Design and experimental validation of the oxazole and thiazole derivatives as potential antivirals against of human cytomegalovirus.

QSAR studies of a set of previously synthesized azole derivatives tested against human cytomegalovirus (HCMV) were performed using the OCHEM web platform. The predictive ability of the classification models has a balanced accuracy (BA) of 73-79%. The validation of the models using an external test set proved that the models can be used to predict the activity of newly designed compounds with a reasonable accuracy within the applicability domain (BA = 76-83%). The models were applied to screen a virtual chemical library with expected activity of compounds against HCMV. The five most promising new compounds were identified, synthesized and their antiviral activities against HCMV were evaluated in vitro. Two of them showed some activity against the HCMV strain AD169. According to the results of docking analysis, the most promising biotarget associated with HCMV is DNA polymerase. The docking of the most active compounds 1 and 5 in the DNA polymerase active site shows calculated binding energies of -8.6 and -7.8 kcal/mol, respectively. The ligand's complexation was stabilized by the formation of hydrogen bonds and hydrophobic interactions with amino acids Lys60, Leu43, Ile49, Pro77, Asp134, Ile135, Val136, Thr62 and Arg137.

Brincidofovir treatment of acyclovir-resistant disseminated varicella zoster virus infection in an immunocompromised host.

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet.

Predictive efficacy of SCID-hu mouse models for treatment of human cytomegalovirus infections.

There are few animal models to evaluate the in vivo activity of new compounds against human cytomegalovirus (HCMV) infections, as virus replication is largely limited to human cells. In our studies, we have utilized SCID mice implanted with human tissue (SCID-hu) and inoculated with HCMV as models for infections of the eye or visceral organs in an immunocompromised host. For the ocular model, fetal human retinal tissue was implanted in the anterior chamber of the SCID mouse eye and inoculated 6-9 weeks later with 2,000-7,500 plaque-forming units (pfu) of HCMV. In the second model, fetal thymus and liver (thy/liv) tissue was implanted under the kidney capsule of SCID mice and inoculated 12-14weeks later with 2,200-9,000 pfu of HCMV. At various times after infection, implant tissues were removed, homogenized, and HCMV titres quantified by plaque assay. The replication of the Toledo strain of HCMV in both models was similar in that viral titres increased through day 21, remained high through day 35, and then gradually decreased. To validate the two models, the efficacy of ganciclovir (GCV) and cidofovir (CDV) was determined in both ocular and thy/liv model implants. In SCID-hu retinal tissue, once daily intraperitoneal (i.p.) treatment with 33 mg GCV per kg significantly reduced viral titres (2-20-fold) between 14 and 28 days after infection. In SCID-hu thy/liv implants, the same treatment regimen reduced viral replication either completely or by 3-5 log10. In retinal implant tissue, i.p. treatment with 25 mg CDV per kg once daily for 14 days, followed by three times weekly for the next 14 days, reduced viral titres by 2-3 log10 between 10 and 42 days after infection. In comparison, once daily i.p. administration of 30 mg CDV per kg completely inhibited HCMV replication in thy/liv implants. These results indicate that both the SCID-hu retinal and SCID-hu thy/liv implant models are useful for determining in vivo activity against HCMV, and appear to be predictive of efficacy for both ocular and systemic infections in humans.

Replication of human cytomegalovirus in severe combined immunodeficient mice implanted with human retinal tissue.

Because human cytomegalovirus (HCMV) infection and replication are limited to human cells, few animal models can be used to specifically examine the biology of HCMV in vivo. In these studies, fetal human retinal tissue was implanted into the anterior chamber of the severe combined immunodeficient (SCID) mouse eye and subsequently was inoculated with HCMV. Viral replication, localized to glial cells in the xenografts, was first detected 7 days after infection. Thereafter, HCMV replication increased to peak levels through days 21-28 and then gradually decreased to undetectable levels by 8 weeks after infection. The clinical isolate Toledo replicated to higher titers than did strain AD169 or Towne. A comparison of implant age indicated that older tissue could support higher levels of HCMV replication than could younger implants. SCID mice implanted with human retinal tissue provide an excellent model for evaluation of HCMV infection of an ocular structure in vivo.

Synthesis and enantioselectivity of the antiviral effects of (R,Z)-,(S,Z)-methylenecyclopropane analogues of purine nucleosides and phosphoralaninate prodrugs: influence of heterocyclic base, type of virus and host cells.

