Thiazolo[4,5-d]pyrimidines. Part II. Synthesis and anti-human cytomegalovirus activity in vitro of certain acyclonucleosides and acyclonucleotides derived from the guanine analogue 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione.

The synthesis and in vitro antiviral activity of certain hydroxyalkoxymethyl, hydroxyalkyl, hydroxyalkenyl and phosphonoalkenyl derivatives of the guanine congener 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione are reported. The compounds of this study were selected for their structural similarity to acyclonucleosides with known anti-herpesvirus activity. 5-Amino-3-[(Z)-4-hydroxy-2-buten-1-yl]thiazolo[4,5-d]pyrimidine-2, 7(3H,6H)- dione was the only member of the series to display significant in vitro activity against human cytomegalovirus (HCMV); however, this compound did not inhibit other herpesviruses, human immunodeficiency virus type 1 or murine cytomegalovirus. It was found to have a cytotoxicity profile similar to that of ganciclovir (DHPG). The antiviral effect was found to be sensitive to the initial viral input and the time of addition during the virus replication cycle. Significantly, the compound was found to have equal anti-HCMV activity, against standard virus strains, to DHPG, but also showed potent activity against DHPG-resistant virus strains, except for a strain mutated in the UL97 (phosphotransferase) gene.

Modified antisense oligonucleotides directed against tumor necrosis factor receptor type I inhibit tumor necrosis factor alpha-mediated functions.

Tumor necrosis factor alpha (TNF alpha), a polypeptide produced by activated macrophages, is a highly pleiotropic cytokine which elicits inflammatory and immunological reactions. The binding of TNF alpha to tumor necrosis factor receptor type I (TNFRI) is considered the initial step responsible for some of the multiple biological functions mediated by TNF alpha. The role of TNF alpha as an inflammatory mediator through human TNFRI makes TNFRI an attractive target for intervention in both acute and chronic inflammatory diseases. In this study, we have identified partial phosphorothioate oligodeoxyribonucleotides (ODNs) containing C-5 propynyl or hexynyl derivatives of 2'-deoxyuridine which specifically inhibited TNFRI and subsequently inhibited the functions of TNF alpha mediated through TNFRI. The most active ODNs were directed against the 3'-poly adenylation signal site on the TNFRI mRNA, and in a cellular assay, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations, in the presence of Cellfectin. The inhibition of gene expression correlated with the binding affinity of the ODN for the target mRNA. The ODNs lowered TNFRI protein levels and TNF alpha-mediated functions by specifically reducing levels of TNFRI mRNA. These anti-TNFRI ODNs offer a novel approach for controlling biological functions of TNF alpha and may be useful as human therapeutic agents for treating diseases in which TNF alpha has been implicated.

Inhibition of herpes simplex virus in culture by oligonucleotides composed entirely of deoxyguanosine and thymidine.

Oligodeoxynucleotides (ODNs) composed entirely of deoxyguanosine and thymidine, but not specifically designed to act as antisense agents, were able to significantly inhibit herpes simplex virus growth in acute infection assay systems. The guanosine/thymidine (GT) ODNs which demonstrated this antiviral activity contained either natural phosphodiester (PO) or phosphorothioate (PS) modified internucleoside linkages. In all experiments, the antiviral activity of the ODNs was enhanced when the backbone was modified to contain the PS linkages. When present during the time of virus addition, the ODNs were able to block the adsorption of virus to Vero cells. In this assay the PS-containing ODNs had ID50 values of approximately 0.020 microM for HSV-2 and of 0.3 microM for HSV-1. When these same PS-containing ODNs were used against HSV-2 in single-cycle viral yield assays, designed to minimize the effects due to external blockage of virus, the ID50 values rose to 0.2 microM. Analysis of viral DNA obtained 14 h post-HSV-2 infections in the single-cycle assay, revealed a decrease in replicated viral DNA in cells treated with PS-ODNs. Analysis of viral mRNA obtained 4 h post-HSV-2 infection revealed, in cells treated with the PS-ODNs, a decrease in measurable HSV-2 alpha- and beta-mRNAs. Although the mechanism of action of the antiviral activity (beyond adsorption blocking) is not fully understood, the toxicity of these compounds was low, giving high therapeutic indices for the GT-rich PS-ODNs. The good therapeutic index of GT-ODNs make this a class of compounds which warrant investigation as therapeutic agents to be used against herpes viruses.

Induction of carcinoembryonic antigen and its crossreactive gene products in human colonic adenomas and carcinomas by transforming growth factor-beta.

The inducing effect of transforming growth factor-beta (TGF-B) on carcinoembryonic antigen (CEA) secretion and the cellular expression of CEA and CEA crossreactive glycoproteins (CEA-GLY) was examined from a panel of human colonic cell lines with different phenotypic classification. This panel included carcinomas with dissimilar differentiation characteristics and metastatic behavior, and premalignant adenomas derived from colonic polyps. A great degree of heterogeneity was observed in the endogenous levels of CEA secretion and the cellular expression of CEA and CEA-GLY species. The response profiles of the different cell lines to TGF-B treatment were also found to be heterogenous. However, TGF-B was able to induce CEA secretion and up-modulated the cellular expression of CEA and CEA-GLY from a majority of the cells tested. More importantly, TGF-B was able to exert these effects on carcinoma cells that secrete or express minimal or nondetectable amounts of these glycoproteins. These biologic modifying effects of TGF-B may have potential in augmenting the efficacy of CEA as a colon cancer marker, and in antibody-directed radioimaging and therapeutics. Further investigation in vivo in an experimental animal model system is warranted.