Introduction: Nucleoside analogues have a special place among the most effective antiviral drugs and the study of fluorinated nucleoside sugars has led to the development of novel promising chemotherapeutic agents. Our work is dedicated to the determination of cytotoxicity and antiviral activity towards HSV-1, HSV-2 and HAdV5 of such modified nucleosides.
Methodology: Cells and viruses: cell culture MDBK (bovine kidney), adenovirus serotype 5 (HAdV5), herpes simplex virus 1 and 2 (HSV-1/US, HSV-2/BH) were used.
Tested substances: analogs of nucleoside based on 5-tosyl-6-(trifluoromethyl)uracile (G26), namely 2-N-substituted-4-tosyl-5-(perfluoroalkyl)-1,2,3-triazoles (G8 and G9); polyfluoro-substituted thiopeptide analogs, namely t-butyl(tetrafluoropropanethioyl)-L-alaninate (10S-20), methyl(tetrafluoropropanethioyl)-L-phenylalaninate (10S-21), methyl(tetrafluoropropanethioyl)-D-tryptophanate (10S-22), sodium (polyfluoroalkanethioyl)-L-phenylalaninate (10S-23 and 10S-24).
Cytotoxicity of the compounds was determined by MTT-test or neutral red dye (NR) according to standard protocols. Antiviral effect was determined using PCR, MTT-test and cytomorphological methods. This method was used to identify adenovirus infected cells containing specific virus inclusion. Cells were treated with the compounds in the growth medium after virus adsorption at non-toxic concentrations.
Results: According to the MTT test results, CC50 values of compounds G8 and G9 were 887 and 990 mg/ml, respectively. Other compounds demonstrated the following CC50 values, G26: 532 mg/ml (MTT) and 985 mg/ml (NR); 10S-22: 290 mg/ml (MTT) and 554 mg/ml (NR). Compound 10S-23 showed CC50 value of >1000 mg/ml (MTT, NR). CC50 values obtained using NR were higher than the previous results for the compounds 10S-20, 10S-21, 10S-24 (2022, 1585, and 2519 mg/ml, respectively). CC50 values assessed via MTT test for these compounds were lower – 653, 394 and >1000 mg/ml, respectively. Inhibition of HAdV5 reproduction was demonstrated for three compounds G8, G26 and 10S-23. PCR revealed inhibition of virus reproduction (13 %) for compound G8 at a concentration 62 mg/ml, 95-100 % for G26 at concentrations 31-62 mg/ml and 31 % for 10S-23 at a concentration of 36 mg/ml. The results of cytomorphologycal method showed the following values of EC50 for the compounds: 16, 120, 90 mg/ml, respectively. Using MTT-test it was shown that compounds G8, G9, 10S-20, 10S-23 and 10S-24 suppressed HSV-1/US reproduction by 50 % at concentrations of 50, 59, 62, 71, 35 mg/ml, respectively. Compounds 10S-21 and 10S-22 had lower efficiency and suppressed virus reproduction by 37% and 22%, respectively. Compounds G8, G9, G26 suppressed HSV-2/BH reproduction by 50% at the concentrations of 56, 71 and 44 mg/ml, respectively. 10S-23 suppressed only 12% of the virus at the concentration of 62 mg/ml. Therefore, two compounds G8 and 10S-23 inhibited the reproduction of all viruses with different efficiency. The compound G9 was active only in relation to both types of herpes. G26 suppressed reproduction of HSV-2/BH and HAdV5, whereas compounds 10S-20 and 10S-24 were active only in relation to reproduction HSV-1/US.
Conclusion: The tested compounds emerged as promising hits for further in-depth in vitro and in vivo studies
