HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer.

BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome ß5 + ß2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits ß5 + ß1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.

Effects of individual drug and combination antiretroviral therapy on trophoblast proliferation.

BACKGROUND: Combination antiretroviral therapy (cART) has been reported to reduce perinatal transmission of human immunodeficiency virus (HIV) and improve maternal survival outcomes. Recent studies have associated in-utero exposure to cART drugs with adverse outcomes such as pre-eclampsia, preterm delivery, low birth weight and small-for-gestational-age births. However, the exact molecular mechanisms underlying cART-induced adverse pregnancy outcomes remain poorly defined. OBJECTIVES: To investigate the effects of cART drugs on trophoblast proliferation in the HTR-8/SVneo cell line. STUDY DESIGN: HTR-8/SVneo cells were exposed to tenofovir (0.983-9.83 µM), emtricitabine (0.809-8.09 µM) and efavirenz (0.19-1.09 µM), the individual drugs of the first-line single tablet cART regimen termed 'Atripla', and zidovudine (1.12-1.12 µM), lamivudine (0.65-6.5 µM), lopinavir (0.32-3.2 µM) and ritonavir (0.69-6.9 µM), the individual drugs of the second-line single tablet cART regimen termed 'Aluvia'. The cells were treated for 24, 48, 72 and 96 h, and trophoblast proliferation was assessed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide assay. RESULTS: Two-way analysis of variance showed a significant dose-dependent decrease (p < 0.05) in trophoblast proliferation in response to individual and combined drug components of first- and second-line antiretroviral therapy. CONCLUSIONS: First- and second-line cART drugs inhibit trophoblast proliferation, and may contribute to placenta-mediated adverse pregnancy outcomes in patients with HIV.

Lopinavir enhances anoikis by remodeling autophagy in a circRNA-dependent manner.

Macroautophagy/autophagy-mediated anoikis resistance is crucial for tumor metastasis. As a key autophagy-related protein, ATG4B has been demonstrated to be a prospective anti-tumor target. However, the existing ATG4B inhibitors are still far from clinical application, especially for tumor metastasis. In this study, we identified a novel circRNA, circSPECC1, that interacted with ATG4B. CircSPECC1 facilitated liquid-liquid phase separation of ATG4B, which boosted the ubiquitination and degradation of ATG4B in gastric cancer (GC) cells. Thus, pharmacological addition of circSPECC1 may serve as an innovative approach to suppress autophagy by targeting ATG4B. Specifically, the circSPECC1 underwent significant m6A modification in GC cells and was subsequently recognized and suppressed by the m6A reader protein ELAVL1/HuR. The activation of the ELAVL1-circSPECC1-ATG4B pathway was demonstrated to mediate anoikis resistance in GC cells. Moreover, we also verified that the above pathway was closely related to metastasis in tissues from GC patients. Furthermore, we determined that the FDA-approved compound lopinavir efficiently enhanced anoikis and prevented metastasis by eliminating repression of ELAVL1 on circSPECC1. In summary, this study provides novel insights into ATG4B-mediated autophagy and introduces a viable clinical inhibitor of autophagy, which may be beneficial for the treatment of GC with metastasis.

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.

Investigation of antiviral substances in Covid 19 by Molecular Docking: In Silico Study.

