Although BRACO19 is a potent G-quadruplex binder, its potential for clinical usage is hindered by its low selectivity towards DNA G-quadruplex over duplex. High-resolution structures of BRACO19 in complex with neither single-stranded telomeric DNA G-quadruplexes nor B-DNA duplex are available. In this study, the binding pathway of BRACO19 was probed by 27.5 µs molecular dynamics binding simulations with a free ligand (BRACO19) to a DNA duplex and three different topological folds of the human telomeric DNA G-quadruplex (parallel, anti-parallel and hybrid). The most stable binding modes were identified as end stacking and groove binding for the DNA G-quadruplexes and duplex, respectively. Among the three G-quadruplex topologies, the MM-GBSA binding energy analysis suggested that BRACO19′s binding to the parallel scaffold was most energetically favorable. The two lines of conflicting evidence plus our binding energy data suggest conformation-selection mechanism: the relative population shift of three scaffolds upon BRACO19 binding (i.e., an increase of population of parallel scaffold, a decrease of populations of antiparallel and/or hybrid scaffold). This hypothesis appears to be consistent with the fact that BRACO19 was specifically designed based on the structural requirements of the parallel scaffold and has since proven effective against a variety of cancer cell lines as well as toward a number of scaffolds. In addition, this binding mode is only slightly more favorable than BRACO19s binding to the duplex, explaining the low binding selectivity of BRACO19 to G-quadruplexes over duplex DNA. Our detailed analysis suggests that BRACO19′s groove binding mode may not be stable enough to maintain a prolonged binding event and that the groove binding mode may function as an intermediate state preceding a more energetically favorable end stacking pose; base flipping played an important role in enhancing binding interactions, an integral feature of an induced fit binding mechanism.

Human telomeric DNA G-quadruplex has been identified as a good therapeutic target in cancer treatment. G-quadruplex-specific ligands that stabilize the G-quadruplex have great potential to be developed as anticancer agents. Two crystal structures (an apo form of parallel stranded human telomeric G-quadruplex and its holo form in complex with BRACO19, a potent G-quadruplex ligand) have been solved, yet the binding mechanism and pathway remain elusive. In this study, we simulated the binding of a free BRACO19 molecule to the apo form of the G-quadruplex using the latest AMBER DNA (OL15) and ligand (GAFF2) force fields. Three binding modes have been identified: top stacking, bottom intercalation, and groove binding. Bottom intercalation (51% of the population) resembles the bottom binding pose in the complex crystal structure very well. The groove binding mode is less stable than the bottom binding mode and is likely to be an intermediate state leading to the bottom binding mode. A flip-insertion mechanism was observed in the bottom intercalation mode, during which flipping of the bases outward makes space for ligand insertion, after which the bases flip back to increase the stability of the complex. In addition to reproducing the base-flipping behavior for some loop residues upon ligand binding, the direct alignment type of the ATAT-tetrad was observed in our simulations for the first time. These successes provide initial support for using this combination of the OL15 and GAFF2 force fields to study quadruplex-ligand interactions.