Caroll B. Hartline

Enhanced Inhibition of Orthopoxvirus Replication In Vitro by Alkoxyalkyl Esters of Cidofovir and Cyclic Cidofovir

ABSTRACT The nucleotide phosphonates cidofovir (CDV) and cyclic cidofovir (cCDV) are potent antiviral compounds when administered parenterally but are not well absorbed orally. These compounds have been reported to have activity against orthopoxvirus replication in vitro and in animal models when administered parenterally or by aerosol. To obtain better oral activity, we synthesized a novel series of analogs of CDV and cCDV by esterification with two long-chain alkoxyalkanols, 3-hexadecyloxy-1-propanol (HDP-CDV; HDP-cCDV) or 3-octadecyloxy-1-ethanol (ODE-CDV; ODE-cCDV). Their activities were evaluated and compared with those of CDV and cCDV in human foreskin fibroblast (HFF) cells infected with vaccinia virus (VV) or cowpox virus (CV) using a plaque reduction assay. The 50% effective concentrations (EC50s) against VV in HFF cells for CDV and cCDV were 46.2 and 50.6 μM compared with 0.84 and 3.8 μM for HDP-CDV and HDP-cCDV, respectively. The EC50s for ODE-CDV and ODE-cCDV were 0.20 and 1.1 μM, respectively. The HDP analogs were 57- and 13-fold more active than the parent nucleotides, whereas the ODE analogs were 231- and 46-fold more active than the unmodified CDV and cCDV. Similar results were obtained using CV. Cytotoxicity studies indicated that although the analogs were more toxic than the parent nucleotides, the selective index was increased by 4- to 13-fold. These results indicate that the alkoxyalkyl esters of CDV and cCDV have enhanced activity in vitro and need to be evaluated for their oral absorption and efficacy in animal models.

Alkoxyalkyl Esters of Cidofovir and Cyclic Cidofovir Exhibit Multiple-Log Enhancement of Antiviral Activity against Cytomegalovirus and Herpesvirus Replication In Vitro

ABSTRACT The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir (GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be partially explained by a much greater cell penetration of the novel analogs. 1-O-Hexadecyloxypropyl-CDV, 1-O-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evaluation for treatment and prevention of CMV and herpes simplex virus infections in humans.

In Vitro Activities of Benzimidazole d- and l-Ribonucleosides against Herpesviruses

ABSTRACT Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8) are responsible for a number of clinical manifestations in both normal and immunocompromised individuals. The parent benzimidazole ribonucleosides evaluated in this series, 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB) and maribavir (1263W94), are potent and selective inhibitors of human CMV replication. These nucleosides act by two different mechanisms. BDCRB blocks the processing and maturation of viral DNA, whereas 1263W94 inhibits the viral enzyme pUL97 and interferes with DNA synthesis. In the present study, we have evaluated the in vitro antiviral activity of BDCRB, an analog, GW275175X (175X), and 1263W94 against the replication of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6, and HHV-8. By using various methodologies, significant activity was observed against human CMV and EBV but not against HSV-1, HSV-2, VZV, HHV-6, or HHV-8. Plaque reduction assays performed on a variety of laboratory and clinical isolates of human CMV indicated that all strains, including those resistant to ganciclovir (GCV) and foscarnet, were sensitive to all three benzimidazole ribonucleosides, with mean 50% effective concentration values of about 1 to 5 μM compared to that of GCV at 6 μM. The toxicity of these compounds in tissue culture cells appeared to be similar to that observed with GCV. These results demonstrate that the benzimidazole ribonucleosides are active against human CMV and EBV and suggest that they may be useful for the treatment of infections caused by these herpesviruses.

Ether Lipid-Ester Prodrugs of Acyclic Nucleoside Phosphonates: Activity against Adenovirus Replication In Vitro

The acyclic nucleoside phosphonate cidofovir (CDV) and its closely related analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine ([S]-HPMPA) have been reported to have activity against many adenovirus (AdV) serotypes. A new series of orally active ether lipid-ester prodrugs of CDV and of (S)-HPMPA that have slight differences in the structure of their lipid esters were evaluated, in tissue-culture cells, for activity against 5 AdV serotypes. The results indicated that, against several AdV serotypes, the most active compounds were 15-2500-fold more active than the unmodified parent compounds and should be evaluated further for their potential to treat AdV infections in humans.

Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis

ABSTRACT Recombinant human cytomegaloviruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly refractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.

