A new class of dual-targeted antivirals: monophosphorylated acyclovir prodrug derivatives suppress both human immunodeficiency virus type 1 and herpes simplex virus type 2

Abstract

Background: Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) are responsible for 2 intersecting epidemics in which the disease caused by 1 virus facilitates the transmission of and pathogenesis by the other. Therefore, suppression of one virus infection will affect the other. Acyclovir, a common antiherpetic drug, was shown to directly suppress both viruses in coinfected tissues. However, both antiviral activities of acyclovir are dependent on phosphorylation by the nucleoside kinase activity of coinfecting human herpesviruses.

Methods: We developed acyclovir ProTides, monophosphorylated acyclovir with the phosphate group masked by lipophilic groups to allow efficient cellular uptake, and investigated their antiviral potential in cell lines and in human tissues ex vivo.

Results: Acyclovir ProTides suppressed both HIV-1 and HSV-2 at median effective concentrations in the submicromolar range in ex vivo lymphoid and cervicovaginal human tissues and at 3-12 micromol/L in CD4(+) T cells. Acyclovir ProTides retained activity against acyclovir-resistant HSV-2.

Conclusions: Acyclovir ProTides represent a new class of antivirals that suppress both HIV-1 and HSV-2 by directly and independently blocking the key replicative enzymes of both viruses. Further optimization of such compounds may lead to double-targeted antivirals that can prevent viral transmission and treat the 2 synergistic diseases caused by HIV-1 and HSV-2. To our knowledge, the acyclovir ProTides described here represent the first example of acyclic nucleoside monophosphate prodrugs being active against HIV-1.

Figure 1. Generic structure of ACV ProTides

ACV ProTides consist of monophosphorylated ACV and of aryloxy and aminoacyl ester groups linked to the phosphate moiety.

Figure 2. ACV ProTides inhibit HIV-1 replication in human lymphoid tissues

Human tonsillar tissues (27 blocks of tissue from each of n donors for each experimental condition) were infected with HIV-1_LAI.04 and treated with ACV ProTide Cf2648 or Cf2649 at various concentrations. Anti-HIV-1 activities of ProTides were evaluated by comparing viral replication in drug-treated and untreated donor-matched tissues. A. Presented is HIV inhibition at different concentrations of ProTides as defined by the following formula: Inhibition = (1 − R_ProTide/R_contr)×100% where R_ProTide and R_contr are the amounts of p24 accumulated in the medium over the 12 day-culture period in ProTide-treated and donor-matched untreated cultures, respectively. The 50% effective concentration for each ACV ProTide (EC₅₀) was estimated. EC₅₀ for Cf2648 and Cf2649 are respectively 1 ± 0.4μM and 0.14 ± 0.03μM. Presented are means ± SEM of the results with tissues from four to five donors. B. Presented are flow-cytometry density plots of HIV-1_LAI.04-infected CD8⁻ T cells at day 12 after HIV-1 infection of a representative experiment. Isolated cells from donor-matched uninfected, HIV-infected, and HIV-infected ACV ProTide Cf2649 treated tissues were stained for CD3, CD4, CD8 and CD45RA surface markers as well as for intracellular p24.

Figure 3. ACV ProTides suppress HIV-1 replication in human cervicovaginal tissues and in HSV-2-coinfected tonsillar tissues

A. ACV ProTides suppress HIV-1 replication in human cervicovaginal tissues. Blocks of human cervicovaginal tissue were infected with HIV-1_BaL and treated or not (control) with the ACV ProTide Cf2649 for 12 days at the concentration of 1μM. Each measurement represented the amount of HIV-1 accumulated in culture medium over a 3-day period and presented as percentage of the maximal p24 concentration in untreated control tissues to account for the variation in absolute replication levels in tissues from different donors. Presented in the cumulative curve are means ± SEM of the results with tissues from four donors. B. Blocks of human tonsillar tissue were coinfected with HIV-1_LAI.04, and HSV-2 (G). Tissues were treated or not with the ACV ProTide Cf2648 at 1μM. Replication of HIV-1 _LAI was evaluated by p24_gag core antigen release in pooled medium bathing 27 tissue blocks using a bead-based assay. Presented are means ± SEM of percent of maximal cumulative p24 production. C. Blocks of human tonsillar tissue were coinfected with HIV-1_LAI.04, and HSV-2 (G). Tissues were treated or not with the ACV ProTide Cf2648 at 1μM. Replication of HSV-2 was monitored by real time PCR for viral DNA accumulated in the culture media. Presented are means ± SEM of maximal of cumulative production of genome equivalent concentration.