineticstudiesconductedinalbinoSpragueDawleyratsshowed that DRV/RIT-loadedNiMDSs containing 17% w/w drug loading based on dry weight significantlyincreasedthe oral bioavailabilityof DRVby 2.3-foldwith respect to both theunprocessedandthenanonized DRV/RIT combinations that showed statistically similar performance. Moreover, they highlighted the limited advantage of only drugnanonizationto improve the oral pharmacokinetics of protease inhibitors and the potential of our novel delivery approach to improve the oral pharmacokinetics of nanonized poorly water-soluble drugs displaying pH-dependent solubility., Statement of Significance: Protease inhibitors (PIs) are gold-standard drugs in many ARV cocktails. Darunavir (DRV) is the latest approved PI and it is included in the 20th WHO Model List of Essential Medicines. PIs poorly-water soluble at intestinal pH and more soluble under gastric conditions. Drug nanonization represents one of the most common nanotechnology strategies to increase dissolution rate of hydrophobic drugs and thus, their oral bioavailability. For instance, pure drug nanosuspensions became the most clinically relevant nanoformulation. However, according to the physicochemical properties of PIs, nanonization does not appear as a very beneficial strategy due to the fast dissolution rate anticipated under the acid conditions of the stomach and their uncontrolled recrystallization and precipitation in the small intestine that might result in the formation of particles of unpredictable size and structure (e.g., crystallinity and polymorphism) and consequently, unknown dissolution rate and bioavailability. In this work, we developed a sequential nanoprecipitation method for the production of pure nanoparticles of DRV and its boosting agent ritonavir in a clinically relevant 8:1 wt ratio using alginate as viscosity stabilizer and used this nanosuspension to produce a novel kind of nanoparticle-in-microparticle delivery system that was fully characterized and the pharmacokinetics assessed in rats. The most significant points of the current manuscript are., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC 50 s) were 2.5 to 30 nM against wild-type HIV-1 NL4-3 , 0.3 to 6.7 nM against HIV-2 EHO , and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIV MDR ). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIV DRV r p51 ), with EC 50 s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC 50 s of >1,000 nM) against HIV DRV r p51 Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2' Cp-Abt group forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIV MDR strains (HIV 11MIX ), HIV 11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV 11MIX failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2' Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1 NL4-3 and a wide spectrum of HIV MDR strains., (Copyright © 2018 American Society for Microbiology.)

Darunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIV DRV R P51 ). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIV DRV R P51 are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIV I54M and rHIV I84V , respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIV V32I also developed high-level DRV resistance. However, wild-type HIV NL4-3 (rHIV WT ) failed to acquire V32I and did not develop DRV resistance. Compared to rHIV WT , rHIV V32I was highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIV WT with DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1's DRV resistance development and elucidate, at least in part, a mechanism of DRV's high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution. IMPORTANCE Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1's drug resistance. However, the mechanism(s) of the DRV's high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro -selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIV WT ), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIV WT , strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1's resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

Aims: Patients receiving darunavir are advised to take it concomitantly with food. The objectives of the present cross-sectional study were to evaluate the actual concomitant food intake of patients visiting an HIV outpatient clinic., Methods: Sixty participants treated with darunavir/ritonavir once daily were subjected to a food recall questionnaire concerning their last concomitant food intake with darunavir. Darunavir trough concentrations were calculated., Results: The median food intake was 507 (0-2707) kcal; protein intake, 20 (0-221)g; carbohydrate intake, 62 (0-267)g; fat intake: 14 (0-143)g; and dietary fibre: 4 (0-30)g. Twenty-five patients (42%) ingested their drug with between-meal snacks. No relationship was found between food intake and trough concentrations., Conclusions: Clear advice on the optimal caloric intake is needed, to avoid high caloric intake in patients who already have an increased risk of cardiovascular disease due to their HIV infection., (© 2017 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir., Competing Interests: The authors have declared that no competing interests exist.