Aims: This paper aimed to investigate the antiviral drugs against Sars-Cov-2 main protease (MPro) using in silico methods. Material and Method: A search was made for antiviral drugs in the PubChem database and antiviral drugs such as Bictegravir, Emtricitabine, Entecavir, Lamivudine, Tenofovir, Favipiravir, Hydroxychloroquine, Lopinavir, Oseltamavir, Remdevisir, Ribavirin, Ritonavir were included in our study. The protein structure of Sars-Cov-2 Mpro (PDB ID: 6LU7) was taken from the Protein Data Bank (www.rcsb. Org) system and included in our study. Molecular docking was performed using AutoDock/Vina, a computational docking program. Protein-ligand interactions were performed with the AutoDock Vina program. 3D visualizations were made with the Discovery Studio 2020 program. N3 inhibitor method was used for our validation. Results: In the present study, bictegravir, remdevisir and lopinavir compounds in the Sars-Cov-2 Mpro structure showed higher binding affinity compared to the antiviral compounds N3 inhibitor, according to our molecular insertion results. However, the favipiravir, emtricitabine and lamuvidune compounds were detected very low binding affinity. Other antiviral compounds were found close binding affinity with the N3 inhibitor. Conclusion: Bictegravir, remdevisir and lopinavir drugs showed very good results compared to the N3 inhibitor. Therefore, they could be inhibitory in the Sars Cov-2 Mpro target.

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

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

Lopinavir and ritonavir act synergistically with azoles against Candida auris in vitro and in a mouse model of disseminated candidiasis.

INTRODUCTION AND OBJECTIVES: The emergence of Candida auris has created a global health challenge. Azole antifungals are the most affected antifungal class because of the extraordinary capability of C. auris to develop resistance against these drugs. Here, we used a combinatorial therapeutic approach to sensitize C. auris to azole antifungals. METHODS AND RESULTS: We have demonstrated the capability of the HIV protease inhibitors lopinavir and ritonavir, at clinically relevant concentrations, to be used with azole antifungals to treat C. auris infections both in vitro and in vivo. Both lopinavir and ritonavir exhibited potent synergistic interactions with the azole antifungals, particularly with itraconazole against 24/24 (100%) and 31/34 (91%) of tested C. auris isolates, respectively. Furthermore, ritonavir significantly interfered with the fungal efflux pump, resulting in a significant increase in Nile red fluorescence by 44%. In a mouse model of C. auris systemic infection, ritonavir boosted the activity of lopinavir to work synergistically with fluconazole and itraconazole and significantly reduced the kidney fungal burden by a 1.2 log (∼94%) and 1.6 log (∼97%) CFU, respectively. CONCLUSION: Our results urge further comprehensive assessment of azoles and HIV protease inhibitors as a novel drug regimen for the treatment of serious invasive C. auris infections.

Beneficial effects of Naringin against lopinavir/ ritonavir-induced hyperlipidemia and reproductive toxicity in male albino rats.

OBJECTIVE: This research work was planned to determine whether Naringin (NG) had any protective effects against lopinavir/ritonavir (LR)-induced alterations in blood lipid levels, hepatotoxicity, and testicular toxicity. MATERIALS AND METHODS: Four groups of six rats each were used for the study: Control (1% ethanol), naringin (80 mg/kg), lopinavir (80 mg/kg)/ritonavir (20 mg/kg), and lopinavir (80 mg/kg)/ritonavir (20 mg/kg) + naringin (80 mg/kg). The drug treatment was continued for 30 days. On the last day, the serum lipid fractions, liver biochemical parameters, testicular antioxidants (enzymatic and non-enzymatic), and the histopathology of the liver and testis tissue were assessed for all rats. RESULTS: Treatment with NG decreased significantly (p<0.05), the baseline serum levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C). But these parameters were significantly (p<0.05) increased in LR-treated animals. Naringin, co-administered with LR, restored the liver and testicular biochemical, morphological, and histological balance. CONCLUSIONS: This study shows that NG can be used as a treatment for LR-induced biochemical and histological changes in the liver and testes and changes in serum lipid levels.

Enterovirus D68 Infection Induces TDP-43 Cleavage, Aggregation, and Neurotoxicity.

