The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication.

Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC50) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.

Reversal of methylmercaptopurine ribonucleoside cytotoxicity by purine ribonucleosides and adenine.

6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.

Cytological effects of sulfur and selenium purine analogues on two transplantable hepatomas and on normal renewing cells in mice.

The effects of 50% lethal doses of three purine analogues, 6-methylmercaptopurine riboside ( MMPR ), 6-thioguanosine (TGR), and 6- selenoguanosine ( SeGR ), on mitotic activity in a slow-growing ( SS1H ) and a fast-growing ( BH3 ) transplantable hepatocellular adenoma in C3H/ StW and BUB mice, respectively, were analyzed statistically. No significant difference in response was found between the two benign hepatomas. MMPR alone effectively reduced mitotic activity in the tumors and did so as efficiently on the first day of treatment as on subsequent days of daily i.p. administrations for up to 10 days. TGR alone and SeGR alone were ineffective in reducing the mitotic index significantly below that of controls. When either TGR or SeGR was injected simultaneously with MMPR , the effect on tumor mitosis resembled that of MMPR alone. The reactions of normal cells of the hosts to these agents were analyzed quantitatively in duodenal epithelium with respect to mitotic activity and to the number of cells present in the crypts. Differences between the two strains of mice were small and, for the most part, not significant. MMPR produced a slight but not significant reduction in duodenal mitotic activity and cell number. TGR alone induced significant decreases in both after 3 and 5 days of treatment. SeGR alone had no effect on the duodena . The effects of a combination of SeGR with MMPR on the duodena differed only slightly from MMPR or SeGR alone, but TGR plus MMPR produced greater inhibition of mitosis than did either administered alone. Our results suggest that MMPR may be a promising chemotherapeutic agent against some types of solid hepatocellular tumors in vivo because it can inhibit mitosis in these tumors effectively, rapidly, and continuously, while its inhibitory effect on normal replicating cells of the host intestine occurs more slowly and only with long-sustained treatment.

Interaction among the distinct effects of adenine and guanine depletion in mouse lymphoma cells.

Toxic guanine depletion was shown previously to result in a dramatic reduction of DNA synthesis, while toxic adenine depletion failed to affect DNA synthesis (M. B. Cohen and W. Sadée, Cancer Res., 43: 1587-1591, 1983). In this study, relative DNA synthesis rates were measured in mouse lymphoma S49 cells over 24 hr after drug exposure and were compared to cell growth curves. DNA synthesis inhibition by mycophenolic acid (guanine starvation) was achieved at lower drug concentrations than was the inhibition of cell growth. This result further supports the hypothesis (reference above) that guanine starvation specifically affects cells in S phase while it allows cells with full DNA complement to divide. In contrast, L-alanosine (adenine starvation) failed to affect DNA synthesis for at least 24 hr at a concentration that inhibits cell growth by 80%. The dramatically different effects of guanine and adenine starvation on DNA synthesis can thus be used to assess the magnitude of each when blocking early de novo purine biosynthesis by 6-methyl-mercaptopurine ribonucleoside (6- MMPR ). The results suggest that, although 6- MMPR effects primarily resemble those of guanine depletion, adenine starvation measurably contributes to the overall toxicity of 6- MMPR . Drug combination experiments with L-alanosine, mycophenolic acid, and 6- MMPR suggest that the basic mechanisms underlying the toxic effects of guanine and adenine starvation act synergistically.

Relationship between phosphorylation and cytotoxicity of 2-chloroadenosine and 6-methylmercaptopurine riboside in human cells.

2-Chloroadenosine but not 6-methylmercaptopurine riboside was phosphorylated by adenosine kinase negative human B cells, and the adenosine negative cells were resistant to 6-methylmercaptopurine riboside but not to 2-chloroadenosine. Phosphorylation of 6-methylmercaptopurine was totally dependent on adenosine kinase, but 2-chloroadenosine seemed to be phosphorylated by other enzyme(s) as well. The cytotoxicity of both of these analogs depends on the phosphorylation.