Comparative Activities of Lipid Esters of Cidofovir and Cyclic Cidofovir against Replication of Herpesviruses In Vitro

ABSTRACT Cidofovir (CDV) is an effective therapy for certain human cytomegalovirus (HCMV) infections in immunocompromised patients that are resistant to other antiviral drugs, but the compound is not active orally. To improve oral bioavailability, a series of lipid analogs of CDV and cyclic CDV (cCDV), including hexadecyloxypropyl-CDV and -cCDV and octadecyloxyethyl-CDV and -cCDV, were synthesized and found to have multiple-log-unit enhanced activity against HCMV in vitro. On the basis of the activity observed with these analogs, additional lipid esters were synthesized and evaluated for their activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus, murine cytomegalovirus, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8. Using several different in vitro assays, concentrations of drug as low as 0.001 μM reduced herpesvirus replication by 50% (EC50) with the CDV analogs, whereas the cCDV compounds were generally less active. In most of the assays performed, the EC50 values of the lipid esters were at least 100-fold lower than the EC50 values for unmodified CDV or cCDV. The lipid analogs were also active against isolates that were resistant to CDV, ganciclovir, or foscarnet. These results indicate that the lipid ester analogs are considerably more active than CDV itself against HSV, VZV, CMV, EBV, HHV-6, and HHV-8 in vitro, suggesting that they may have potential for the treatment of infections caused by a variety of herpesviruses.

Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

ABSTRACT Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinase-dependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

Comparison of the Antiviral Activities of Alkoxyalkyl and Alkyl Esters of Cidofovir against Human and Murine Cytomegalovirus Replication In Vitro

ABSTRACT Alkoxyalkyl esters of cidofovir (CDV) have substantially greater antiviral activity and selectivity than unmodified CDV against herpesviruses and orthopoxviruses in vitro. Enhancement of antiviral activity was also noted when cyclic CDV was esterified with alkoxyalkanols. In vitro antiviral activity of the most active analogs against human cytomegalovirus (HCMV) and orthopoxviruses was increased relative to CDV up to 1,000- or 200-fold, respectively. Alkyl chain length and linker structure are important potential modifiers of antiviral activity and selectivity. In this study, we synthesized a series of alkoxyalkyl esters of CDV or cyclic CDV with alkyl chains from 8 to 24 atoms and having linker moieties of glycerol, propanediol, and ethanediol. We also synthesized alkyl esters of CDV which lack the linker to determine if the alkoxyalkyl linker moiety is required for activity. The new compounds were evaluated in vitro against HCMV and murine CMV (MCMV). CDV or cyclic CDV analogs both with and without linker moieties were highly active against HCMV and MCMV, and their activities were strongly dependent on chain length. The most active compounds had 20 atoms esterified to the phosphonate of CDV. Both alkoxypropyl and alkyl esters of CDV provided enhanced antiviral activities against CMV in vitro. Thus, the oxypropyl linker moiety is not required for enhanced activity. CDV analogs having alkyl ethers linked to glycerol or ethanediol linker groups also demonstrated increased activity against CMV.

In Vitro Activity and Mechanism of Action of Methylenecyclopropane Analogs of Nucleosides against Herpesvirus Replication

ABSTRACT We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC50) levels of ≤50 μM. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC50s of 0.5 to 44 μM, and all were active against MCMV by PR (0.3 to 54 μM). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 μM). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 μM), and three compounds were active against HHV-8 (5.5 to 16 μM). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC50s of 0.7 to 8 μM. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.

Oral Activity of a Methylenecyclopropane Analog, Cyclopropavir, in Animal Models for Cytomegalovirus Infections

ABSTRACT We reported previously that purine 2-(hydroxymethyl)methylenecyclopropane analogs have good activity against cytomegalovirus infection. A second-generation analog, (Z)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}guanine (ZSM-I-62, cyclopropavir [CPV]), has particularly good activity against murine and human cytomegaloviruses (MCMV and HCMV) in vitro. To determine the oral activity of this compound in vivo, BALB/c or severe combined immunodeficient (SCID) mice infected with MCMV and two models using SCID mice implanted with human fetal tissue and subsequently infected with HCMV were used. In MCMV-infected normal mice, CPV at 10 mg/kg of body weight was highly effective in preventing mortality when administered at 24, 48, or 72 h post-viral inoculation and reduced titers of virus in tissues of SCID mice by 2 to 5 log10. In one HCMV model, human fetal retinal tissue was implanted into the anterior chamber of the mouse eye and inoculated with the Toledo strain of HCMV, and in the second, human fetal thymus and liver tissues were implanted under the kidney capsule of mice and then inoculated with HCMV. In general, replication of HCMV in both types of implant tissue increased from 7 through 21 to 28 days and then gradually decreased to undetectable levels by 8 weeks postinfection. Oral treatment with 45 or 15 mg of CPV/kg initiated 24 h after infection was highly effective in reducing replication to undetectable levels in both models and was generally more effective than ganciclovir. These data indicate that the methylenecyclopropane analog, CPV, was highly efficacious in these four animal models and should be evaluated for use in HCMV infections in humans.