Intracellular phosphorylation of carbocyclic 3-deazaadenosine, an anti-Ebola virus agent.

摘要

Carbocyclic 3-deazaadenosine (C-c3Ado) is a potent inhibitor of Ebola virus in mice by infrequent dosing, even though its half life in plasma is only 23-28 min. This prompted studies to determine whether C-c3Ado undergoes intracellular metabolism to derivatives that may promote in vivo activity. In cells, radiolabelled compound readily underwent metabolism to monophosphate, diphosphate and triphosphate (C-c3ATP) forms, with C-c3ATP being the major metabolite detected. A non-polar metabolite was also detected both inside and outside treated cells. The retention time of C-c3ATP was similar but not identical to ATP on a strong anion exchange high performance liquid chromatography (HPLC) column or on a DEAE-Sephadex open column. C-c3ATP and ATP were susceptible to degradation to their respective nucleosides by bovine alkaline phosphatase. Intracellular formation of C-c3ATP reached a plateau by about 4 h after treatment of monkey (Vero 76) and mouse (Balb/3T3 clone A31) cells with 10 or 100 microM extracellular compound. Phosphorylation was linearly dose responsive at 1, 3 and 10 microM. However, the extent of phosphorylation decreased with increasingly higher concentrations (30, 100 and 300 microM). When compound was removed from the medium, the nucleoside cleared the cells within 1 min, whereas C-c3ATP had a half life of decay of 2-3 h in five cell lines. Phosphorylation of C-c3Ado to C-c3ATP was not inhibited by cotreatment of cells (at a 20:1 ratio) with adenosine, guanosine, inosine, xanthosine, cytidine or uridine. There was no evidence of incorporation of C-c3Ado (10 microM) into macromolecules of cells over 72 h, whereas adenosine was readily incorporated. C-c3ATP may represent a form of C-c3Ado that might contribute to extending its intracellular half life or otherwise exhibit antiviral activity and/or toxicity.