We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC 50 ] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC 50 ] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1 NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC 50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: To assess the impact of the loss-of-function CYP3A5*3 allele (rs776746, 6986A>G SNP) on darunavir (DRV) plasma concentrations., Methods: 135 HIV-1 infected patients treated with DRV-based therapy were included in the study and plasma samples were obtained immediately before drug intake in order to determine DRV trough concentrations using an ultra performance liquid chromatography method (UPLC) with diode-array detection (DAD). Noteworthy is the fact that in 16 (11.9%) patients, etravirine (ETR) was combined with DRV. CYP3A5 genotypes were determined using real time PCR method (TaqMan® genotyping assay). The patients were then classified into CYP3A5 expressors (CYP3A5*1 allele carriers) and non-expressors (CYP3A5*3 homozygous). Subsequently, the association between DRV plasma trough concentration ([DRV]plasma) and CYP3A5 genotype-based expression status was analyzed., Results: 45% of the patients were classified as CYP3A5 expressors. In the whole cohort, mean [DRV]plasma was not different between CYP3A5 expressors and non-expressors (1894ng/ml [CI95%: 1566-2290] versus 1737ng/ml [CI95%: 1468-2057], p = 0.43). However, in the subgroup of the 16 patients receiving DRV combined with ETR, a significantly lower [DRV]plasma was observed for CYP3A5 expressors when compared to non-expressors (1385ng/ml [CI95%:886.3-2165] versus 3141ng/ml [CI95%:2042-4831], p = 0.007)., Conclusions: Interaction between DRV and ETR is partly mediated by CYP3A5 polymorphism with lower DRV plasma trough concentrations in CYP3A5 expressors suggesting a specific ETR-driven CYP3A5 activation only in CYP3A5 expressors. Consequently, these patients might be more at risk of infra-therapeutic [DRV]plasma. This potentially important observation is a good illustration of a genotype-based drug interaction, which could also have considerable consequences if translated to other CYP3A5-metabolized drugs. Further investigations are thus needed to confirm this association and to explore its clinical impact, mainly in the African population among whom CYP3A5 expressors are more frequent, before recommending systematic CYP3A5 pre-emptive genotyping for DRV-ETR co-administration., Competing Interests: The authors have declared that no competing interests exist.

Patients with human immunodeficiency virus type 1 (HIV-1) who receive antiretroviral therapy (ART) often achieve increased survival and improved quality of life. In this respect, monotherapy with darunavir/ritonavir (mDRV/r) can be a useful treatment strategy. This prospective study analyses the effect of mDRV/r on sperm quality and viral load in a group of 28 patients who had previously been given conventional ART and who had recorded a viral load <20 copies/mL for at least six months. These patients were given mDRV/r at a dose of 800/100 mg for 48 weeks. At baseline (V0), CD4, CD8, FSH, LH and testosterone levels were measured, together with HIV-1 viral load in plasma and semen. In addition, seminal fluid quality was studied before mDRV/r treatment was prescribed. At week 48 (V1), HIV-1 viral load in plasma and semen and the quality of the seminal fluid were again measured. The results obtained indicate that at V0, 10% of the patients with ART had a positive viral load in seminal fluid (>20 copies/ml), and that at V1, after mDRV/r treatment, this figure had fallen to 3%. The quality of seminal fluid was close to normal in 57% of patients at V0 and in 62% at V1. We conclude that, similar to ART, mDRV/r maintains HIV-1 viral load in most patients, and that there is no worsening in seminal fluid quality.

