Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

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.

FMO-guided design of darunavir analogs as HIV-1 protease inhibitors.

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.

Rational design, synthesis and biological evaluation of novel HIV-1 protease inhibitors containing 2-phenylacetamide derivatives as P2 ligands with potent activity against DRV-Resistant HIV-1 variants.

A novel kind of potent HIV-1 protease inhibitors, containing diverse hydroxyphenylacetic acids as the P2-ligands and 4-substituted phenyl sulfonamides as the P2' ligands, were designed, synthesized and evaluated in this work. Majority of the target compounds exhibited good to excellent activity against HIV-1 protease with IC50 values below 200 nM. In particular, compound 18d with a 2-(3,4-dihydroxyphenyl) acetamide as the P2 ligand and a 4- methoxybenzene sulfonamide P2' ligand exhibited inhibitory activity IC50 value of 0.54 nM, which was better than that of the positive control darunavir (DRV). More importantly, no significant decline of the potency against HIV-1DRVRS (DRV-resistant mutation) and HIV-1NL4_3 variant (wild type) for 18d was detected. The molecular docking study of 18d with HIV-1 protease (PDB-ID: 1T3R, www.rcsb.org) revealed possible binding mode with the HIV-1 protease. These results suggested the validity of introducing phenol-derived moieties into the P2 ligand and deserve further optimization which was of great value for future discovery of novel HIV-1 protease.

Dynamics of Low-Level Viremia and Immune Activation after Switching to a Darunavir-Based Regimen.

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.

Enteral administration of crushed rilpivirine in a patient with HIV: A case report.

Antiretroviral therapy administration is challenging in patients with HIV requiring enteral nutrition. There are limited pharmacokinetic data available regarding the absorption of crushed rilpivirine (RPV) and its impact on drug bioavailability, plasma concentrations and, consequently, the efficacy of treatment. We present the case of a 60-year-old woman with HIV diagnosed with squamous cell carcinoma who needed enteral administration of antiretroviral therapy following the insertion of a gastrotomy tube in September 2018. Initially, the patient was treated with a daily dose of RPV 25 mg, dolutegravir 50 mg and emtricitabine 200 mg. The treatment was later intensified with darunavir boosted with ritonavir. RPV and dolutegravir were crushed, dissolved in water and administered via a percutaneous endoscopic gastrostomy tube. Therapeutic drug and viral load monitoring determined the adequacy of enteral antiretroviral dosing. RPV plasma concentrations remained within the expected therapeutic range of 43-117 ng/mL, with only 1 below the currently used 50 ng/mL efficacy threshold. After the treatment intensification with darunavir boosted with ritonavir, the patient achieved an undetectable viral load. While we observed satisfactory RPV plasma concentrations, it is essential to maintain strict monitoring of administration method, plasma concentrations and virological responses when initiating treatment with crushed RPV. Hence, additional pharmacokinetic data are necessary to ensure the effective enteral administration of RPV and to establish the best antiretroviral dosing regimens.

Machine learning-guided design of potent darunavir analogs targeting HIV-1 proteases: A computational approach for antiretroviral drug discovery.

In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.

A detailed characterization of drug resistance during darunavir/ritonavir monotherapy highlights a high barrier to the emergence of resistance mutations in protease but identifies alternative pathways of resistance.

BACKGROUND: Maintenance monotherapy with ritonavir-boosted darunavir has yielded variable outcomes and is not recommended. Trial samples offer valuable opportunities for detailed studies. We analysed samples from a 48 week trial in Cameroon to obtain a detailed characterization of drug resistance. METHODS: Following failure of NNRTI-based therapy and virological suppression on PI-based therapy, participants were randomized to ritonavir-boosted darunavir (n = 81) or tenofovir disoproxil fumarate/lamivudine +ritonavir-boosted lopinavir (n = 39). At study entry, PBMC-derived HIV-1 DNA underwent bulk Protease and Reverse Transcriptase (RT) sequencing. At virological rebound (confirmed or last available HIV-1 RNA ≥ 60 copies/mL), plasma HIV-1 RNA underwent ultradeep Protease and RT sequencing and bulk Gag-Protease sequencing. The site-directed mutant T375A (p2/p7) was characterized phenotypically using a single-cycle assay. RESULTS: NRTI and NNRTI resistance-associated mutations (RAMs) were detected in 52/90 (57.8%) and 53/90 (58.9%) HIV-1 DNA samples, respectively. Prevalence in rebound HIV-1 RNA (ritonavir-boosted darunavir, n = 21; ritonavir-boosted lopinavir, n = 2) was 9/23 (39.1%) and 10/23 (43.5%), respectively, with most RAMs detected at frequencies ≥15%. The resistance patterns of paired HIV-1 DNA and RNA sequences were partially consistent. No darunavir RAMs were found. Among eight participants experiencing virological rebound on ritonavir-boosted darunavir (n = 12 samples), all had Gag mutations associated with PI exposure, including T375N, T375A (p2/p7), K436R (p7/p1) and substitutions in p17, p24, p2 and p6. T375A conferred 10-fold darunavir resistance and increased replication capacity. CONCLUSIONS: The study highlights the high resistance barrier of ritonavir-boosted darunavir while identifying alternative pathways of resistance through Gag substitutions. During virological suppression, resistance patterns in HIV-1 DNA reflect treatment history, but due to technical and biological considerations, cautious interpretation is warranted.

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues.

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.

Towards designing of a potential new HIV-1 protease inhibitor using QSAR study in combination with Molecular docking and Molecular dynamics simulations.

