Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments.

Nanjie Deng,Junchao Xia,Clement Lin,Yunting Yin,Kaibo Wang,Lauren Wickstrom,Danzhou Yang,Peng He

doi:10.3390/molecules24081574

Abstract

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 5′-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding.

Highlights

G-quadruplex DNA is a four-stranded non-canonical secondary structure formed in DNA sequences containing consecutive runs of guanines such as d(TTAGGG)n in human telomeric DNA.A G-quadruplex consists of stacked planar building blocks called G-tetrads, containing four guanines connected by a network of Hoogsteen hydrogen bonding [1]
The binding free energies computed using the MM-PB(GB)SA are consistent with experimental binding affinity measurements and NMR titration experiments performed in this study
The results show that, in order to reproduce the experimental ligand selectivity in computation, it is crucial to account for the conformational flexibility in the ligand-DNA complexes in the binding free energy calculation

Summary

Introduction

G-quadruplex DNA is a four-stranded non-canonical secondary structure formed in DNA sequences containing consecutive runs of guanines such as d(TTAGGG)n in human telomeric DNA. A G-quadruplex consists of stacked planar building blocks called G-tetrads, containing four guanines connected by a network of Hoogsteen hydrogen bonding [1]. To form stable G-quadruplex, monovalent cations K+ (or Na+ ) centrally located between the adjacent G-tetrad planes are required [2]. The human telomere is a region of repetitive nucleotide sequences at the ends of chromosomes, which protects the chromosome from degradation [3]. Each cell replication results in a shortening of the telomere, which will lead to apoptosis or programmed cell death when a critical shortening is reached. Intramolecular G-quadruplex formed by the guanine-rich DNA sequences d(TTAGGG)n in telomeres inhibits the telomerase access, and

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