Deazaflavin Inhibitors of Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Specific for the Human Enzyme and Active against Cellular TDP2

Abstract

Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively inhibit the human TDP2 enzyme in a competitive manner both with recombinant and native TDP2. We show that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to the drugs and that key protein residues are responsible for drug resistance. Among them, human residues L313 and T296 confer high resistance when mutated to their mouse counterparts. Moreover, deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in human prostate cancer DU145 cells and TDP2-dependent synergy in TK6 human lymphoblast and avian DT40 cells. Deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors.

Discovery of selective inhibitors of tyrosyl-DNA phosphodiesterase 2 by targeting the enzyme DNA-binding cleft

“…NSC114532 and NSC3198 both inhibited zTDP2 with IC 50 values of 15 and 20 µM, respectively but interestingly, mTDP2 was totally resistant to both compounds up to 111 µM (Figure 3 and Table 1). These results suggest that the TDP2 binding site for both NSC114532 and NSC3198 is not conserved in the mouse enzyme similarly to what was recently observed for the first reported selective TDP2 inhibitor Compound 1 (Figure 2) 22 .…”

“…Concentrations of compounds are 0.5, 1.4, 4.1, 12.3, 37 and 111 µM. Concentrations of the positive control Compound 1 22 are 0.005, 0.017, 0.05, 0.15, 0.46, 1.4 µM.…”

Long-term hepatitis B infection in a scalable hepatic co-culture system

“…To test the effect of pharmacologic inhibition on HBV infection, we utilized two small molecules (JK-3-121 and SV-F-153, Supplementary Fig. 7a) previously shown to efficiently suppress the activity of human TDP2 with high selectivity 40 . To corroborate that JK-3-121 and SV-F-153 indeed had an inhibitory effect on this enzyme, we produced recombinant human TDP2 in E. coli (Supplementary Fig.…”

“…Following established protocols, we obtained high yields of hTDP2, which was purified by nickel affinity column size exclusion chromatography (Supplementary Fig. 7c) 40 . hTDP2 has Mg 2+ -dependent activity on 5′-phosphotyrosylated (5′-Y) termini of single-stranded DNA or on duplex substrates with 5′ overhangs of one to four nucleotides and is thought to be involved in the removal of the viral polymerase from HBV rcDNA (Supplementary Fig.…”

ZATT (ZNF451)–mediated resolution of topoisomerase 2 DNA-protein cross-links

“…In this context, TDP2 modulates cellular (7, 10) and organismal (12) survival following TOP2 targeting anticancer drug treatments, and TDP2 inhibitors hold promise for chemotherapy (13, 14). A critical question in TOP2 biology is how TDP2 accesses the TOP2-DNA phosphotyrosyl chemical bond which is protected within the TOP2 protein shell (15, 16) (Fig.…”