Enterovirus D68 (EV-D68), which causes severe respiratory diseases and irreversible central nervous system damage, has become a serious public health problem worldwide. However, the mechanisms by which EV-D68 exerts neurotoxicity remain unclear. Thus, we aimed to analyze the effects of EV-D68 infection on the cleavage, subcellular translocation, and pathogenic aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in respiratory or neural cells. The results showed that EV-D68-encoded proteases 2A and 3C induced TDP-43 translocation and cleavage, respectively. Specifically, 3C cleaved residue 327Q of TDP-43. The 3C-mediated cleaved TDP-43 fragments had substantially decreased protein solubility compared with the wild-type TDP-43. Hence, 3C activity promoted TDP-43 aggregation, which exerted cytotoxicity to diverse human cells, including glioblastoma T98G cells. The effects of commercially available antiviral drugs on 3C-mediated TDP-43 cleavage were screened, and the results revealed lopinavir as a potent inhibitor of EV-D68 3C protease. Overall, these results suggested TDP-43 as a conserved host target of EV-D68 3C. This study is the first to provide evidence on the involvement of TDP-43 dysregulation in EV-D68 pathogenesis. IMPORTANCE Over the past decade, the incidence of enterovirus D68 (EV-D68) infection has increased worldwide. EV-D68 infection can cause different respiratory symptoms and severe neurological complications, including acute flaccid myelitis. Thus, elucidating the mechanisms underlying EV-D68 toxicity is important to develop novel methods to prevent EV-D68 infection-associated diseases. This study shows that EV-D68 infection triggers the translocalization, cleavage, and aggregation of TDP-43, an intracellular protein closely related to degenerative neurological disorders. The viral protease 3C decreased TDP-43 solubility, thereby exerting cytotoxicity to host cells, including human glioblastoma cells. Thus, counteracting 3C activity is an effective strategy to relieve EV-D68-triggered cell death. Cytoplasmic aggregation of TDP-43 is a hallmark of degenerative diseases, contributing to neural cell damage and central nervous system (CNS) disorders. The findings of this study on EV-D68-induced TDP-43 formation extend our understanding of virus-mediated cytotoxicity and the potential risks of TDP-43 dysfunction-related cognitive impairment and neurological symptoms in infected patients.

Menthol augmented niosomes for enhanced intestinal absorption of lopinavir.

Lopinavir is effective in treatment of HIV infection but experiences low oral bioavailability due to poor solubility, pre-systemic metabolism, and P-gp intestinal efflux. Co-processing with menthol enhanced its dissolution and intestinal permeability. Niosomes comprising Span 60, cholesterol, and poloxamer 407 were formulated in absence and presence of menthol. These were evaluated for size, morphology, entrapment efficiency (EE%), lopinavir release, and intestinal absorption. The later employed in situ rabbit intestinal absorption model. Niosomes were spherical with vesicle size of 140.2 ± 23 and 148.2 ± 27 nm for standard and menthol containing niosomes, respectively. The EE% values were 94.4% and 96.3% for both formulations, respectively. Niosomes underwent slow release during the time course of absorption with menthol hastening lopinavir release, but the release did not exceed 9%. Niosmoal encapsulation enhanced lopinavir intestinal absorption compared with drug solution. This was reflected from the fraction absorbed from duodenum, which was 24.15%, 73.09%, and 83.23% for solution, standard niosomes and menthol containing vesicles, respectively. These values were 34.32%, 80.8%, and 86.56% for the same formulations in case of jejuno-ileum. Lopinavir absorption from niosomes did not depend on release supporting intact vesicle absorption. The study introduced menthol containing niosomes as carriers for enhanced lopinavir intestinal absorption.

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.

Investigating and Resolving Cardiotoxicity Induced by COVID-19 Treatments using Human Pluripotent Stem Cell-Derived Cardiomyocytes and Engineered Heart Tissues.