A series of R and S enantiomers of 2-aminopurine methylenecyclopropane analogues of nucleosides was synthesized. Two diastereoisomeric lipophilic phosphate prodrugs derived from R and S enantiomers of 2,6-diaminopurine analogue were also prepared. Enantioselectivity (diastereoselectivity in case of prodrugs) of in vitro antiviral effects was investigated with human and murine cytomegalovirus (HCMV and MCMV, respectively), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV). Strong differences in enantioselectivity were found between the R and S enantiomers of adenine analogue and enantiomeric 2-aminopurine analogues. Thus, the enantiomers of adenine analogue were equipotent against HCMV but not MCMV, where the S enantiomer is strongly preferred. The same S preference was found throughout the 2-aminopurine series for both HCMV and MCMV. In contrast, R-synadenol in HIV-1 assays was the best agent, whereas the S enantiomers of moderately effective 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine analogues were preferred. Little enantiomeric preference was found for R and S enantiomers of synadenol and the corresponding enantiomers of 2,6-diaminopurine analogue against HBV. A mixed pattern of enantioselectivity was observed for EBV depending on the type of host cells and assay. Against VZV, the R and S enantiomers of adenine analogue were equipotent or almost equipotent, but throughout the series of 2-aminopurine analogues a distinct preference for the S enantiomers was found. The stereoselectivity pattern of both diastereoisomeric prodrugs mostly followed enantioselectivity of the parent analogues. The varying enantioselectivities in the series of purine methylenecyclopropane analogues are probably a consequence of differences in the mechanisms of action in different virus/host cell systems.

In vitro activities of methylenecyclopropane analogues of nucleosides and their phosphoralaninate prodrugs against cytomegalovirus and other herpesvirus infections.

Human cytomegalovirus (HCMV) infection does not generally cause problems in the immunocompetent adult but can result in severe clinical disease in the fetus, neonate, and immunocompromised host. Ganciclovir (GCV), the agent currently used to treat most HCMV infections, has resulted in much therapeutic success; however, efficacy remains suboptimal. Therefore, there is still a need to develop new compounds for use against HCMV infections. In the present study, several Z- and E-series methylenecyclopropane analogues and their phosphoroalaninate prodrugs were tested initially for activity against HCMV, strain AD169, and murine cytomegalovirus (MCMV) in vitro. Many were found to exhibit efficacy comparable to that of GCV against HCMV in plaque assays and were active against MCMV as well. The compounds were also tested for efficacy against herpes simplex virus types 1 and 2, varicella-zoster virus, and Epstein-Barr virus, and some had levels of activity that were comparable to that of acyclovir. In addition, the compounds synguanol (QYL-438) and 2-amino-6-cyclopropylamino analogue (QYL-769) were chosen for further evaluation and were found to be effective against additional laboratory and clinical isolates of HCMV and GCV-resistant isolates. QYL-438 and QYL-769 were found to be nontoxic in human and mouse fibroblasts and were considerably less toxic than GCV in granulocyte macrophage CFUs and erythroid burst-forming units. These results provide evidence for the high activity of some of these methylenecyclopropane analogues against various herpesviruses, particularly HCMV, in tissue culture and suggest that further evaluation is warranted to determine their potential for use in future clinical studies.

Effective treatment of murine cytomegalovirus infections with methylenecyclopropane analogues of nucleosides.

A number of new nucleoside analogues with a Z- or E-methylenecyclopropane structure exhibited significant activity against human and murine cytomegaloviruses (HCMV, MCMV) in tissue culture that was generally comparable to, or greater than, 9-[(1-3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, GCV). Several of these analogues were chosen for further evaluation of therapeutic efficacy utilizing a MCMV infection. Intraperitoneal (i.p.) inoculation of 3-week-old Balb/c mice with 2.0 x 10(5) plaque forming units (pfu) of MCMV results in an acute, lethal infection with rapid virus replication in visceral and glandular tissue, thus, making it an ideal model for identifying compounds that have potential for use in humans. Synadenol (QYL-284A) and synguanol (QYL-438) were administered i.p. once daily for 5 days initiated 6, 24, or 48 h post-viral infection. Significant protection was demonstrated at 50 and 16.7 mg/kg compared to placebo, with efficacy comparable to GCV. When delivered orally once or twice daily at 100 mg/kg per day, QYL-438 was active, but less effective than GCV. In addition, 2-amino-6-methoxypurine analogue (QYL-941) was active at 60 mg/kg administered orally twice daily, comparable to GCV, while it's prodrug (QYL-972) was as effective as GCV at 40 mg/kg when delivered twice daily for 5 days. Additionally, analogue 2-amino-6-cyclopropylaminopurine (QYL-769) was found to be highly efficacious when given orally twice daily for 5 days. Mortality of 0% and 13% was observed at 60 and 20 mg/kg, respectively, which was similar to GCV. Oral treatment with QYL-769 or GCV reduced virus replication in target organs, but neither resulted in complete clearance of MCMV. These data indicate that these new analogues have activity comparable to GCV when given orally to mice and should be evaluated further to assess their potential for use in humans.