The outbreak of SARS-CoV-2 is responsible for the COVID-19 pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design (CADD) approach, we identified and validated 5 potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both the compounds showed antiviral activity with IC 50 values in the micromolar range (MU-UNMC-1: IC 50 = 0.67 μM and MU-UNMC-2: IC 50 = 1.72 μM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC 50 = 9.27 μM & 3.00 μM) and Scotland variant B.1.222 (IC 50 = 2.64 μM & 1.39 μM) respectively. Following these assays, we conducted ‘induced-fit (flexible) docking’ to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir (RDV), the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2 specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir, suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19, infected individuals.
Dear Editor, We demonstrated that SF2523, a dual small molecule inhibitor of PI3K-α/mTOR/BRD4 pathways, can inhibit the replication of SARS-CoV-2 and its emerging variants of concern (VOCs), including Delta and Omicron. Further, we also found that SF2523 acts synergistically with remdesivir (RDV) and MU-UNMC-2 (a small molecule entry inhibitor of SARS-CoV-2). 1 The ongoing COVID-19 pandemic due to the emergence of a novel coronavirus SARS-CoV-2 remains a significant health concern globally. Several vaccine candidates and anti-virals received emergency use authorization. However, these vaccines/anti-virals safety, efficacy and durability remain unknown, especially for the individuals with comorbid conditions, and VOCs, such as Delta, Omicron, BA.2 and Deltacron, which have evolved mutations in the receptor-binding domain of SARS-CoV-2 spike protein may even evade antibodies induced by vaccines or natural infection. 2 Similarly, recently FDA-approved Molnupiravir and PAXLOVID remain sensitive towards VOCs. 3 The indepth understanding of the molecular mechanism of the SARS-CoV-2 lifecycle revealed the interaction of several host factors with viral proteins essential for the reproduction of progeny viruses, such as bromodomain, containing extra-terminal domain proteins (BETs), and the mTOR pathway. 4 Recent studies identified 67 potential interactions between host and viral proteins essential for the SARS-CoV-2 lifecycle, like BRD2/BRD4 with the E protein of SARS-CoV-2, 4 and suggested that BRD2 inhibition downregulates ACE2 expression, blocks the entry of SARS-CoV-2 into host cells and controls hyperactive immune response in COVID-19 patients through downregulation Interferon stimulated genes (ISGs). 5 Targeted therapies that exploit host-virus interaction are likely to be least impacted by the VOCs of SARS-CoV-2 andThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
HIV persists in cellular reservoirs despite effective anti-retroviral therapy with rebound of viremia upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effects of opioids on viral reservoir remains elusive. Herein, we describe a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs were ramped up with morphine (n=10) or saline (n=9) for two weeks to a final dosage of 5mg/kg administered twice daily, which was maintained for seven weeks, and then infected with SIVmac251. Combined anti-retroviral therapy (cART) was initiated in approximately half the animals in each group five weeks post-infection and morphine/saline administration continued for 10 months. Among drug naïve macaques, there were no differences in plasma/CSF viral load nor in cell-associated DNA/RNA loads. However, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA copy numbers, and inducible SIV reservoir in both peripheral blood and lymph nodes (LNs) of morphine-administered RMs compared to saline controls. Further, PBMCs of morphine administered RMs, a reduction in Th1 polarized CD4+ T cells and in LNs there was a reduction in the total Tfh and Th1 like Tfh cells were observed, indicating probably have impact on reduction of viral reservoirs. In distinction to PBMC and LNs, within the CNS size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages of morphine-dependent RMs. These data suggest that morphine plays a role in modulating SIV reservoirs, reduces the CD4+ T-cell reservoir in both peripheral blood and LNs, and increases microglia/macrophage reservoirs in CNS. These findings will aid in understanding of molecular mechanism(s) of opioid-mediated differential modulation of viral reservoirs and evaluation of therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH.Author summaryOpioids are commonly used as well as abused by HIV infected individuals, are known to suppress immune responses. However, their effects on modulating viral reservoir dynamics is not known. Here we developed a morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs and immune cells. We found that there was no difference in viral loads or cell-associated DNA/RNA loads among morphine dependent vs. saline treated control macaques. On the other hand, when macaques were treated with cART, there was a reduction in SIV reservoirs both in periphery and lymphoid tissues in morphine administered RMs, and the size of latent SIV reservoir was higher in the CNS CD11b+ microglia/macrophages as compared to control macaques. Therefore, these new data form macaque models suggest that PLWH who suffering from opioid use disorders have higher reservoirs in CNS as compared to lymphoid system. Thus, through understanding these reservoirs among PLWH who uses opioids are critical for better designing HIV cure strategies.
HIV persists in cellular reservoirs despite effective combined antiretroviral therapy (cART) and there is viremia flare up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remain elusive. Herein, we developed a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine (or saline control) regimen were infected with SIVmac251. The cART was initiated in approximately half the animals five weeks post-infection, and morphine/saline administration continued until the end of the study. Among the untreated RM, we did not find any difference in plasma/CSF or in cell-associated DNA/RNA viral load in anatomical tissues. On the other hand, within the cART suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoir in lymph nodes (LNs) of morphine administered RMs. In distinction to LNs, in the CNS, the size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages in morphine dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues, while increasing the microglia/reservoir size in CNS tissue. The findings from this pre-clinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid dependent PLWH. IMPORTANCE Identification and clearance of HIV reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by co-morbidities that may alter the size of the reservoirs. There is an overlap between the risk factors for HIV and opioid abuse. Opiates have been recognized as prominent co-morbidities in HIV-infected populations. People infected with HIV also abusing opioids have immune modulatory effects and more severe neurological disease. However, the impact of opioid abuse on HIV reservoirs remains unclear. In this study, we used morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. Our studies suggested that people with HIV who abuse opioids had higher reservoirs in CNS than the lymphoid system. Extrapolating the macaque findings in humans suggests that such differential modulation of HIV reservoirs among people living with HIV abusing opioids could be considered for future HIV cure research efforts.
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