Date Published: July 25, 2013
Publisher: Public Library of Science
Author(s): Jan Balzarini, Graciela Andrei, Emanuela Balestra, Dana Huskens, Christophe Vanpouille, Andrea Introini, Sonia Zicari, Sandra Liekens, Robert Snoeck, Antonín Holý, Carlo-Federico Perno, Leonid Margolis, Dominique Schols, Guido Silvestri.
Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4+ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).
Human immunodeficiency virus (HIV) infection is commonly associated with other sexually transmitted infections such as herpes simplex virus type 2 (HSV-2). Such infections with HSV-2 may facilitate the risk of HIV acquisition and often worsen the clinical course of the HIV disease –. In fact, HIV-1 has been recovered frequently from genital herpes lesions in co-infected individuals . Although HSV target cells in tissues are still poorly understood and it is not known whether macrophages are important targets for HSV in vivo, both HIV-1 and HSV-2 can infect macrophages. Cells of the monocyte/macrophage (M/M) lineage reside in genital mucosal tissues and are thought to be reservoirs of HIV-1 in the genital tract , . Also, there is evidence that HSV infection can also stimulate macrophages in vitro and induce HIV-1 replication in these cells . Thus, it would be beneficial if a microbicide has efficient suppressive activity against both HIV-1 and HSV-2. Highly specific drugs, such as the acyclic nucleoside phosphonate (ANP) analogue 9-(2-phosphonylmethoxypropyl)adenine [(R)PMPA; tenofovir] against HIV  and the nucleoside analogue 9-(2-hydroxyethyloxymethyl)guanine (acyclovir; ACV) against HSV , have been developed. Unexpectedly, treatment of HIV-1/HSV-2–coinfected individuals with acyclovir diminishes both HSV-2 and HIV-1 load –, while topically applied tenofovir diminishes transmission not only of HIV-1  but also of HSV-2 . Both drugs have been found to be directly active against these two viruses , , although suppression of HIV-1 by acyclovir and of HSV-2 by tenofovir is suboptimal. It would be advisable if antiviral agents can be designed and developed that concomitantly display pronounced inhibitory activity against both pathogens. Therefore, the different subclasses of acyclic nucleoside phosphonates were revisited because it has been previously shown that several members of the ANPs display potent activity against a variety of DNA viruses, including herpesviruses and hepatitis B virus, and retroviruses –. From a wide screen of hundreds of ANPs, a compound designated 6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine (PMEO-DAPym) (Fig. 1) emerged as a novel prototype drug that concomitantly act as an efficient inhibitor of both HIV-1 and HSV-2 replication, but that was also surprisingly endowed with a capacity to interact with HIV entry. This combination of unique properties in one single molecule makes it a promising new-generation multitargeted antiviral for dual-viral (HSV/HIV) infection therapy and HIV transmission prevention.
We demonstrated the potent anti-HIV and -HSV activity of PMEO-DAPym in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4+ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. In all assay systems the drugs were administered prior to, or at the time of, virus infection which is relevant from a microbicidal application viewpoint, for which the drugs are preferentially already present at the time of infection. However, it might well be possible that these drugs are also effective when exposed shortly after virus infection given the DNA polymerase/HIV reverse transcriptase as being one of the intracellular targets of PMEO-DAPym. In addition to our findings that PMEO-DAPym can efficiently suppress a wide variety of HSV clinical isolates including acyclovir-resistant virus strains, it has previously been demonstrated that PMEO-DAPym suppresses a wide variety of HIV-1 clinical strains belonging to different HIV-1 clades (i.e. A, B, C, A/E, G) . This study also revealed that PMEO-DAPym showed a somewhat more favorable cross-resistance profile to various mutant HIV-1 isolates than adefovir and tenofovir. Likewise, we could demonstrate that the antivirally active PMEO-DAPym diphosphate metabolite potently inhibited mutant HIV-1 RT enzymes that harbor the tenofovir-characteristic K65R and K70R mutations (IC50 in the submicromolar range) (data not shown) explaining the efficient suppression of a variety of (mutant) clinical virus isolates. Thus, PMEO-DAPym may have a rather high genetic barrier suppressing a wide variety of clinical HSV and HIV-1 clade isolates, including clinically relevant mutant viruses.