Cellular Metabolism and Anti-Influenza Activity of 1,3,4-Thiadiazol-2-Ylcyanamide (LY217896)

Abstract

LY217896 (1,3,4-thiadiazol-2-ylcyanamide) is a 2-substituted thiadiazole that is an effective inhibitor of influenza A and B viruses in vitro and in the mouse infection model. The in vitro anti-influenza activity of LY217896 is reversed by a 10-fold excess amount of guanine or guanosine. LY217896 (1 or 10μg ml−1) effected a selective 60% decrease in the levels of intracellular pools of GTP in MDCK cells. The extent of cytotoxicity of LY217896 is positively correlated with the amount of LY217896 metabolite formed intracellularly. A cell line, derived from parental MDCK cells, was selected for resistance to 50 ng of LY217896 per ml. Unlike parental MDCK cells, the resistant cells were able to undergo log phase replication in LY217896 (25 g ml−1) and were unable to metabolize the compound. Furthermore, LY217896 had no antiviral activity against influenza A/Ann Arbor (IC50 >200μg ml−1) or vaccinia virus (IC50 = 13 μg ml−1) in resistant cells. In contrast, LY217896 inhibited influenza A/Ann Arbor (IC50 = 0.5 μg ml−1) or vaccinia virus (IC50 = 0.13 μg ml−1) in the parental MDCK cells. A thiadiazole, with a guanidinyl group in the 2 position, and ribavirin were active in both the parental cells and resistant cells. Nicotinamide (up to 240-fold excess) did not reverse the anti-influenza activity of LY217896 in vitro or in the mouse infection model. A 10-fold excess of nicotinamide reversed the cytotoxicity of 2-aminothiadiazole but not that of LY217896.

Approaches and Strategies for the Treatment of Influenza Virus Infections

“…The mechanism of action of ribavirin is due, in part, to its phosphorylation to the monophosphate, which can then inhibit IMPDH (Streeter et al, 1973(Streeter et al, , 1977Wray et al, 1985). Consistent with this is the observation that the antiviral activity of ribavirin can be reversed by excess guanosine (Streeter et al, 1973;Colacino et al, 1993) and by inosine and xanthosine (Streeter et al, 1973). Mycophenolic acid, which is also an inhibitor of inosine monophosphate dehydrogenase, displays potent anti-HIV activity in vitro by limiting the rate of de novo synthesis of guanosine nucleotides, thereby inhibiting HIV reverse transcriptase activity (Ichimura & Levy, 1995).…”

“…One was that influenza virus variants could not be selected in vitro for resistance to LY217896 or its sodium salt (Hayden et al, 1990; JC Tang & JM Colacino, personal communication) suggesting the lack of a virus-specific target. The other was that the antiviral activity could be reversed by adding a molar excess of either guanosine or guanine (Colacino et al, 1993). Previously, a related compound, 2-amino-1,3,4-thiadiazole, was shown to inhibit Sindbis virus replication in vitro (Malinoski & Stollar, 1981) and it was thought that this compound is converted to the mononucleotide form intracellularly, and as such it is a competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) with respect to the natural substrate, IMP (Nelson et al, 1977).…”

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

“…1), is effective in vitro against influenza A and B viruses, respiratory syncytial virus (RSV), and parainfluenza virus type 3 (PIV3), and is therapeutically effective in the mouse model of infection (Colaoino et al, 1990) as well as in cotton rats infected with RSV or PIV3 (Wyde et al, 1990). However, there appeared to be little antiviral selectivity when the anti-influenza IC so (0.7Ilg ml") was compared to the oytotoxic concentration (1-3 Ilg mr') in rapidly dividing uninfected cells (Colacino et aI., 1990;Hayden et al, 1990;Colacino et al, 1993). Recently, the major metabolite of LY217896 was identified ( Fig.…”

“…Previously, it was shown that LY217896 effected a seleotive reduction of GTP pools in MOCK cells but not in LY'MOCK cells (MOCK cells selected for resistance to the toxicity of LY217896), which did not metabolize LY217896 (Colacino et al, 1993). It was proposed that a metabolite of LY217896 inhibits IMPOH resulting ultimately in the reduction of GTP pools (Colacino et al, 1990;.…”

“…While II formed in cell incubations was found mostly in the incubation medium, significant amounts of radioactivity were also found within BS-C-1 cells after incubation with [14C]_LY217896 (Colacino et al, 1993;Ehlhardt et al, 1993). The majority of this intracellular material was identified as II (80-90% of [ 14C]in fractions after gradient SAX analysis; retention time 11-12min in Fig.2a), whereas very little LY217896 « 1%) was found in these cells.…”