Protease Inhibitor Drugs Research Articles

Retrospective evaluation of "Rods and Rings" pattern detected in the anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) test.

Anti-rods and rings (anti-RR) antibodies have recently been described as a cytoplasmic pattern in IIF-based screening of autoantibodies on HEp-2 cells and ICAP has named it as AC-23. It is most frequently related to drug-induced antibody generation. This study aimed to investigate the clinical significance of AC-23 positivity and its relevance to the diagnosis and/or follow-up of the associated diseases and/or drug use. A multicenter retrospective study was conducted among 10 hospitals from six different provinces in Türkiye from January 2017 to December 2021. The laboratory data and clinical information of 600 patients with positive anti-RR antibodies out of 547.558 HEp-2 IIF ANA samples were analyzed. The distribution of AC-23 positive patients by year indicated a steady increase between 2017-2021. Anti-RR prevalence in post-COVID-19 period was significantly higher than that of pre-COVID-19 period (p=0.00). Concomitant ANA positivity was detected in 56.5% of patients, the most common patterns being AC-4 and AC-5 (41.1%). The most frequent pathology among the anti-RR positive patients was an autoimmune disease (19.83%); 28.57% of which had rheumatoid arthritis and 17.65% autoimmune liver disease. Among the 600 patients, 65 (10.83%) were diagnosed as hepatitis C virus (HCV) infection. Available data for 38 of the HCV patients revealed that 71.05% of them had a history of interferon alfa+ribavirin and 28.95% of them had a history of NS3/4/5A/5B polymerase inhibitor or protease inhibitor drug use. Significant increase in the rate of anti-RR positivity was observed in the post-COVID-19 period when compared to pre-COVID-19 period (p:0.00). This is the first multicenter study in Türkiye about the clinical association of anti-RR antibodies which may be ignored during routine HEp-2 IIF testing. Pathologies other than HCV should be taken into consideration in terms of the possible role of anti-RR in autoimmune diseases and other pathologies. The preliminary data obtained in this study suggest that anti-RR antibody development might also be associated to COVID-19, supporting the several previous data related to the potential of viruses triggering the formation of autoantibodies. Large-scale prospective studies should elucidate the clinical significance of RR pattern and determine its role in patient diagnosis and follow-up.

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases.

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented an enormous challenge to health care systems and medicine. As a result of global research efforts aimed at preventing and effectively treating SARS-CoV-2 infection, vaccines with fundamentally new mechanisms of action and some small-molecule antiviral drugs targeting key proteins in the viral cycle have been developed. The most effective small-molecule drug approved to date for the treatment of COVID-19 is PaxlovidTM, which is a combination of two protease inhibitors, nirmatrelvir and ritonavir. Nirmatrelvir is a reversible covalent peptidomimetic inhibitor of the main protease (Mpro) of SARS-CoV-2, which enzyme plays a crucial role in viral reproduction. In this combination, ritonavir serves as a pharmacokinetic enhancer, it irreversibly inhibits the cytochrome CYP3A4 enzyme responsible for the rapid metabolism of nirmatrelvir, thereby increasing the half-life and bioavailability of nirmatrelvir. In this tutorial review, we summarize the development and pharmaceutical chemistry aspects of Paxlovid, covering the evolution of protease inhibitors, the warhead design, synthesis and the mechanism of action of nirmatrelvir, as well as the synthesis of ritonavir and its CYP3A4 inhibition mechanism. The efficacy of Paxlovid to novel virus mutants is also overviewed.

HIV-1 envelope facilitates the development of protease inhibitor resistance through acquiring mutations associated with viral entry and immune escape.

