The Intracellular Formation of a Mononucleotide of the Anti-Influenza Agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896)

Abstract

LY217896 is a substituted thiadiazole compound with anti-influenza activity in vitro and in the mouse model of infection. LY297336 is a ribosylated (N-4) derivative of LY217896. A highly polar intracellular metabolite of LY217896 was isolated by HPLC, and mass spectral analysis and treatment of the metabolite with alkaline phosphatase showed that it was a monophosphate (LY307987) derived from LY217896. The formation of LY307987 was inhibited by 43 and 63% when 10 μm of LY217896 was incubated with 100 μM of 8-aminoguanosine (8AGuo) and guanine (Gua), respectively, whereas inosine (Ino) and hypoxanthine (Hx) had no effect on the formation of LY307987. LY217896 inhibited the incorporation of [14C]-Hx into nucleic acids in cells which metabolize LY217896; however, LY217896 did not inhibit the formation of inosine 5′-monophosphate (IMP) from Hx in a cell-free HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-catalysed reaction. Incubation of MDCK cells with 10 μm of LY217896 resulted in an 8-fold increase in the level of intracellular IMP. At 100 μm, neither LY217896 nor LY297336 inhibited inosine 5′-monophosphate dehydrogenase (IMPDH) and only cellular extracts which contained intracellular metabolites of LY217896 inhibited IMPDH. Quantification of the 5-phosphorylribose pyrophosphate (PRPP) levels in BS-C-1, MDCK, and MCN cells showed a positive correlation between PRPP concentration and cellular metabolism of LY217896. Combination studies of LY217896 with 2′,3′-dideoxyinosine (ddlno) or 2′,3′-dideoxyguanosine (ddGuo) showed that LY217896 enhanced the antiretroviral activities of these dideoxynucleosides, which is consistent with an inhibitory effect on IMPDH.