Combining Electrospray Mass Spectrometry (ESI-MS) and Computational Techniques in the Assessment of G-Quadruplex Ligands: A Hybrid Approach to Optimize Hit Discovery

Abstract

Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand-GQ interactions.

Figure 1

(A) BRACO-19 interacts with GQ via stacking (PDB ID 3CE5). (B) Distamycin is a GQ groove-binding agent interacting with GQ with a 2:1 stoichiometry (PDB ID 2JT7). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, California (supported by NIH P41 RR-01081).

Figure 2

Comparison of the spectra resulting from the acquisition of the same sample (10 μM human telomeric GQ forming sequence 5′-AGGGTTAGGGTTAGGGTTAGGGT-3′ in 150 mM ammonium acetate, negative ionization mode) with (A) LTQ Orbitrap Velos and (B) LCQ fleet ion trap instruments.

Figure 3

Carbazole derivative BMVC forms (A) 1:1 (PDB ID 6JJ0) and (B) 2:1 (PDB ID 6O2L) complexes with the c-MYC GQ.

Figure 4

Example of the dissociation study carried out by our group. A ligand–GQ complex (5′-AGGGTTAGGGTTAGGGTTAGGGT-3′ human telomeric sequence), marked with a star in the spectrum, undergoes fragmentation by loss of the ligand. In the inset, the plot of the relative intensity of the complex against the collision energy (eV) is depicted.

Figure 5

Functional forms for potential energy for AMBER and CHARMM. For both, the first sum regards covalent bonds, while the last one regards the Lennard-Jones and charge–charge interactions. The image was adapted from the Amber20 manual.