There is increasing evidence supporting a role for HIV-1 envelope in the development of Protease Inhibitor drug resistance, and a recent report from our group suggested that Env mutations co-evolve with Gag-Protease mutations in the pathway to Lopinavir resistance. In this study, we investigated the effect of co-evolving Env mutations on virus function and structure. Co-receptor usage and n-linked glycosylation were investigated using Geno2Pheno as well as tools available at the Los Alamos sequence database. Molecular dynamics simulations were performed using Amber 18 and analyzed using Cpptraj, and molecular interactions were calculated using the Ring server. The results showed that under Protease Inhibitor drug selection pressure, the envelope gene modulates viral entry by protecting the virus from antibody recognition through the increased length and number of N-glycosylation sites observed in V1/V2 and to some extent V5. Furthermore, gp120 mutations appear to modulate viral entry through a switch to the CXCR4 coreceptor, induced by higher charge in the V3 region and specific mutations at the coreceptor binding sites. In gp41, S534A formed a hydrogen bond with L602 found in the disulfide loop region between the Heptad Repeat 1 and Heptad Repeat 2 domains and could negatively affect the association of gp120-gp41 during viral entry. Lastly, P724Q/S formed both intermolecular and intramolecular interactions with residues within the Kennedy loop, a known epitope. In conclusion, the results suggest that mutations in envelope during Protease Inhibitor treatment failure are related to immune escape and that S534A mutants could preferentially use the cell-to-cell route of infection.

Lopinavir-Loaded Self-Nanoemulsifying Drug Delivery System for Enhanced Solubility: Development, Characterisation and Caco-2 Cell Uptake.

The antiretroviral protease inhibitor drug, lopinavir (LPV), is used to treat HIV-1 infection. LPV is known to have limited oral bioavailability, which may be attributed to its poor aqueous solubility, low efficacy and high first-pass metabolism. Self-nanoemulsifying drug delivery systems (SNEDDS) for LPV have been developed and optimised to counter the current issues. The titration method was used to prepare LPV-loaded SNEDDS (LPV-SNEDDS). Six different pseudo-ternary phase diagrams were constructed to identify the nanoemulsifying region. The developed formulations were chosen in terms of globule size < 100 nm, dispersity ≤ 0.5, dispersibility (Grade A) and% transmittance > 85. Heating-cooling cycle, freeze-thaw cycle, and centrifugation studies were performed to confirm the stability of the developed SNEDDS. The final LPV-SNEDDS (L-14) droplet size was 58.18 ± 0.62 nm, with polydispersity index, zeta potential, and entrapment efficiency (EE%) values of 0.326 ± 0.005, -22.08 ± 1.2 mV, and 98.93 ± 1.18%, respectively. According to high-resolution transmission electron microscopy (HRTEM) analysis, the droplets in the optimised formulation were < 60 nm in size. The selected SNEDDS released nearly 99% of the LPV within 30 min, which was significantly (p < 0.05) higher than the LPV-suspension in methylcellulose (0.5% w/v). It indicates the potential use of SNEDDS to enhance the solubility of LPV, which eventually could help improve the oral bioavailability of LPV. The Caco-2 cellular uptake study showed a significantly (p < 0.05) higher LPV uptake from the SNEEDS (LPV-SNEDDS-L-14) than the free LPV (LPV-suspension). The LPV-SNEDDS could be a potential carrier for LPV oral delivery.

The Impact of Certain Pharmacogenetic Differences on the Metabolism of Antiretroviral Drugs Used in A Black South African Population.

