Anti - herpesvirus Activity of Two Novel 4 ’ - Thiothymidine Derivatives KAY - 2 - 41 and KAH - 39 - 149 is 1 dependent on viral and cellular thymidine

24 The emergence of drug-resistant herpesviruses represents a significant problem in clinical practice, primarily 25 in immunocompromised patients. Furthermore, effective antiviral therapy against gammaherpesvirus26 associated diseases is lacking. Here, we present two thiothymidine derivatives, KAY-2-41 and KAH-39-149, 27 with different spectrum of antiviral activity compared to reference anti-herpetic drugs, showing inhibitory 28 activities against herpes simplex virus, varicella zoster virus, and particularly against Epstein-Barr virus with 29 high selectivity in vitro. While KAY-2-41and KAH-39-149-resistant herpesviruses harbored mutations in 30 the viral thymidine kinase (TK), these mutations conferred only low levels of resistance to these drugs, but 31 high levels to other TK-dependent drugs. Also, antiviral assays in Hela TK-deficient cells showed lack of 32 KAY-2-41 and KAH-39-149 activity against HSV-1 and HSV-2 TK-deficient mutants. Furthermore, 33 enzymatic TK assays showed the ability of HSV-1 TK, VZV TK, cellular TK1 and TK2 to recognize and 34 phosphorylate KAY-2-41 and KAH-39-149. These results demonstrated that the compounds depend on both 35 viral and host TKs to exert their antiviral activity. Additionally, the antiviral efficacy of KAH-39-149 36 proved superior to that of KAY-2-41 in a mouse model of gammaherpesvirus infection, highlighting the 37 potential of this class of antiviral agents for further development as selective therapeutics against Epstein38