Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: selection and characterization of mutant isolates

Abstract

Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2. These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three-to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype. Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (K d) for R1-R2 subunit association and/or reduced activity (k cat). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.

Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase

“…A structural determination of E. coli R1 confirmed this location [7] and identified a specific α-helical region that interacted with the E. coli R2 C-terminal peptide. Two HSV-1 R1 residues, Pro-1090 and Ala-1091, located within the proposed R2 interaction helix, were found to be mutated in viruses that developed resistance to peptidomimetic inhibitors of HSV-1 RR subunit interaction [21], providing direct evidence for the role of this R1 region in R2 binding. Amino acids in the C-termini of HSV-1 and EHV-4 R1 demonstrate an exceptionally high degree of conservation : there are only 17 non-conserved residues within the C-terminal 100 residues.…”

“…Rational drug design based on Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu has generated peptidomimetic antiviral compounds that are effective in animal models of infection [19,20] and HSV-1 RR has provided a model for the study of peptides that disrupt protein-protein interactions as a route for antiviral drug development. However, HSV-1 mutants that are resistant to peptidomimetic inhibitors develop in tissue culture [21] and there is a need to develop alternative inhibitors of this essential virus enzyme. In addition to its role in RR, HSV-1 R1 has another, as yet unidentified, function [22,23] that resides in a unique N-terminal extension of approx.…”

Modern Ethiotropic Chemotherapy of Herpesvirus Infections: Advances, New Trends and Perspectives. Alphaherpesviruses (Part Ii)

“…In vitro селекционирован вирусный штамм, резистентный к BILD 733 (пептиду, аналогичному BILD 1633 SE). Показано, что мутации, обусловливающие резистентность, локализованы на С-конце субъединицы R1 (A1091S и P1090L) [12]. BILD 1633 SE не влияет на активность РР человека в концентрации до 250 мкМ, в отношении ВПГ-1 in vitro он в 10 раз эффективнее, чем AЦВ (ИД 50 0,35-0,46 мкМ, ХТИ около 35); активность соединения не зависит от чувствительности вируса к АЦВ и ФМК.…”

Current and Potential Treatments for Ubiquitous but Neglected Herpesvirus Infections

“… 108 Follow-up studies by researchers at Boehringer Ingelheim developed peptidomimetic compounds, such as BILD 1263 ( 34 , Figure 10 ), that improved potency over shorter synthetic peptides by over 200 000-fold and that displayed in vivo efficacy in a mouse ocular model of infection: the first such example for a PPI antagonist. 109 The evolution of viral resistance to related compounds and identification of the sites on the R1 subunit of the HSV-1 reductase sufficient for resistance (A1091S and P1090L) were also described. 109b…”