Genetic polymorphism of drug-metabolising enzymes and transporters may influence the effect and toxicity of antiretroviral drugs. To determine and compare the minimum allele frequency of 20 single nucleotide polymorphisms (SNPs) with possible involvement in the metabolism of the antiretroviral drugs with other populations. To investigate the influence of these variants on Reverse transcriptase, Protease and Integrase strand transfer inhibitor drugs. DNA samples were collected from 1489 subjects. All SNPs with a gene call score of > 0.6 were selected for genotyping. The R package calculated call rates, MAF and Hardy-Weinberg equilibrium (HWE), test p-values, and Chi-squared analysis were performed on the data. The Fisher's exact test compared the allele frequencies between the populations. The highest similarities in minimum allele frequency (MAF) were between the Prospective Urban and Rural Epidemiological group (PURE), a Black population in South Africa, and the Yoruba and Luhya populations in Africa. The following SNPs were identified with a possible effect on metabolism: CYP2B6 rs28399494 (MAF 11%) is indicated in the toxicity of Efavirenz and Nevirapine. CYP3A5 rs776746 (MAF 17%) and CYP3A4 rs2749674 (MAF 23%) both cause an increase in the metabolism of the protease inhibitors. The very low MAF values for both SCL01B1 rs4149056 (MAF 0.6%) and ABCC rs717620 (MAF 2.8%) are indications that OATP1B1 transport function and glomerular filtration tempo will not be compromised. The high MAF value of 30% for UGTA1 rs10929302 can result in hyperbilirubinemia, which can decrease the clearance of Dolutegravir. These results show a possibility of kidney protection and an increase in bilirubin in this population.

B-266 Analytical Performance Comparison of Two Genotyping Assays for HIV-1 Drug Resistance Using Sanger Sequencing

Abstract Background The first case of Acquired Immunodeficiency Syndrome (AIDS) was registered in the United States in early 1980. Two years later, was identified the Human Immunodeficiency Virus (HIV-1), which causes AIDS. Compared to other diseases, the treatment of AIDS achieved an important advance in just over 30 years, in which several drugs were used and replaced. However, resistance to antiretrovirals (ARVs) remains a problem in clinical practice. There are about 20 approved HIV-1 treatment drugs belonging to four different classes: Protease Inhibitors (PI), Nucleoside Reverse Transcriptase Inhibitors (NRTI), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI), and Integrase Strand Transfer Inhibitor (INSTI). ARVs resistance is caused by mutations in molecular targets of these drugs: protease, reverse transcriptase, and integrase enzymes. Virus genotyping that includes these regions is extremely important to identify the resistance mutations to ARVs therapies, being able to direct treatments. Thus, this study aimed to evaluate the analytical performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase compared to in-house Sanger genotyping method. Methods Viral RNA isolation from 20 individuals was performed by QIAamp Viral RNA Kit (Qiagen), according to the manufacturer’s instructions. HIV-1 genotyping was performed using both methods: in-house Sanger genotyping and Applied Biosystems HIV-1 Genotyping Kit with Integrase (Thermo Fisher), according to the manufacturer’s instructions. All results were submitted to SeqScape software to evaluate sequencing metrics. The consensus sequence was generated for each sample and analyzed at the online Stanford HIVdb Program (version 9.4) for drug resistance evaluation. Results The 54 mutations analyzed were distributed among the targets: PI (27.8%; 15/54), NRTI (24.1%; 13/54), NNRTI (22.2%; 12/54), and INSTI (25.9%; 14/54). The in-house method evaluated the susceptibility to PI drugs (ATV, DRV, FPV, IDV, LPV, NFV, SQV, and TPV), NRTI (ABC, AZT, D4T, DDI, FTC, 3TC, and TDF), and NNRTI (DOR, EFV, ETR, NVP, and RPV). In addition to protease and reverse transcriptase targets, the commercial kit also evaluated INSTI (BIC, CAB, DTG, EVG, and RAL) drugs. Four of 20 individuals evaluated had viral loads below 1000 copies/mL and have no results in both methods, as expected due to the detection limit of both assays. Furthermore, due to sequencing limitations using the in-house methodology, three samples were not included in the analysis as it didn't show any results. It was observed 92.3% agreement (n = 12/13) in the analyzed samples. A sensitivity of 100% and precision of 99% were found in the commercial kit using the in-house kit as a reference. The evaluation of the intra-assay (repeatability) and inter-assay (reproducibility) indicated 100% of agreement. Conclusions The Applied Biosystems HIV-1 Genotyping Kit with Integrase showed satisfactory performance in the detection of the main antiretroviral resistance mutations.

