Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIα

Abstract

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme/DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to that of etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme/DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-terminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: (1) interfacial poison and (2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA.

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

“…As a poison, it stabilises TopoII:DNA complexes, whereas as an inhibitor ETP interacts with the catalytic site of TopoII, decreasing the number of active cleavage complexes [307]. ETP acts as a poison by stabilizing the cleavage complex of TopoII via decoupling the key catalytic residues, thus preventing the religation of cleaved DNA ends [308].…”

“…100× more efficient at inhibiting TopoII than ETP. ETP-quinone can block binding of the enzyme to DNA by stabilisation of the N-terminal clamp [307]. In cases where the enzyme still binds to DNA, the metabolite can stabilise the enzyme:DNA complex by inhibiting the religation step thus leading to higher levels of DSBs [307].…”

“…ETP-quinone can block binding of the enzyme to DNA by stabilisation of the N-terminal clamp [307]. In cases where the enzyme still binds to DNA, the metabolite can stabilise the enzyme:DNA complex by inhibiting the religation step thus leading to higher levels of DSBs [307]. The ETP-catechol metabolite works similarly to the parent compound but can also be oxidised to the quinone [311].…”

Targeting DNA Replication Stress and DNA Double-Strand Break Repair for Optimizing SCLC Treatment

“…Mechanistically, TOP2 cleaves double-stranded DNA to allow passage of an intact DNA duplex 150 through the TOP2-linked DSB [56]. Etoposide, a TOP2-specific inhibitor, stabilizes TOP2 cleavage 151 complexes, thus inducing DSBs and subsequent cell death if not repaired [57]. Besides, TOP1 reduces 152 DNA torsional stress created by twisting and supercoiling during DNA replication progression.…”

Infusão transdérmica de fármaco no tratamento do melanoma murino B16F10

“…Os efeitos celulares e moleculares do etoposídeo resultam da sua complexação com a enzima topoisomerase II (Deweese et al, 2009). Ao impedir que essa enzima religue as moléculas de ácido nucleico clivadas, o fármaco induz quebras nas fitas do DNA, resultando na ativação das vias ATM (Ataxia telangiectasia mutated) e ATR (ATM-RAD3-related gene), envolvidas nos mecanismos de reparo do DNA (Wu et al, 2011;Gibson et al, 2016;Vesela et al, 2017). Ocorre então paralização do ciclo celular entre as fases S e G2/M, havendo remodelação da cromatina e bloqueio da replicação do DNA, com subsequente indução da morte celular por apoptose, que é desencadeada principalmente devido à ativação da via intrínseca, mas também através da sinalização mediada pelo receptor de morte CD95 (Zhao et al, 2012;Vesela et al, 2017 A radiofrequência fracionada cria uma matriz de microcanais de pequeno porte na pele (Kam et al, 2012).…”