Validating TDP1 as an Inhibition Target for Lipophilic Nucleoside Derivative in Human Cells

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important DNA repair enzyme and its functioning is considered as one of the possible reasons for tumor resistance to topoisomerase 1 (TOP1) poisons such as topotecan. Thus, TDP1 inhibitors in combination with topotecan may improve the effectiveness of anticancer therapy. TDP1 acts somehow in a phospholipase manner, depleting the phosphodiester bond between lipophilic tyrosine residue and 3' end of DNA; therefore, lipophilic molecules bearing aromatic substituents can interact with TDP1 and even possess high inhibitory activity, which is evidenced by data from the literature. Previously, we identified lipophilic nucleoside derivative (compound <b>6d</b>, IC₅₀ = 0.82 µM) as an effective inhibitor of the purified enzyme TDP1 that enhances the cytotoxic, DNA-damaging, and antitumor effects of topotecan. However, the role of TDP1 inhibition in this synergistic effect remained not fully understood. In the present study, we have tested the hypothesis of a TDP1-dependent mechanism of action for compound <b>6d</b>, showing that it sensitizes wild-type A549 lung cancer cells, but not TDP1 knockout cells, to the cytotoxic effects of topotecan. The sensitizing effect was absent in non-cancerous HEK293A cells regardless of TDP1 status. Additionally, we analyzed the effect of compound <b>6d</b> and topotecan on the expression level of <i>TOP1</i> and <i>TDP1</i> to determine whether the observed synergy was due to direct TDP1 inhibition and/or changes in regulation of these enzymes. The data obtained shows that compound <b>6d</b> did not affect <i>TDP1</i> gene expression level in HEK293A and A549 WT cells. Thus, compound <b>6d</b> most probably does not suppress the transcription or mRNA stability of TDP1, and the synergistic action of <b>6d</b> with topotecan is related to TDP1 inhibtion.