Susceptibilities of Human Cytomegalovirus Clinical Isolates and Other Herpesviruses to New Acetylated,Tetrahalogenated Benzimidazole d-Ribonucleosides

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl) benzimidazole (Cl₄RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl₄RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl₄RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV- and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCV-sensitive clinical isolates and slightly active against other herpesviruses.Human cytomegalovirus (HCMV) is one of eight human herpesviruses and is a serious, life-threatening, opportunistic pathogen in immunocompromised patients (organ recipients or AIDS patients) (8, 19). HCMV is widespread, with a seroprevalence throughout the world of up to 100% in adults. To date, nearly all anti-HCMV drugs for systemic treatment are inhibitors of the viral DNA polymerase (2, 13, 21, 22). Due to the low bioavailability, number of side effects, dose-dependent toxicity, and appearance of resistances caused by the current available drugs, development of new antiviral compounds which have a different mode of action is needed. Consequently, to broaden therapy of HCMV infections and to circumvent current mechanisms of drug resistance, an inhibitor of HCMV terminase would be of great value, because it would act subsequently to DNA synthesis and block the first steps in viral maturation.Viral replication includes cleavage of newly synthesized, concatemeric DNA into unit-length genomes and packaging into preformed procaspids. These processes occur in or in close proximity to replication centers in the nucleus. Enzymes involved in the packaging process are responsible for duplex nicking and insertion of the DNA into the procapsids (1, 4, 9, 10), the so-called terminases. The HCMV terminase consists of two subunits, one subunit encoding pUL56 and the other pUL89 (5-7); each protein has a different function. Whereas subunit pUL56 mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required for duplex nicking and enhancement of the UL56-associated ATPase activity (15, 23, 24). The hydrolysis of ATP has multiple functions during the packaging process. It is also involved in the formation of the packaging complex. The anti-HCMV benzimidazole d-nucleosides, 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidazole (BDCRB) and 2,5,6-trichloro-1-(β-d-ribofuranosyl) benzimidazole (TCRB), developed in the laboratories of Townsend, Drach, and coworkers (12, 26), target the HCMV terminase (18, 27). However, even though these are excellent inhibitors of HCMV infection in cell culture, BDCRB is not metabolically stable in vivo. To overcome this problem, various analogs have been synthesized by Townsend and coworkers. Recently, by using a new bioluminometric assay, we found the two most active new compounds were 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl) benzimidazole (Cl₄RB) (16). Since DNA packaging is ATP-dependent and the portal protein has no enzymatic activity, the interaction of the terminase subunit pUL56 with the portal protein pUL104 is essential. Therefore, analyses were undertaken to investigate the effects of the compounds on this interaction. By coimmunoprecipitation, we identified a direct interaction between pUL56 and pUL104 that was specifically inhibited by Cl₄RB but not by the virologically inactive control CDMRB or by the other tetrahalogenated benzimidazole BTCRB (11). Furthermore, electron microscopy (EM) demonstrated that the formation of infectious particles was inhibited (16). Since the compounds represent promising antivirals, we performed analysis with ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates and different herpesviruses.     

Verletzungen Gewaltsamer Tod aus physikalischer Ursache

International Journal of Legal Medicine -     

Über Adrenergische und Cholinergische Nerven

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Zwei Studien über Herpes Zoster

Zusammenfassung In den ersten Tagen läßt sich an Herpes Zoster-Kranken eine geringe Blutdrucksenkung feststellen, die mit dem Freiwerden der H.-Substanz in der Peripherie in Zusammenhang gebracht wird. Die spätere Pigmentierung der Haut läßt sich durch Vitamin C-Gaben nicht beeinflussen.