The effect of HIV protease gene mutations to protease inhibitor drugs resistance in Papua patients: In silico analysis

The use of phenotypic assay requires laborious work to culture HIV isolates to observe the phenotypic change of the virus in the presence of antiretroviral drugs. The genotypic approach may rely on the secondary data of documented mutations that are responsible for phenotypic alterations to antiretroviral-resistant HIV. HIV genomes were extracted from patients’ plasma, which was subsequently subjected to RT-PCR and Sanger sequencing. The obtained HIV genome sequencing data were analyzed for mutation detection. Three-dimensional (3D) structures of mutant HIV protease were constructed using FoldX software. The binding affinity of the mutant HIV protease with protease inhibitor drugs (Saquinavir, Ritonavir, Nelfinavir, Indinavir, and Lopinavir) was analyzed using AutoDock Vina. There were 90 patients involved in this study. The patients attended the Voluntary Counseling Test (VCT) of Mitra Masyarakat Hospital in Mimika, Papua, Indonesia. Among recruited subjects, the HIV genomes corresponding to the protease-encoded gene of 30 patients were successfully sequenced. There was only one patient (RSMM_70) infected with HIV harboring minor mutations (L10V, I15V, M36I, and R41K) in the protease-encoded gene that was not a new finding mutation. The 3D structure showed that the hydrophobicity and stability of mutant HIV protease were different from the wild genotype. Docking analysis showed decreasing binding affinity of the mutant HIV protease to the protease inhibitor drugs, which may lead to the alteration of inhibitory effectiveness. In silico docking, the analysis may provide an alternative approach to predict the effect of minor mutations in the HIV protease gene on the effectiveness of protease inhibitor drugs.

Proteolysis and Deficiency of α1-Proteinase Inhibitor in SARS-CoV-2 Infection.

The SARS-CoV-2 pandemic had stimulated the emergence of numerous publications on the α1-proteinase inhibitor (α1-PI, α1-antitrypsin), especially when it was found that the regions of high mortality corresponded to the regions with deficient α1-PI alleles. By analogy with the data obtained in the last century, when the first cause of the genetic deficiency of α1-antitrypsin leading to elastase activation in pulmonary emphysema was proven, it can be supposed that proteolysis hyperactivation in COVID-19 may be associated with the impaired functions of α1-PI. The purpose of this review was to systematize the scientific data and critical directions for translational studies on the role of α1-PI in SARS-CoV-2-induced proteolysis hyperactivation as a diagnostic marker and a therapeutic target. This review describes the proteinase-dependent stages of viral infection: the reception and penetration of the virus into a cell and the imbalance of the plasma aldosterone-angiotensin-renin, kinin, and blood clotting systems. The role of ACE2, TMPRSS, ADAM17, furin, cathepsins, trypsin- and elastase-like serine proteinases in the virus tropism, the activation of proteolytic cascades in blood, and the COVID-19-dependent complications is considered. The scientific reports on α1-PI involvement in the SARS-CoV-2-induced inflammation, the relationship with the severity of infection and comorbidities were analyzed. Particular attention is paid to the acquired α1-PI deficiency in assessing the state of patients with proteolysis overactivation and chronic non-inflammatory diseases, which are accompanied by the risk factors for comorbidity progression and the long-term consequences of COVID-19. Essential data on the search and application of protease inhibitor drugs in the therapy for bronchopulmonary and cardiovascular pathologies were analyzed. The evidence of antiviral, anti-inflammatory, anticoagulant, and anti-apoptotic effects of α1-PI, as well as the prominent data and prospects for its application as a targeted drug in the SARS-CoV-2 acquired pneumonia and related disorders, are presented.