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.

Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance.

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.

Promising Repurposed Antiviral Molecules to Combat SARS-CoV-2: A Review.

COVID-19, an extremely transmissible and pathogenic viral disease, triggered a global pandemic that claimed lives worldwide. To date, there is no clear and fully effective treatment for COVID-19 disease. Nevertheless, the urgency to discover treatments that can turn the tide has led to the development of a variety of preclinical drugs that are potential candidates for probative results. Although most of these supplementary drugs are constantly being tested in clinical trials against COVID-19, recognized organizations have aimed to outline the prospects in which their use could be considered. A narrative assessment of current articles on COVID-19 disease and its therapeutic regulation was performed. This review outlines the use of various potential treatments against SARS-CoV-2, categorized as fusion inhibitors, protease inhibitors, and RNA-dependent RNA polymerase inhibitors, which include antiviral drugs such as Umifenovir, Baricitinib, Camostatmesylate, Nafamostatmesylate, Kaletra, Paxlovide, Darunavir, Atazanavir, Remdesivir, Molnupiravir, Favipiravir, and Ribavirin. To understand the virology of SARS-CoV-2, potential therapeutic approaches for the treatment of COVID-19 disease, synthetic methods of potent drug candidates, and their mechanisms of action have been addressed in this review. It intends to help readers approach the accessible statistics on the helpful treatment strategies for COVID-19 disease and to serve as a valuable resource for future research in this area.

Evaluation of darunavir-derived HIV-1 protease inhibitors incorporating P2' amide-derivatives: Synthesis, biological evaluation and structural studies.

We report here the synthesis and biological evaluation of darunavir derived HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. The P2' 4-amino functionality was modified to make a number of amide derivatives to interact with residues in the S2' subsite of the HIV-1 protease active site. Several compounds exhibited picomolar enzyme inhibitory and low nanomolar antiviral activity. The X-ray crystal structure of the chloroacetate derivative bound to HIV-1 protease was determined. Interestingly, the active chloroacetate group converted to the acetate functionality during X-ray exposure. The structure revealed that the P2' carboxamide functionality makes enhanced hydrogen bonding interactions with the backbone atoms in the S2'-subsite.

Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal.

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.

Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

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 (3CLpro) 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 3CLpro 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-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro 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 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

Beyond darunavir: recent development of next generation HIV-1 protease inhibitors to combat drug resistance.

We report our recent development of a conceptually new generation of exceptionally potent non-peptidic HIV-1 protease inhibitors that displayed excellent pharmacological and drug-resistance profiles. Our X-ray structural studies of darunavir and other designed inhibitors from our laboratories led us to create a variety of inhibitors incorporating fused ring polycyclic ethers and aromatic heterocycles to promote hydrogen bonding interactions with the backbone atoms of HIV-1 protease as well as van der Waals interactions with residues in the S2 and S2' subsites. We have also incorporated specific functionalities to enhance van der Waals interactions in the S1 and S1' subsites. The combined effects of these structural templates are critical to the inhibitors' exceptional potency and drug-like properties. We highlight here our molecular design strategies to promote backbone hydrogen bonding interactions to combat drug-resistance and specific design of polycyclic ether templates to mimic peptide-like bonds in the HIV-1 protease active site. Our medicinal chemistry and drug development efforts led to the development of new generation inhibitors significantly improved over darunavir and displaying unprecedented antiviral activity against multidrug-resistant HIV-1 variants.

Discovery of Novel HIV Protease Inhibitors Using Modern Computational Techniques.

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

HIV-1 protease with 10 lopinavir and darunavir resistance mutations exhibits altered inhibition, structural rearrangements and extreme dynamics.

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 Ki 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.

Multiple Molecular Dynamics Simulations and Energy Analysis Unravel the Dynamic Properties and Binding Mechanism of Mutants HIV-1 Protease with DRV and CA-p2.

PRS17, 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 PRS17 with inhibitor Darunavir (DRV), two complexes of WT PR and PRS17 with substrate analogue CA-p2, two unligand WT PR and PRS17), 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 PRS17 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.

Mechanism of darunavir binding to monomeric HIV-1 protease: a step forward in the rational design of dimerization inhibitors.

HIV protease (HIVPR) is a key target in AIDS therapeutics. All ten FDA-approved drugs that compete with substrates in binding to this dimeric enzyme's active site have become ineffective due to the emergence of drug resistant mutants. Blocking the dimerization interface of HIVPR is thus being explored as an alternate strategy. The latest drug, darunavir (DRV), which exhibited a high genetic barrier to viral resistance, is said to have a dual mode of action - (i) binding to the dimeric active site, and (ii) preventing the dimerization by binding to the HIVPR monomer. Despite several reports on DRV complexation with dimeric HIVPR, the mode and mechanism of the binding of DRV to the HIVPR monomer are poorly understood. In this study, we utilized all-atomic MD simulations and umbrella sampling techniques to identify the best possible binding mode of DRV to the monomeric HIVPR and its mechanism of association. The results suggest that DRV binds between the active site and the flap of the monomer, and the flap plays a crucial role in directing the drug to bind and driving the other protein domains to undergo induced fit changes for stronger complexation. The obtained binding mode of DRV was validated by comparing with various mutational data from clinical isolates to reported in vitro mutations. The identified binding pose was also able to successfully reproduce the experimental Ki value in the picomolar range. The residue-level information extracted from this study could accelerate the structure-based drug designing approaches targeting HIVPR dimerization.

Evaluation of COVID-19 protease and HIV inhibitors interactions.

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.