Antonín Holý

Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines

Various 3-hydroxy-2-phosphonylmethoxypropyl (HPMP) and 2-phosphonylmethoxyethyl (PME) derivatives of purine [adenine (A), guanine (G), 2,6-diaminopurine (DAP), 2-monoaminopurine (MAP), hypoxanthine (HX)] and pyrimidine [cytosine (C), uracil (U), thymine (T)] have been evaluated for their antiviral properties. PMEDAP, (S)-HPMPA [and the cyclic phosphonate thereof, (S)-cHPMPA)], (S)-HPMPC, PMEG, PMEA, HPMPG and HPMPDAP proved to be effective inhibitors of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). (S)-HPMPA and (S)-cHPMPA were the most effective inhibitors of varicella-zoster virus (VZV), and (S)-HPMPC was the most effective inhibitor of cytomegalovirus (CMV). Against adenovirus (types 2, 3 and 4) and vaccinia virus again (S)-HPMPA and (S)-cHPMPA showed the greatest inhibitory activity. As a rule, the PME derivates were much less inhibitory to VZV, CMV, vaccinia and adenovirus than the HPMP derivatives. However, PMEA, PMEDAP and PMEMAP showed marked and selective activity against the human immunodeficiency virus (HIV). (S)-HPMPA was selected for further evaluation in animal model infections. It proved efficacious in the topical treatment of HSV-1 keratitis in rabbits and cutaneous HSV-1 infection in hairless mice, and in the systemic treatment of both HSV-1 and vaccinia virus infections in mice.

Acyclic nucleoside phosphonates: a key class of antiviral drugs

Almost 20 years after the broad antiviral activity spectrum of the first acyclic nucleoside phosphonates was described, several of these compounds have become important therapies for DNA virus and retrovirus infections. Here, we review the discovery and development of acyclic nucleoside phosphonates, focusing on cidofovir and its potential in the treatment of various herpes-, papilloma-, polyoma-, adeno- and pox-virus infections, adefovir for the treatment of hepatitis B and tenofovir for the treatment of AIDS and the prevention of HIV infections.

(S)-9-(2,3-Dihydroxypropyl)adenine: An Aliphatic Nucleoside Analog with Broad-Spectrum Antiviral Activity

(S)-9-(2,3-Dihydroxypropyl)adenine, a novel nucleoside analog, the sugar moiety of which is replaced by an aliphatic chain, inhibits the replication in vitro of several DNA and RNA viruses, including vaccinia, herpes simplex (types 1 and 2), measles, and vesicular stomatitis. It is also effective in vivo in reducing the mortality rate of mice inoculated intranasally with vesicular stomatitis virus.

Synthesis and Cytostatic Activity of Substituted 6-Phenylpurine Bases and Nucleosides: Application of the Suzuki−Miyaura Cross-Coupling Reactions of 6-Chloropurine Derivatives with Phenylboronic Acids

The Suzuki-Miyaura reaction of protected 6-chloropurine and 2-amino-6-chloropurine bases and nucleosides with substituted phenylboronic acids led to the corresponding protected 6-(substituted phenyl)purine derivatives 6-9. Their deprotection yielded a series of substituted 6-phenylpurine bases and nucleosides 10-13. Significant cytostatic activity (IC(50) 0.25-20 micromol/L) in CCRF-CEM, HeLa, and L1210 cell lines was found for several 6-(4-X-substituted phenyl)purine ribonucleosides 12 (X = H, F, Cl, and OR), while the 6-phenylpurine and 2-amino-6-phenylpurine bases 10 and 11, as well as 2-amino-6-phenylpurine ribosides 13, were entirely inactive against these cell lines.

Acyclic nucleotide analogues: synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro.

