Insights into the action mechanism of azacyanines: Their topoisomerase iiα inhibition potential and nucleic acid selectivity

Thesis Type: Postgraduate

Institution Of The Thesis: Middle East Technical University, Graduate School of Natural and Applied Sciences, Biochemistry, Turkey

Approval Date: 2018

Thesis Language: English


Supervisor: Özgül Persil Çetinkol


Topoisomerase II alpha (Topo IIα) is one of the essential enzymes in cell viability. It is required for cell cycle progression due to its role in regulating the topological constraints during DNA replication and transcription. Due to its role, Topo IIα became one of the extensively exploited targets in chemotherapy, and there is an on-going research in design of small molecules targeting topoisomerase’s catalytic activity at different stages using different mechanisms. Within the scope of this thesis first, the effects of five azacyanine derivatives on Topoisomerase IIα enzyme (Topo IIα) at molecular level have been assessed by using an in-vitro Topoisomerase Drug Screening kit. Three of the azacyanines (Azamethyl, Azaethyl and Azaisobutyl) used were benzimidazole derivatives differing from each other in terms of the alkyl chain length on the benzimidazole ring and two of them were benzothiazoles (Aza4 and Aza5). Second, the affinity and selectivity of these compounds towards different nucleic acid sequences and structures have been investigated by using competition dialysis method. Our primary goal was to accentuate azacyanines as probable Topo IIα inhibitors, and provide some mechanistic explanations for their effects. Our results revealed that three of the five azacyanine molecules investigated in here decreases the catalytic activity of Topo IIα. Detailed investigation of the effect of Azamethyl on Topo IIα revealed that the effect on Topo IIα was concentration dependent, such that the higher Azamethyl concentrations led to stronger effect as determined by the intensity of the DNA bands in ethidium bromide (EtBr) stained agarose gels. Moreover, when used at the same concentrations (50.0 µM), Azamethyl showed stronger effect than Etoposide which is a well-known Topo IIα inhibitor being used in chemotherapy. Even better, the efficiency of 50.0 μM Azamethyl was higher than 500.0 μM Etoposide. To shed light onto the mechanism of Topo IIα catalytic activity change, we further investigated the affinity and selectivity of azacyanines towards different nucleic acid sequences and structures via competition dialysis. We included three more azacyanines (Azapropyl, Azabutyl and Azaisopropyl) in our competition dialysis setup to understand the effect of the chain length and branching on the benzimidazole ring in nucleic acid binding affinity and selectivity. Our results revealed that the azacyanines were highly selective towards triple helical nucleic acid structure poly(dA).[poly(dT)]2. More importantly, their affinity and selectivity towards poly(dA).[poly(dT)]2 was decreasing with the increasing linear alkyl chain length, and increasing branching. The order of affinity was Azamethyl > Azaethyl > Azapropyl > Azabutyl > Azaisobutyl > Azaisopropyl. Altogether, the findings presented in here demonstrate the potential of azacyanines to inhibit Topo IIα’s catalytic activity.