Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions.

Sung-Kun Kim,Edward S Kim,Austin P Dinkel,Aaron Castro,Xiaoyun Liu,Miguel Castro

doi:10.1371/journal.pone.0147234

Sung-Kun Kim, Edward S Kim + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0147234

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Abstract

Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2’,4’-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2’,4’-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2’,4’-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2’,4’-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2’,4’-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2’,4’-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2’,4’-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

Highlights

DNA replication is an essential process for the proliferation of all forms of life
In an attempt to explore the possibility of inhibition by the nucleoside analogs based on locked nucleic acids (LNAs) and bridged nucleic acids (BNAs) (NC-NMe), this work used Taq DNA polymerase as a target polymerase, a critical component of PCR
An initial attempt to test inhibition for the nucleosides was made by monitoring an increase in the product amounts detected by using SYBR Green in the quantitative real-time PCR (qPCR) process

Summary

Introduction

DNA replication is an essential process for the proliferation of all forms of life. The importance of the inhibition of DNA replication is hardly understated in modern therapeutic strategy against numerous diseases including viral infections and cancer. DNA replication is in the active site of DNA polymerase, which can be classified into at least five different families based on amino acid sequence comparison and crystal structure analyses [1,2]. As the antiviral drug Acyclovir, a guanosine mimic and suicide inhibitor of Herpes simplex virus, binds to the active site of the viral DNA polymerase [8]. Following the generation of LNA, bridged nucleic acids (BNAs) such as 2’,4’-BNACOC and 3’-, and 5’-amino-2’,4’-BNAs were developed with enhanced binding abilities and nuclease resistance [19,20,19,21]. In an attempt to explore the possibility of inhibition by the nucleoside analogs based on LNA and BNA (NC-NMe), this work used Taq DNA polymerase as a target polymerase, a critical component of PCR. Molecular dynamics (MD) simulations offered detailed binding interaction after initial docking

Materials and Methods

Results and Discussion

Conclusions