Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Artemy D Beniaminov,Mikhail A Livshits,Vladimir A Mitkevich,Olga K Mamaeva,Galina V Chashchina,Dmitry N Kaluzhny,Anna K Shchyolkina,Alexander A Shtil,Anna N Tevyashova,Olga I Kechko

doi:10.3390/ijms21155299

Abstract

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug–DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA–DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

Highlights

Antibiotics of the aureolic acid family include structurally close olivomycin A (OA), chromomycin A3 (Chro) and mithramycin (Mith) [1]
To resolve the inconsistency between the results of electrophoretic mobility gel shift assay (EMSA) and equilibrium studies in solution, we investigated the kinetics of OA–DNA complex formation
Contrary to the isothermal titration calorimetry (ITC) results for another antibiotic, Mithramycin, that supported entropically driven binding to DNA [29], we found the enthalpy driving nature of OA’s interaction with DNA

Summary

Introduction

Antibiotics of the aureolic acid family include structurally close olivomycin A (OA), chromomycin A3 (Chro) and mithramycin (Mith) [1]. These agents have been considered as antitumor drug candidates due to their potent cytotoxicity for cultured cells [1,2] Binding of these compounds to GC-rich DNA stretches and, in particular, the prevention of binding of the transcription factors such as SP1 and ETS to DNA provided a mechanistic justification for the therapeutic perspective of the aureolic acid derived agents [3,4,5]. The chemical structures of OA and Chro are highly similar (Figure 1A); minor differences include methyl groups at position 7 of the aglycon moiety and the acyl residue in sugar E The antibiotics of this group are able to bind to double-stranded DNA, with a strong preference for G/C rich regions, and are believed to exert their antibacterial and antitumor activities via the inhibition of replication and transcription [6,7], other targets have been considered [8]. Six GC-specific hydrogen bo3nodf 1s5 between Chro and DNA provide specificity to the GGCC sequence

Methods

Results

Conclusion