In Silico Analyzes for the Inhibition of HIV Protease by Ritonavir and Indinavir

Introduction: This research was conducted to investigate the molecular interaction of HIV protease inhibitor drugs using molecular docking. HIV protease is responsible for processing gag and gag-polyproteins during virion maturation. The activity of this enzyme is essential against viral infections and has beneficial therapeutic effects on HIV treatment.&#x0D; Materials and Methods: To meet the aim of the study, indinavir and ritonavir were selected as HIV Protease inhibitor drugs. The necessary information on molecular docking was collected through information servers, such as Drug bank and Program database (PDB). Then, molecular docking was performed using Molegro virtual docker software. In order to check the stability of the resulting complex structure and its cellular penetration, a molecular dynamics simulation was run for 50 nanoseconds using GROMACS2019.6 package and Amber99SB force force field. During the molecular dynamics simulation, root mean square deviations (RMSD), root mean square fluctuations (RMSF), the radius of gyration (RG), hydrogen bonds, and distance between ligands and complex were investigated.&#x0D; Results: The obtained results indicated that the RMSD of the complex of the ligands and HIV protease at the end of 50 nanoseconds had a linear slope. Hydrogen bonds decreased at beginning of simulation but they increase at the end of simulation However RG was decreased at the end of the simulation Also the RMSF was decreased at the end of simulation rather than beginning of simulation, So all the obtained results showing the stability and strength of the structure.&#x0D; Conclusion: Molecular docking method can indicate the relationship between structure-activity and the effectiveness of ligands on HIV protease based on the level of interaction between the ligands and the receptors.

Improving Drug Sensitivity of HIV-1 Protease Inhibitors by Restriction of Cellular Efflux System in a Fission Yeast Model.

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

Efficacy and safety of dolutegravir or darunavir in combination with lamivudine plus either zidovudine or tenofovir for second-line treatment of HIV infection (NADIA): week 96 results from a prospective, multicentre, open-label, factorial, randomised, non-inferiority trial

WHO guidelines recommend dolutegravir plus two nucleoside reverse transcriptase inhibitors (NRTIs) for second-line HIV therapy, with NRTI switching from first-line tenofovir to zidovudine. We aimed to examine whether dolutegravir is non-inferior to darunavir, the best-in-class protease inhibitor drug, and whether maintaining tenofovir in second-line therapy is non-inferior to switching to zidovudine. In this prospective, multicentre, open-label, factorial, randomised, non-inferiority trial (NADIA), participants with confirmed HIV first-line treatment failure (HIV-1 RNA ≥1000 copies per mL) were recruited at seven clinical sites in Kenya, Uganda, and Zimbabwe. Following a 2 × 2 factorial design and stratified by site and screening HIV-1 RNA concentration, participants were randomly assigned (1:1:1:1) to receive a 96-week regimen containing either dolutegravir (50 mg once daily) or ritonavir-boosted darunavir (800 mg of darunavir plus 100 mg of ritonavir once daily) in combination with either tenofovir (300 mg once daily) plus lamivudine (300 mg once daily) or zidovudine (300 mg twice daily) plus lamivudine (150 mg twice daily). The NRTI drugs allocated by randomisation were administered orally in fixed-dose combination pills; other drugs were administered orally as separate pills. The previously reported primary outcome was the proportion of participants with a plasma HIV-1 RNA concentration of less than 400 copies per mL at 48 weeks. Here, we report the main secondary outcome: the proportion of participants with a plasma HIV-1 RNA concentration of less than 400 copies per mL at 96 weeks (non-inferiority margin 12%). We analysed this outcome and safety outcomes in the intention-to-treat population, which excluded only those who were randomly assigned in error and withdrawn before receiving trial drugs. This study was registered at ClinicalTrials.gov, NCT03988452, and is complete. Between July 30 and Dec 18, 2019, we screened 783 patients and enrolled 465. One participant was randomly assigned in error and immediately withdrawn. The remaining 464 participants were randomly assigned to receive either dolutegravir (n=235) or ritonavir-boosted darunavir (n=229) and to receive lamivudine plus either tenofovir (n=233) or zidovudine (n=231). At week 96, 211 (90%) of 235 participants in the dolutegravir group and 199 (87%) of 229 participants in the darunavir group had HIV-1 RNA less than 400 copies per mL (percentage point difference 2·9, 95% CI -3·0 to 8·7), indicating non-inferiority. Nine (4%) participants (all in the dolutegravir group) developed dolutegravir resistance; no participants developed darunavir resistance (p=0·0023). In the other randomised comparison, 214 (92%) of 233 patients in the tenofovir group and 196 (85%) of 231 patients in the zidovudine group had HIV-1 RNA less than 400 copies per mL (percentage point difference 7·0, 95% CI 1·2 to 12·8), showing non-inferiority and indicating the superiority of tenofovir (p=0·019). The proportions of participants with any grade 3-4 adverse event were similar between the dolutegravir (26 [11%]) and darunavir (28 [12%]) groups and between the tenofovir (22 [9%]) and zidovudine (32 [14%]) groups. There were no deaths related to study medication. Dolutegravir-based and darunavir-based regimens maintain good viral suppression during 96 weeks; dolutegravir is non-inferior to darunavir but is at greater risk of resistance in second-line therapy. Tenofovir should be continued in second-line therapy, rather than being switched to zidovudine. Janssen.

