Immunosuppressants Exert Differential Effects against Pan-Coronavirus Infection and Distinct Combinatory Antiviral Activity with Molnupiravir and Nirmatrelvir

ABSTRACT

Introduction: There are seven types of coronaviruses that are known to infect humans, including three highly pathogenic members MERS-CoV, SARS-CoV-1 and SARS-CoV-2, and four seasonal coronaviruses, including NL63, 229E, OC43 and HKU1. Immunocompromised populations, such as organ transplant recipients receiving immunosuppressive medications and patients with inflammatory bowel disease treated with immunosuppressive or immunomodulatory therapies, are in general more susceptible to coronavirus infections. However, little is known how immunosuppressants directly affect coronavirus infection. Aims & Methods: This study aims to profile the effects of immunosuppressants and the combination of immunosuppressants with oral antiviral drug molnupiravir and nirmatrelvir on pan-coronavirus infection in cell culture models. Different coronaviruses (including wild type, delta and omicron variants of SARS-CoV-2, NL63, 229E and OC43) were used in cell culture models. The effects of immunosuppressants including dexamethasone, budesonide, prednisolone, sulfasalazine, aminosalicylates, 6-thioguanine, azathioprine, 6-mercaptopurine, methotrexate, tacrolimus, cyclosporine A, mycophenolic acid (MPA), rapamycin, everolimus, tofacitinib and figotinib on coronavirus infections, and the combination with molnupiravir and nirmatrelvir were studied in cell culture. Result(s) To profile the effect of immunosuppressants, we first tested all these regimens at 1 muM on wild type SARS-CoV-2, and then validated on different coronaviruses. We found dexamethasone and aminosalicylates can significantly stimulate the replication of these coronaviruses. For example, treatment with 1 muM dexamethasone significantly increased virus RNA level of wild type SARS-CoV-2 by 137+/-40%. 6-thioguanine, MPA, tofacitinib and figotinib treatment inhibited viral replication in a dosedependent manner in all tested coronaviruses. 6-thioguanine at 1 muM concentration had already potently inhibited the replication of wild type SARS-CoV-2 by 95+/-16%. The half maximum effective concentration (EC50) of tofacitinib against wild type SARS-CoV-2 replication was 0.60 muM and the half maximum cytotoxic concentration (CC50) was above 30 muM, which resulted in a selective index (SI) above 50. Combination treatment is often used to enhance antiviral efficacy, avoid drug resistance development in clinical applications. We evaluated the combined antiviral effects of 6-thioguanine, MPA and tofacitinib with molnupiravir and nirmatrelvir in wild type SARS-CoV-2 infection model. The combination of tofacitinib with molnupiravir had a moderate additive effect with the ZIP synergy score of 7, whereas the other two combinations had no such effect. However, the combination of MPA, 6-thioguanine, and tofacitinib with nirmatrelvir resulted in strong synergistic effect, and the ZIP synergy scores were 31, 22 and 12 respectively. Conclusion(s) These results demonstrated that different immunosuppressants had differential effects on coronavirus replication. 6-thioguanine, MPA, tofacitinib and figotinib were identified as potent inhibitors against pan-coronavirus infections. Although combination of MPA, 6-thioguanine, and tofacitinib with molnupiravir exerted moderate additive effect, combination with nirmatrelvir exerted strong synergistic antiviral activity. Thus, these findings provide as an important reference for clinicians to choose the optimal immunosuppressants for coronavirus infected immunocompromised patients or when they are under antiviral treatment of molnupiravir or nirmatrelvir.