Inhibition of Herpesvirus Replication by 5-Substituted 4′-Thiopyrimidine Nucleosides

124

114

The molecular mechanisms of herpes simplex virus (HSV) resistance to antiviral drugs interfering with viral DNA synthesis reported so far rely on the presence of mutations within UL23 (thymidine kinase [TK]) and UL30 (DNA polymerase) genes. The interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objectives of this work were to describe extensively the natural polymorphism of UL23 TK and UL30 DNA polymerase among HSV-1 and HSV-2 strains and the amino acid changes potentially associated with HSV resistance to antivirals. The sequence analysis of the full-length UL23 and UL30 genes was performed. Ninety-four drugsensitive clinical isolates (43 HSV-1 and 51 HSV-2) and 3 laboratory strains (KOS, gHSV-2, and MS2) were studied for natural polymorphism, and 25 clinical isolates exhibiting phenotypic traits of resistance to antivirals were analyzed for drug resistance mutations. Our results showed that TK and DNA polymerase are highly conserved among HSV strains, with a weaker variability for HSV-2 strains. This study provided a precise map of the natural polymorphism of both viral enzymes among HSV-1 and HSV-2 isolates, with the identification of 15 and 51 polymorphisms never previously described for TK and DNA polymerase, respectively, which will facilitate the interpretation of genotypic antiviral-resistant testing. Moreover, the genotypic characterization of 25 drug-resistant HSV isolates revealed 8 new amino acid changes located in TK and potentially accounting for acyclovir (ACV) resistance.Herpes simplex virus type 1 (HSV-1) and HSV-2 are responsible for a variety of clinical manifestations (10). In immunocompetent individuals, the symptoms are usually self-limited, whereas severe diseases, sometimes life-threatening, may occur in immunocompromised patients (14,17,26). The discovery of acyclovir (ACV), almost 30 years ago, represents a milestone in the management of HSV infections. The antiviral activity and selectivity of ACV is based on the phosphorylation to its monophosphate form by the virus-encoded thymidine kinase (TK). Then ACV monophosphate is further phosphorylated by cellular thymidilate kinases to the triphosphate form and is incorporated into the growing DNA chain by the viral DNA polymerase, thereby inhibiting replication through chain termination. The decreased activity of TK confers HSV resistance to ACV, resulting in the inability of the drug to inhibit viral replication. Alternative drugs, like foscarnet (FOS), are effective without the requirement of phosphorylation by viral TK. FOS inhibits directly the viral DNA polymerase as a substrate analogue of the pyrophosphate formed during DNA synthesis (23).HSV TK is a 376-amino-acid protein, encoded by the UL23 gene, containing an ATP binding site (codons 51 to 63), a nucleoside binding site (codons 168 to 176 for HSV-1 and 169 to 177 for HSV-2), and a highly conserved cysteine residue at position 336 for HSV-1 and 337 for HSV-2 (2,...

Section: Discussionmentioning

confidence: 88%

Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals

Burrel

Deback

Agut

et al. 2010

124

114

“…For combined efficacy and animal studies, strains E-377 and MS were used to evaluate efficacy against HSV-1 and HSV-2, respectively. The provenance and phenotypic and genotypic characteristics of each of these viruses were reported recently (26). Mutations in these isolates thought to confer resistance to ACV are in the TK open reading frame.…”

Section: Methodsmentioning

confidence: 99%

CMX001 Potentiates the Efficacy of Acyclovir in Herpes Simplex Virus Infections

Prichard

Kern

Hartline

et al. 2011

Self Cite

Although acyclovir (ACV) has proven to be of value in the therapy of certain herpes simplex virus (HSV) infections, there is a need for more effective therapies, particularly for serious infections in neonates and immunocompromised individuals, where resistance to this drug can be problematic. CMX001 is an orally bioavailable lipid conjugate of cidofovir that is substantially less nephrotoxic than the parent drug and has excellent antiviral activity against all the human herpesviruses. This compound retains full antiviral activity against ACV-resistant laboratory and clinical isolates. The combined efficacy of CMX001 and ACV was evaluated in a new real-time PCR combination assay, which demonstrated that the combination synergistically inhibited the replication of HSV in cell culture. This was also confirmed in murine models of HSV infection, where the combined therapy with these two drugs synergistically reduced mortality. These results suggest that CMX001 may be effective in the treatment of ACV-resistant HSV infections and as an adjunct therapy in individuals with suboptimal responses to ACV.

“…Other laboratories have demonstrated the inhibitory potential of 5-substituted 4=-thiopyrimidine nucleoside derivatives against poxviruses (27,29), despite the fact that the narrow substrate specificity of poxvirus TK remains a challenge for selecting potent nucleoside analogues. Our study demonstrated that OPVs (VACVs, CPXVs, and CML1) are efficiently inhibited by KAY-2-41 in vitro at concentrations in the submicromolar range.…”

Section: Discussionmentioning

confidence: 99%

“…In 2003, a series of 5-substituted 2=-deoxy-4=-thiopyrimidine nucleosides was also shown to retain good antiviral activity against some herpesviruses, with the 2=-deoxy-5-ethyl-4=-thiouridine derivative being the most potent against alphaherpesviruses (28). The antiviral efficacy of 5-substituted 4=-thiopyrimidine against alphaherpesviruses and OPVs was reported in two studies (27,29). The compound 5-iodo-4=-thio-2=-deoxyuridine (4=-thioIDU) inhibited both VACV and CPXV replication at concentrations in the nanomolar range (27).…”

mentioning

confidence: 99%

KAY-2-41, a Novel Nucleoside Analogue Inhibitor of OrthopoxvirusesIn VitroandIn Vivo

Duraffour

Drillien

Haraguchi

et al. 2014

The availability of adequate treatments for poxvirus infections would be valuable not only for human use but also for veterinary use. In the search for novel antiviral agents, a 1=-methyl-substituted 4=-thiothymidine nucleoside, designated KAY-2-41, emerged as an efficient inhibitor of poxviruses. In vitro, KAY-2-41 was active in the micromolar range against orthopoxviruses (OPVs) and against the parapoxvirus orf. The compound preserved its antiviral potency against OPVs resistant to the reference molecule cidofovir. KAY-2-41 had no noticeable toxicity on confluent monolayers, but a cytostatic effect was seen on growing cells. Genotyping of vaccinia virus (VACV), cowpox virus, and camelpox virus selected for resistance to KAY-2-41 revealed a nucleotide deletion(s) close to the ATP binding site or a nucleotide substitution close to the substrate binding site in the viral thymidine kinase (TK; J2R) gene. These mutations resulted in low levels of resistance to KAY-2-41 ranging from 2.7-to 6.0-fold and cross-resistance to 5-bromo-2=-deoxyuridine (5-BrdU) but not to cidofovir. The antiviral effect of KAY-2-41 relied, at least in part, on activation (phosphorylation) by the viral TK, as shown through enzymatic assays. The compound protected animals from disease and mortality after a lethal challenge with VACV, reduced viral loads in the serum, and abolished virus replication in tissues. In conclusion, KAY-2-41 is a promising nucleoside analogue for the treatment of poxvirus-induced diseases. Our findings warrant the evaluation of additional 1=-carbon-substituted 4=-thiothymidine derivatives as broad-spectrum antiviral agents, since this molecule also showed antiviral potency against herpes simplex virus 1 in earlier studies.