Repurposing of FDA-approved drugs as potential inhibitors of the SARS-CoV-2 main protease: Molecular insights into improved therapeutic discovery

With numerous infections and fatalities, COVID-19 has wreaked havoc around the globe. The main protease (Mpro), which cleaves the polyprotein to form non-structural proteins, thereby helping in the replication of SARS-CoV-2, appears as an attractive target for antiviral therapeutics. As FDA-approved drugs have shown effectiveness in targeting Mpro in previous SARS-CoV(s), molecular docking and virtual screening of existing antiviral, antimalarial, and protease inhibitor drugs were carried out against SARS-CoV-2 Mpro. Among 53 shortlisted drugs with binding energies lower than that of the crystal-bound inhibitor α-ketoamide 13 b (−6.7 kcal/mol), velpatasvir, glecaprevir, grazoprevir, baloxavir marboxil, danoprevir, nelfinavir, and indinavir (−9.1 to −7.5 kcal/mol) were the most significant on the list (hereafter referred to as the 53-list). Molecular dynamics (MD) simulations confirmed the stability of their Mpro complexes, with the MMPBSA binding free energy (ΔGbind) ranging between −124 kJ/mol (glecaprevir) and −28.2 kJ/mol (velpatasvir). Despite having the lowest initial binding energy, velpatasvir exhibited the highest ΔGbind value for escaping the catalytic site during the MD simulations, indicating its reduced efficacy, as observed experimentally. Available inhibition assay data adequately substantiated the computational forecast. Glecaprevir and nelfinavir (ΔGbind = −95.4 kJ/mol) appear to be the most effective antiviral drugs against Mpro. Furthermore, the remaining FDA drugs on the 53-list can be worth considering, since some have already demonstrated antiviral activity against SARS-CoV-2. Hence, theoretical pKi (Ki = inhibitor constant) values for all 53 drugs were provided. Notably, ΔGbind directly correlates with the average distance of the drugs from the His41–Cys145 catalytic dyad of Mpro, providing a roadmap for rapid screening and improving the inhibitor design against SARS-CoV-2 Mpro.