Current antiretroviral (ARV) therapy for the treatment of human immunodeficiency virus (HIV-1)-infected patients provides long-term control of viral load (VL). Darunavir (DRV) is a nonpeptidomimetic protease inhibitor approved for use with a ritonavir booster (DRV/r). This study evaluated the effectiveness of DRV/r in combination with other ARV agents in routine clinical practice in Italy. In this descriptive observational study, data on utilization of DRV/r, under the conditions described in the marketing authorization, were collected from June 2009 to December 2012. Effectiveness (VL <50 copies/mL), tolerability, and durability in four patient groups (two DRV/r-experienced, one ARV-experienced DRV/r-naïve, and one ARV-naïve) were analyzed. Secondary objectives included immunological response, safety, and persistence/discontinuation rates. In total, 875 of 883 enrolled patients were included in the analysis: of these, 662 (75.7%) completed the follow-up until the end of 2012 and 213 (24.3%) withdrew from the study earlier. Initial DRV dose was 600 mg twice daily (67.1%) or 800 mg once daily (32.9%). Only 16 patients (1.8%) withdrew from the study due to virological failure. Virological response proportions were higher in patients virologically suppressed at study entry versus patients with baseline VL ≥50 copies/mL in each ARV-experienced group, while there was no consistent difference across study groups and baseline VL strata according to baseline CD4(+) cell count. CD4(+) cell count increased from study entry to last study visit in all the four groups. DRV/r was well tolerated, with few discontinuations due to study-emergent nonfatal adverse events (3.0% overall, including 2.1% drug-related) or deaths (3.0% overall, all non-drug-related); 35.3% of patients reported ≥1 adverse events. These observational data show that DRV/r was effective and well tolerated in the whole patient population described here. The DRV/r-containing regimen provided viral suppression in a high percentage of patients in all groups, with low rates of discontinuation due to virological failure.

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

The binding mechanism of HIV-1 protease monomers leading to the catalytically competent dimeric enzyme has been investigated by means of state-of-the-art atomistic simulations. The emerging picture allows a deeper understanding of experimental observations and reveals that water molecules trapped at the interface have an important role in slowing down the kinetics of the association process. Unexpectedly, a cryptic binding pocket is identified at the interface of the complex, corresponding to a partially bound dimer that lacks enzymatic function. The pocket has a transient nature with a lifetime longer than 1 μs, and it displays very favorable druggability features. Docking as well as MM-GBSA free-energy calculations further support the possibility to target the new binding site by means of inhibitors able to prevent the complete dimerization by capturing the inactive conformation. This discovery could open the way to the rational design of a new class of anti-HIV drugs.

Unlabelled: We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR., Importance: Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) ., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a kcat of 7.4 s(-1), KM of 15 μM, and an increase in fluorescence intensity of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of HIV-1 protease. These properties enabled the enzyme concentration in an activity assay to be reduced to 25 pM, which is close to the Kd value of the protease dimer. By fitting inhibition data to Morrison's equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.

Anti-retroviral (ARV) -based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues.

We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC50, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

Background: In South Korea, about 20 types of antiretroviral drugs are used in the treatment of patients with human immunodeficiency virus/acquired immune deficiency syndrome. Since 2010, raltegravir, etravirine, and darunavir have been spotlighted as new drugs for highly active antiretroviral therapy (HAART)-experienced adults with resistant HIV-1 in South Korea. In this study, we investigated potential susceptibility of pseudoviruses derived from treatment-experienced Korean patients to etravirine vs efavirenz and to darunavir vs amprenavir and indinavir using a modified single-round assay., Methods: Pseudoviruses derived from nine treatment-experienced patients infected with HIV-1 were investigated by comparison with the wild-type strain pNL4-3. The 50% inhibitory concentration (IC50) values were calculated and drug susceptibility was compared. The intensity of genotypic drug resistance was classified based on the 'SIR' interpretation of the Stanford data base., Results: Drug susceptibility was generally higher for etravirine and darunavir compared with efavirenz, amprenavir, and indinavir in pseudoviruses derived from treatment-experienced patients. Pseudoviruses derived from patients KRB4025 and KRB8014, who exhibited long-term use of protease inhibitors, showed an outside of tested drug concentration, especially for amprenavir and indinavir. However, they exhibited a lower fold-change in resistance to darunavir., Conclusions: Etravirine and darunavir have been used in HAART since 2010 in South Korea. Therefore, these antiretroviral drugs together with other newly introduced antiretroviral drugs are interesting for the optimal treatment of patients with treatment failure. This study may help to find a more effective HAART in the case of HIV-1 infected patients that have difficulty being treated.