ABSTRACT The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(β- d -ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(β- l -ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs. We found that the erythrofuranosyl analog and the α-lyxofuranosyl analog acted late in the viral replication cycle, similar to BDCRB. In marked contrast, the α-5′-deoxylyxofuranosyl analog of 2,5,6-trichloro-1-(β- d -ribofuranosyl)benzimidazole (compound UMJD1311) acted early in the replication cycle, too early to be consistent with either mechanism. Similar to other reports on early acting inhibitors of herpesviruses, compound 1311 was multiplicity of infection dependent, an observation that could not be reproduced with UV-inactivated virus. HCMV isolates resistant to BDCRB and maribavir were sensitive to compound 1311, as were viruses resistant to ganciclovir, cidofovir, and foscarnet. The preincubation of host cells with compound 1311 and removal prior to the addition of HCMV did not produce an antiviral cellular response. We conclude that this newly discovered early mode of action occurs at a stage of viral replication after entry to cells but prior to viral DNA synthesis, thereby strongly suggesting that the trisubstituted benzimidazole nucleoside series possesses three distinct biochemical modes of action for inhibition of HCMV replication.

This study describes the extent of cross-resistance and interactions for selected inhibitors of human cytomegalovirus (HCMV) DNA synthesis and DNA processing. HCMV isolates resistant to the benzimidazole d-ribonucleoside viral DNA processing inhibitors TCRB and BDCRB were sensitive to BAY 38-4766, a non-nucleoside inhibitor of viral DNA processing. This indicates that these two drug types have distinct interactions with the products of HCMV genes UL56 and UL89 required for viral DNA cleavage and packaging. These virus isolates also were sensitive to ganciclovir (GCV) but slightly resistant to the l-benzimidazole ribonucleoside viral DNA synthesis inhibitor 1263W94. Virus resistant to 1263W94 remained sensitive to BDCRB, GCV, and BAY 38-4766. Examination of drug–drug interactions in cell culture assays measuring inhibition of HCMV replication revealed strong synergism for the combination of BDCRB with 1263W94, and for combinations of 1263W94 with cidofovir (CDV) and foscarnet (PFA), but not with GCV. Combinations of GCV with CDV and PFA were synergistic as well. The combination of GCV with 1263W94 showed additive antiviral interactions, whereas, a combination of BAY 38-4766 with GCV showed antagonism. Interaction of BDCRB with BAY 38-4766 showed a mixed pattern of synergy and antagonism. The antiviral synergy observed between GCV and PFA or CDV serves to validate clinical combination therapies for these drugs. Antagonism seen for BAY 38-4766 with GCV indicates that these two drugs are unlikely to be useful for combination therapies. Notably, 1263W94 demonstrated greater synergy in combination with PFA or CDV than did GCV, suggesting some promise for this benzimidazole l-riboside in such combination therapies.

ABSTRACTWe have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC50; 14 μM) similar to that of roscovitine; it was competitive with respect to ATP (Ki= 14 μM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC50s, 32 to 100 μM) but not its antiviral efficacy (IC50s, 0.02 to 0.3 μM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC50, 38 μM) and HCMV (IC50, 40 μM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.

Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys(18) (CWK(18)) resulting in the formation of nonreducible (PEG-VS-CWK(18)) and reducible (PEG-SS-CWK(18)) PEG-peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by (1)H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK(18), resulting in DNA condensates with a mean diameter of 80-90 nm and zeta (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0. 05-2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK(18) DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK(18) DNA condensates was over two orders of magnitude lower than PEG-SS-CWK(18) DNA condensates and three orders of magnitude lower than AlkCWK(18) DNA condensates. PEG-VS-CWK(18) efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.

An unusual lectin possessing two distinctly different types of carbohydrate combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha 1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Gal beta 1,4GlcNAc-) or lacto-N-biose (i.e. Gal beta 1,3GlcNAc-) groups at its three nonreducing termini.

Two glycopeptides were synthesized by attaching purified glycosylamines (N-glycans) to a 20 amino acid peptide. Triantennary and Man9 Boc-tyrosinamide N-glycans were treated with trifluoroacetic acid to remove the Boc group and expose a tyrosinamide amine. The amine group was coupled with iodoacetic acid to produce N-iodoacetyl-oligosaccharides. These were reacted with the sulfhydryl group of a cysteine-containing peptide (CWK18), resulting in the formation of glycopeptides in good yield that were characterized by 1H NMR and ESIMS. Both glycopeptides were able to bind to plasmid DNA and form DNA condensates of approximately 110 nm mean diameter with zeta potential of +31 mV. The resulting homogeneous glycopeptide DNA condensates will be valuable as receptor-mediated gene-delivery agents.

The N-linked oligosaccharide from soy bean agglutinin (Man9) was isolated on a preparative scale following derivatization with Boc-tyrosine. The procedure utilized preparative hydrazinolysis to release the oligosaccharide and yielded multi-micromol quantities of Boc-tyrosine-Man9 which was characterized by 1H NMR and ES-MS.