Potential Associations of Mutations within the HIV-1 Env and Gag Genes Conferring Protease Inhibitor (PI) Drug Resistance

An increasing number of patients in Africa are experiencing virological failure on a second-line antiretroviral protease inhibitor (PI)-containing regimen, even without resistance-associated mutations in the protease region, suggesting a potential role of other genes in PI resistance. Here, we investigated the prevalence of mutations associated with Lopinavir/Ritonavir (LPV/r) failure in the Envelope gene and the possible coevolution with mutations within the Gag-protease (gag-PR) region. Env and Gag-PR sequences generated from 24 HIV-1 subtype C infected patients failing an LPV/r inclusive treatment regimen and 344 subtype C drug-naïve isolates downloaded from the Los Alamos Database were analyzed. Fisher’s exact test was used to determine the differences in mutation frequency. Bayesian network probability was applied to determine the relationship between mutations occurring within the env and gag-PR regions and LPV/r treatment. Thirty-five mutations in the env region had significantly higher frequencies in LPV/r-treated patients. A combination of Env and Gag-PR mutations was associated with a potential pathway to LPV/r resistance. While Env mutations were not directly associated with LPV/r resistance, they may exert pressure through the Gag and minor PR mutation pathways. Further investigations using site-directed mutagenesis are needed to determine the impact of Env mutations alone and in combination with Gag-PR mutations on viral fitness and LPV/r efficacy.

Enteric Coronavirus Infection and Treatment Modeled With an Immunocompetent Human Intestine-On-A-Chip.

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.

Arjunetin as a promising drug candidate against SARS-CoV-2: molecular dynamics simulation studies

Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant. Our previous studies have shown that, ethanolic extract of T. arjuna bark exhibits radical scavenging anti-oxidant activity and also effectively inhibited catalase activity. In this study, oleanane triterpenoids type compounds viz., oleanolic acid, arjunolic acid, arjunolitin, arjunetin were isolated from ethanolic bark extract as bio-active compound and their structures were elucidated using 1H, 13C NMR, HR-ESIMS, IR. Of the various compounds, Arjunetin showed significant inhibition of catalase activity as compared to the other compounds. Based on the structural similarity between arjunetin and current antiviral drugs, we propose that arjunetin might exhibit antiviral activity. Molecular docking and molecular dynamics studies showed that arjunetin binds to the binds to key targets of SARS-CoV-2 namely, 3CLpro, PLpro, and RdRp) with a higher binding energy values (3CLpro, −8.4 kcal/mol; PLpro, −7.6 kcal/mol and RdRp, −8.1 kcal/mol) as compared with FDA approved protease inhibitor drugs to Lopinavir (3CLpro, −7.2 kcal/mole and PLpro −7.7 kcal/mole) and Remdesivir (RdRp −7.6 kcal/mole). To further investigate this, we performed 200–500 ns molecular dynamics simulation studies. The results transpired that the binding affinity of Arjunetin is higher than Remdesivir in the RNA binding cavity of RdRp. Based on structural similarity between arjunetin and Saikosaponin (a known antiviral agents) and based on our molecular docking and molecular dynamic simulation studies, we propose that arjunetin can be a promising drug candidate against Covid-19. Communicated by Ramaswamy H. Sarma

Single-Agent and Fixed-Dose Combination HIV-1 Protease Inhibitor Drugs in Fission Yeast (Schizosaccharomyces pombe).

Successful combination antiretroviral therapies (cART) eliminate active replicating HIV-1, slow down disease progression, and prolong lives. However, cART effectiveness could be compromised by the emergence of viral multidrug resistance, suggesting the need for new drug discoveries. The objective of this study was to further demonstrate the utility of the fission yeast cell-based systems that we developed previously for the discovery and testing of HIV protease (PR) inhibitors (PIs) against wild-type or multi-PI drug resistant M11PR that we isolated from an infected individual. All thirteen FDA-approved single-agent and fixed-dose combination HIV PI drugs were tested. The effect of these drugs on HIV PR activities was tested in pure compounds or formulation drugs. All FDA-approved PI drugs, except for a prodrug FPV, were able to suppress the wild-type PR-induced cellular and enzymatic activities. Relative drug potencies measured by EC50 in fission yeast were discussed in comparison with those measured in human cells. In contrast, none of the FDA-approved drugs suppressed the multi-PI drug resistant M11PR activities. Results of this study show that fission yeast is a reliable cell-based system for the discovery and testing of HIV PIs and further demonstrate the need for new PI drugs against viral multi-PI resistance.