Several N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl) (HPMP) and N-(2-phosphonylmethoxyethyl) (PME) derivatives of purine bases (adenine, guanine, 2-aminoadenine, 3-deazaadenine) and cytosine inhibit the growth of various DNA viruses. PME-derivatives (PMEA, PMEG and PMEDAP) are also active against retroviruses. Both types of nucleotide analogues undergo phosphorylation by cellular nucleotide kinases to their mono- and diphosphates. The phosphorylation with crude extracts of L-1210 cells is potentiated by an ATP-regenerating system. HPMPA is phosphorylated faster than PMEA with or without the ATP-regenerating system. The HPMP and PME analogues inhibit several virus-encoded target enzymes and their cellular counterparts: (1) HSV-1 DNA polymerase is inhibited by the diphosphates of the PME series; the virus-encoded enzyme is more sensitive than HeLa DNA pol alpha and beta. PMEApp terminates the growing DNA chain; it specifically replaces dATP. HPMPApp also acts as an alternative substrate of dATP, but, in contrast with PMEApp, it permits limited chain growth. (2) Diphosphates of both series inhibit HSV-1 ribonucleotide reductase; the greatest inhibition of CDP reduction to dCDP is exhibited by HPMPApp and PMEApp. The enzyme isolated from a PMEA-resistant HSV-1 mutant proved less sensitive to PMEApp, hydroxyurea and HPMPApp. (3) Diphosphates of PME derivatives efficiently inhibit AMV(MAV) reverse transcriptase. (4) The purine HPMP and PME analogues and, even more so, their monophosphate derivatives inhibit purine nucleoside phosphorylase from L-1210 cells.

Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection

There is concern that variola virus, the aetiological agent of smallpox, may be used as a biological weapon. For this reason several countries are now stockpiling (vaccinia virus-based) smallpox vaccine. Although the preventive use of smallpox vaccination has been well documented, little is known about its efficacy when used after exposure to the virus. Here we compare the effectiveness of (1) post-exposure smallpox vaccination and (2) antiviral treatment with either cidofovir (also called HPMPC or Vistide) or with a related acyclic nucleoside phosphonate analogue (HPMPO–DAPy) after lethal intratracheal infection of cynomolgus monkeys (Macaca fascicularis) with monkeypox virus (MPXV). MPXV causes a disease similar to human smallpox and this animal model can be used to measure differences in the protective efficacies of classical and new-generation candidate smallpox vaccines. We show that initiation of antiviral treatment 24 h after lethal intratracheal MPXV infection, using either of the antiviral agents and applying various systemic treatment regimens, resulted in significantly reduced mortality and reduced numbers of cutaneous monkeypox lesions. In contrast, when monkeys were vaccinated 24 h after MPXV infection, using a standard human dose of a currently recommended smallpox vaccine (Elstree-RIVM), no significant reduction in mortality was observed. When antiviral therapy was terminated 13 days after infection, all surviving animals had virus-specific serum antibodies and antiviral T lymphocytes. These data show that adequate preparedness for a biological threat involving smallpox should include the possibility of treating exposed individuals with antiviral compounds such as cidofovir or other selective anti-poxvirus drugs.

Efficacy of phosphonylmethoxyalkyl derivatives of adenine in experimental herpes simplex virus and vaccinia virus infections in vivo.

The phosphonylmethoxyalkyl derivatives (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) were evaluated for their in vivo efficacies in several animal model infections, i.e., mice infected intravenously with vaccinia virus and mice infected intracutaneously, intraperitoneally, or intracerebrally with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) or thymidine kinase-deficient (TK-) HSV-1. (S)-HPMPA inhibited the development of tail lesions caused by vaccinia virus if it was administered intraperitoneally or subcutaneously at a dosage as low as 5 mg/kg per day. All three compounds completely suppressed the development of skin lesions and the mortality associated therewith in hairless or athymic nude mice inoculated intracutaneously with HSV-1 or TK- HSV-1, if they were administered topically at a concentration as low as 0.1%; when (S)-HPMPA was applied topically at a concentration of greater than or equal to 0.3%, it completely abrogated mortality resulting from intracutaneous HSV-2 infection. Most dramatic were the effects shown by the compounds in mice inoculated intracerebrally with HSV-1, HSV-2, or TK- HSV-1, in which all three compounds given intraperitoneally at a dose of 50 or 100 mg/kg per day effected a significant reduction in the mortality rate of HSV-1-infected mice. The mortality of mice infected intracerebrally with HSV-2 or TK- HSV-1 was significantly reduced even when (S)-HPMPA was given at doses as low as 10 mg/kg per day. These data point to the great potential of the phosphonylmethoxyalkylpurines for both topical and parenteral treatment of HSV-1, HSV-2, and TK- HSV-1 infections. Images