Coronavirus disease 2019 continues to spread worldwide. Given the urgent need for effective treatments, many clinical trials are ongoing through repurposing approved drugs. However, clinical data regarding the cardiotoxicity of these drugs are limited. Human pluripotent stem cell-derived cardiomyocytes (hCMs) represent a powerful tool for assessing drug-induced cardiotoxicity. Here, by using hCMs, it is demonstrated that four antiviral drugs, namely, apilimod, remdesivir, ritonavir, and lopinavir, exhibit cardiotoxicity in terms of inducing cell death, sarcomere disarray, and dysregulation of calcium handling and contraction, at clinically relevant concentrations. Human engineered heart tissue (hEHT) model is used to further evaluate the cardiotoxic effects of these drugs and it is found that they weaken hEHT contractile function. RNA-seq analysis reveals that the expression of genes that regulate cardiomyocyte function, such as sarcomere organization (TNNT2, MYH6) and ion homeostasis (ATP2A2, HCN4), is significantly altered after drug treatments. Using high-throughput screening of approved drugs, it is found that ceftiofur hydrochloride, astaxanthin, and quetiapine fumarate can ameliorate the cardiotoxicity of remdesivir, with astaxanthin being the most prominent one. These results warrant caution and careful monitoring when prescribing these therapies in patients and provide drug candidates to limit remdesivir-induced cardiotoxicity.

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.

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Molnupiravir in COVID-19: A Scoping Review.

BACKGROUND: COVID-19, first detected in Wuhan, China, has evolved into a lifethreatening pandemic spread across six continents, with the global case count being more than 243 million, and mortality over 4.95 million, along with causing significant morbidity. It has initiated an era of research on repurposed drugs such as hydroxychloroquine, lopinavir/ritonavir, corticosteroids, remedesivir, ivermectin, alongside selective antivirals to treat or prevent COVID- 19. Molnupiravir is an orally available emerging antiviral drug considered highly promising for COVID-19. METHODS AND RESULTS: We have performed a scoping review for the use of molnupiravir against SARS-CoV-2 and COVID-19. It acts by inhibiting RNA-dependent RNA polymerase (RdRp), and exhibits broad-spectrum antiviral activity. Preclinical studies have evaluated the therapeutic efficacy as well as prophylactic activity of molnupiravir against SARS CoV-2 in various animal models that include ferrets, hamsters, mice, immunodeficient mice implanted with human lung tissue and cell cultures, in various doses ranging from 5-300 mg/kg, and results have been encouraging. Initial evidence of safety and efficacy from early phase clinical studies has been encouraging too, and recent results from a large phase 3 global trial have shown significant benefits among symptomatic outpatients. Other late-phase clinical trials are still underway with the aim of establishing molnulpiravir as a therapeutic option for COVID-19, particularly for non-hospitalized patients. CONCLUSION AND RELEVANCE: On the basis of the limited evidence available as of now, molnupiravir could prove to be a promising oral therapy, worthy of further exploration of its utility for both treatment and prevention of COVID-19 in humans. Elaborate clinical evaluation is further warranted to confirm whether the results are replicable to the clinical scenario among outpatients to reduce the chance of progression to more severe disease.

Exposure of human immune cells, to the antiretrovirals efavirenz and lopinavir, leads to lower glucose uptake and altered bioenergetic cell profiles through interactions with SLC2A1.

SLC2A1 mediates glucose cellular uptake; key to appropriate immune function. Our previous work has shown efavirenz and lopinavir exposure inhibits T cell and macrophage responses, to known agonists, likely via interactions with glucose transporters. Using human cell lines as a model, we assessed glucose uptake and subsequent bioenergetic profiles, linked to immunological responses. Glucose uptake was measured using 2-deoxyglucose as a surrogate for endogenous glucose, using commercially available reagents. mRNA expression of SLC transporters was investigated using qPCR TaqMan™ gene expression assay. Bioenergetic assessment, on THP-1 cells, utilised the Agilent Seahorse XF Mito Stress test. In silico analysis of potential interactions between SLC2A1 and antiretrovirals was investigated using bioinformatic techniques. Efavirenz and lopinavir exposure was associated with significantly lower glucose accumulation, most notably in THP-1 cells (up to 90% lower and 70% lower with efavirenz and lopinavir, respectively). Bioenergetic assessment showed differences in the rate of ATP production (JATP); efavirenz (4 µg/mL), was shown to reduce JATP by 87% whereas lopinavir (10 µg/mL), was shown to increase the overall JATP by 77%. Putative in silico analysis indicated the antiretrovirals, apart from efavirenz, associated with the binding site of highest binding affinity to SLC2A1, similar to that of glucose. Our data suggest a role for efavirenz and lopinavir in the alteration of glucose accumulation with subsequent alteration of bioenergetic profiles, supporting our hypothesis for their inhibitory effect on immune cell activation. Clarification of the implications of this data, for in vivo immunological responses, is now warranted to define possible consequences for these, and similar, therapeutics.

HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.

Hospital compounding to face shortage: A case study of the development of a lopinavir-ritonavir oral suspension during the first wave of SARS-COV-2 in France.

The potential usefulness of lopinavir-ritonavir on Covid 19 infection during the first wave of contamination in France had boosted Kaletra® syrup prescription to the point of causing its national shortage. In the intensive care units of Parisian hospitals in charge of patients with life-threatening viral contamination, caregivers had to resort to lopinavir-ritonavir-based tablets, crushing them and then dispersing the powder in milk to facilitate administration by nasogastric tube. The difficulties and poor control of this degraded mode, which does not always ensure control of the amount of the drug in the prepared dose and may induce insufficient antiviral exposure, led us to develop in a very short time, while ensuring quality control proportional to the risk, a liquid form as an alternative to Kaletra® oral solution shortage. For this purpose, we describe this compounding formulation and its preparation process, while justifying the quality control strategy adapted to the risk as well as its chemical and physical stability. Based on the chemical and physical studies, the preparation was showed to be stable during at least 2 months between +2°C and +8°C and 1 week at room temperature. This has resulted in the design of kits that include multi-dose packaging and a measuring device and contain the appropriate quantities of drugs to ensure at least one week's treatment for each patient, during which time the kit in use can be stored at room temperature. The intensive care team used this treatment under conditions that they considered well adapted until the imported specialty became available.

Combination of QSAR, molecular docking, molecular dynamic simulation and MM-PBSA: analogues of lopinavir and favipiravir as potential drug candidates against COVID-19.

Pandemic COVID-19 infections have spread throughout the world. There is no effective treatment against this disease. Viral RNA-dependent RNA polymerase (RdRp) catalyzes the replication of RNA from RNA and the main protease (Mpro) has a role in the processing of polyproteins that are translated from the RNA of SARS-CoV-2, and thus these two enzymes are strong candidates for targeting by anti-viral drugs. Small molecules such as lopinavir and favipiravir significantly inhibit the activity of Mpro and RdRp in vitro. Studies have shown that structurally modified lopinavir, favipiravir, and other similar compounds can inhibit COVID-19 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). In this study, lopinavir and its structurally similar compounds were chosen to bind the main protease, and favipiravir was chosen to target RNA-dependent RNA polymerase. Molecular docking and the quantitative structure-activity relationships (QSAR) study revealed that the selected candidates have favorable binding affinity but less druggable properties. To improve the druggability, four structural analogues of lopinavir and one structural analogue of favipiravir was designed by structural modification. Molecular interaction analyses have displayed that lopinavir and favipiravir analogues interact with the active site residues of Mpro and RdRp, respectively. Absorption, distribution, metabolism, excretion and toxicity (ADMET) properties, medicinal chemistry profile, and physicochemical features were shown that all structurally modified analogues are less toxic and contain high druggable properties than the selected candidates. Subsequently, 50 ns molecular dynamics simulation of the top four docked complexes demonstrated that CID44271905, a lopinavir analogue, forms the most stable complex with the Mpro. Further MMPBSA analyses using the MD trajectories also confirmed the higher binding affinity of CID44271905 towards Mpro. In summary, this study demonstrates a new way to identify leads for novel anti-viral drugs against COVID-19. Communicated by Ramaswamy H. Sarma.