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir’s antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.

Increased acquired protease inhibitor drug resistance mutations in minor HIV-1 quasispecies from infected patients suspected of failing on national second-line therapy in South Africa

BackgroundHIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs.MethodsDuring 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient’s treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database.ResultsStatistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p = 0.042) and integrase inhibitor RAM (4%; 2/56; p = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation (n = 13) in protease and K65R (n = 5), K103N (n = 7) and M184V (n = 5) in reverse transcriptase.ConclusionsHTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in < 20% of the population. Deep sequencing could be of greater value in detecting acquired resistance mutations early.

HIV-1 Protease Inhibitors Slow HPV16-Driven Cell Proliferation through Targeted Depletion of Viral E6 and E7 Oncoproteins.

Simple SummaryHigh-risk strains of human papillomavirus (HPV) such as HPV16 cause anogenital and oropharyngeal cancers, with HPV-associated cervical cancer being the fourth most common cancer worldwide and a major cause of death for women. Anti-HPV vaccines are available but coverage is low, and while the vaccines prevent infection they are not therapeutic for treating cancers in individuals already infected with high-risk HPV. Instead, current treatments for HPV-associated cancers include surgery, chemotherapy, and radiation, and affect diseased and healthy tissues alike. Accordingly, there is a dire need for better, molecularly targeted strategies. Herein, we report the novel finding that at least four protease inhibitor drugs currently used to treat HIV/AIDS—lopinavir, ritonavir, nelfinavir, and saquinavir—cause marked and selective depletion of HPV16 E6 and E7 oncoproteins in HPV-positive cancer cells and a 3-D cell culture model for HPV16-positive epithelial pre-cancer (NIKS16 cells). E6 and E7 depletion correlated with anticancer phenotypes including increased p53 protein levels and induction of cell growth arrest in CaSki cells as well as slowed epithelium growth and reversal of HPV16-driven phenotypes consistent with inhibition of the cellular autophagy pathway in the NIKS16 model. Based on this progress in vitro, we propose that HIV protease inhibitors, already known to have anticancer properties, be studied further in the context of developing a targeted therapy for HPV16-driven cancers and pre-cancers.High-risk human papillomavirus strain 16 (HPV16) causes oral and anogenital cancers through the activities of two viral oncoproteins, E6 and E7, that dysregulate the host p53 and pRb tumor suppressor pathways, respectively. The maintenance of HPV16-positive cancers requires constitutive expression of E6 and E7. Therefore, inactivating these proteins could provide the basis for an anticancer therapy. Herein we demonstrate that a subset of aspartyl protease inhibitor drugs currently used to treat HIV/AIDS cause marked reductions in HPV16 E6 and E7 protein levels using two independent cell culture models: HPV16-transformed CaSki cervical cancer cells and NIKS16 organotypic raft cultures (a 3-D HPV16-positive model of epithelial pre-cancer). Treatment of CaSki cells with some (lopinavir, ritonavir, nelfinavir, and saquinavir) but not other (indinavir and atazanavir) protease inhibitors reduced E6 and E7 protein levels, correlating with increased p53 protein levels and decreased cell viability. Long-term (>7 day) treatment of HPV16-positive NIKS16 raft cultures with saquinavir caused epithelial atrophy with no discernible effects on HPV-negative rafts, demonstrating selectivity. Saquinavir also reduced HPV16′s effects on markers of the cellular autophagy pathway in NIKS16 rafts, a hallmark of HPV-driven pre-cancers. Taken together, these data suggest HIV-1 protease inhibitors be studied further in the context of treating or preventing HPV16-positive cancers.