Antiviral activity of selected acyclic nucleoside analogues against human herpesvirus 6

Human herpesvirus 6 (HHV-6) was examined in vitro for its sensitivity to a broad range of nucleoside analogues, including acyclovir (ACV), ganciclovir (GCV), penciclovir (PCV), buciclovir (BCV), brivudin (BVDU), the N7-isomer of 6-deoxyganciclovir (S2242), foscarnet (phosphonoformic acid, PFA), and several acyclic nucleoside phosphonate (ANP) analogues such as (S)-HPMPA, (S)-HPMPC, PMEA and PMEDAP. Antiviral efficacy was monitored microscopically by the inhibitory effect of the compounds on HHV-6-induced cytopathic effect in human T-lymphoblastoid HSB-2 cells. In addition, a newly developed immunofluorescence/flow cytometric assay (FACS) was used to determine HHV-6-specific antigen expression. A close correlation was observed between the antiviral data obtained by the microscopic assay and the flow cytometric assay. Marked antiviral efficacy was noted for S2242, PFA and the ANP analogues (S)-HPMPA, (S)-HPMPC, (S)-cHPMPC, (S)-3-deaza-HPMPA, (S)-3-deaza-cHPMPA, (S)-HPMPG and (R)-HPMPG. Also, PMEA and PMEDAP proved highly active against HHV-6 infection, whereas (S)-FPMPA and (R)-PMPDAP were inactive. ACV was only slightly protective against HHV-6, and no activity was found for GCV, PCV, BCV and BVDU. Overall, the efficacy of the nucleoside analogues against HHV-6 appeared to correlate with their efficacy against human cytomegalovirus (HCMV).

Antivaccinia Activities of Acyclic Nucleoside Phosphonate Derivatives in Epithelial Cells and Organotypic Cultures

ABSTRACT Organotypic “raft” cultures of epithelial cells allow the reconstitution of a skin equivalent that is easily infectible with different viruses with cutaneous tropism. Among these, poxvirus and particularly vaccinia virus (VV) are good candidates for use in antiviral tests, giving histological pictures comparable to those observed in humans infected with smallpox. Therefore, we decided to evaluate a series of phosphonate derivatives for their ability to inhibit VV growth in epithelial cell monolayers, and the most powerful derivatives were tested in the organotypic cultures. The most active compound was 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA], followed by 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine, cyclic (S)-HPMPA, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC; cidofovir, Vistide], and cyclic (S)-HPMPC. Cidofovir, which is on the market for the treatment of human cytomegalovirus retinitis in immunocompromised patients, is potentially a good candidate for the treatment of a poxvirus outbreak, in the absence of any vaccination.

Efficacy of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine in various models of herpes simplex virus infection in mice.

The phosphonylmethoxyalkyl derivative (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) was evaluated for its in vivo efficacy in several model infections for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and thymidine kinase-deficient (TK-) HSV-1 in mice. In hairless mice infected intracutaneously with HSV-1 or HSV-2, HPMPC completely suppressed all manifestations of the disease (skin lesions, paralysis of the hind legs, and mortality) if it was administered topically at a concentration of as low as 0.1, 0.3, or 1%. Similarly, HPMPC completely suppressed TK- HSV-1 infection in athymic nude mice if it was administered topically at 0.1 or 0.3% or intraperitoneally at 100 or 250 mg/kg/day. HPMPC was also effective against intraperitoneal HSV infection if it was given orally at a dose of 50 mg/kg/day or higher. In mice inoculated intracerebrally with HSV-2, intraperitoneal HPMPC treatment achieved a significant and dose-dependent protection at doses ranging from 5 to 400 mg/kg/day. The protective effect of HPMPC (at 200 mg/kg/day) was accompanied by a complete inhibition of virus multiplication in the brain. In all models of infections studied, the efficacy of HPMPC proved to be superior to that of acyclovir. The most remarkable feature of HPMPC was that a single administration of the compound, even as late as 4 days after infection, conferred significant protection against HSV-1 or HSV-2 infection. Topical or systemic HPMPC treatment is efficacious in murine models of HSV-1, HSV-2, and TK- HSV infections.