KSHV targeted therapy: an update on inhibitors of viral lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses.

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-ß-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase.

Antiherpesvirus activities of two novel 4'-thiothymidine derivatives, KAY-2-41 and KAH-39-149, are dependent on viral and cellular thymidine kinases.

The emergence of drug-resistant herpesviruses represents a significant problem in clinical practice, primarily in immunocompromised patients. Furthermore, effective antiviral therapies against gammaherpesvirus-associated diseases are lacking. Here, we present two thiothymidine derivatives, KAY-2-41 and KAH-39-149, with different spectra of antiviral activity from those of the reference antiherpetic drugs, showing inhibitory activities against herpes simplex virus, varicella-zoster virus (VZV), and particularly against Epstein-Barr virus, with high selectivity in vitro. While KAY-2-41- and KAH-39-149-resistant herpesviruses were found to harbor mutations in the viral thymidine kinase (TK), these mutations conferred only low levels of resistance to these drugs but high levels to other TK-dependent drugs. Also, antiviral assays in HeLa TK-deficient cells showed a lack of KAY-2-41 and KAH-39-149 activities against herpes simplex virus 1 (HSV-1) and HSV-2 TK-deficient mutants. Furthermore, enzymatic TK assays showed the ability of HSV-1 TK, VZV TK, and cellular TK1 and TK2 to recognize and phosphorylate KAY-2-41 and KAH-39-149. These results demonstrate that the compounds depend on both viral and host TKs to exert antiviral activity. Additionally, the antiviral efficacy of KAH-39-149 proved to be superior to that of KAY-2-41 in a mouse model of gammaherpesvirus infection, highlighting the potential of this class of antiviral agents for further development as selective therapeutics against Epstein-Barr virus.

Evaluation of novel acyclic nucleoside phosphonates against human and animal gammaherpesviruses revealed an altered metabolism of cyclic prodrugs upon Epstein-Barr virus reactivation in P3HR-1 cells.

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.