Quantitative characterization of human oncogene promoter G‐quadruplex DNA‐ligand interactions using a combination of mass spectrometry and capillary electrophoresis

Abstract

Human c‐KIT oncogene is known to regulate cell growth and proliferation, and thus, acts as a probable target in the treatment of gastrointestinal tumors (GIST). To identify small molecule ligands which can specifically bind with the G‐quadruplex (G4) in the c‐KIT promoter region as potential antitumor agents, we propose the combination of electrospray ionization–mass spectrometry (ESI‐MS), capillary electrophoresis frontal analysis (CE‐FA), and Taylor dispersion analysis (TDA) to accurately investigate the G4/ligands binding properties. First, ESI–MS was used for initial screening of natural products (NPs). CE‐FA was then used to calculate specific binding constants and the stoichiometry of the native state binding pair in solution. Next, TDA, a micro‐capillary flow technique was used to examine the effect of the ligand binding on the diffusivity and particle size of the c‐KIT G4. Two of the screened NPs, scopolamine butylbromide (L1) and isorhamnetin‐3‐O‐neohesperidoside (L3), were found to specifically bind to the c‐KIT G4 with binding constants of around 104 M–1 and 1:1 stoichiometry in a free solution. TDA data showed that ligand binding (both L1 and L3) induced the c‐KIT strands to fold into a tightly structured G4 with a decreased hydrodynamic radius. These ligands have the potential to be drug candidates for the regulation of c‐KIT gene transcription by stabilizing the G4 structure. This methodology not only increased the speed of analysis but also improved its accuracy and specificity compared with the conventional binding approaches.

Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review

“…Most often, we encounter the determination of binding parameters between proteins and drugs [47,52,57,[145][146][147][148]. However, CE-FA was used to characterize the interactions of glycosaminoglycans-protein [149], macrophage-drug [150], heme-insulin [151], platelets-small molecules [152], CD-small molecules [153], ss-DNA-small molecules [49], antigen-adjuvant [58], polymer-copolymer [59,60,154], and protein-polysaccharide [55].…”

“…For clarity, here is a brief list of ACE modes appearing in the literature. Mobility shift ACE (ms‐ACE) which is often simply referred as ACE [45–49], partial‐filling ACE (PF‐ACE) which is also referred to as plug–plug kinetic capillary electrophoresis [50, 51], pressure‐assisted ACE (PA‐ACE) [52], pressure‐assisted partial‐filling ACE (PA‐PF‐ACE), Hummel–Dreyer method [53, 54], vacancy peak method [55], vacancy ACE [56], capillary electrophoresis frontal analysis (CE‐FA) mode [47, 48, 57], and pressure‐assisted CE‐FA (PA‐CE‐FA) [3, 49] are examples of ACE modes. Other modes like continuous capillary electrophoresis frontal analysis (FACCE) [58–60], which is also called continuous nonequilibrium capillary electrophoresis of equilibrium mixtures, nonequilibrium capillary electrophoresis of equilibrium mixtures [61], equilibrium capillary electrophoresis of equilibrated mixtures, sweeping capillary electrophoresis, macroscopic approach to studying kinetics at equilibrium, short sweep capillary electrophoresis, short sweep capillary electrophoresis of equilibrium mixtures [44], moment analysis‐based ACE (MA‐ACE) [62], ideal‐filter capillary electrophoresis [63], and protein cross‐linking capillary electrophoresis [64, 65], are commonly used.…”

“…For clarity, here is a brief list of ACE modes appearing in the literature. Mobility shift ACE (ms-ACE) which is often simply referred as ACE [45][46][47][48][49], partial-filling ACE (PF-ACE) which is also referred to as plug-plug kinetic capillary electrophoresis [50,51], pressure-assisted ACE (PA-ACE)…”

Capillary Electrophoresis: A Three-Year Literature Review

“…CE-frontal analysis (CE-FA) is an attractive method for the characterization of protein–drug interactions. − It involves separating the ligand from the protein and the ligand–protein complex after introducing a relatively large volume of an equilibrated sample mixture into the buffer-filled capillary. Wang et al determined the binding constants between five ligands and the G-quadruplex based on CE-FA . The hydrodynamic radii of the complex at different ligand concentrations were further obtained via Taylor dispersion analysis (TDA).…”

Binding of Quercetin Derivatives toward G-Tetrads as Studied by the Survival Yield Method

“…Quercetin binding to G-quadruplexes has been studied by spectroscopic methods (e.g., NMR, circular dichroism), calorimetric titration, molecular docking, and density functional theory. − On the other hand, quercetin conjugates binding to G-quadruplexes has not been widely studied. Wang et al have studied G-quadruplex interactions with isorhamnetin-3-O-neohesperidoside by using capillary electrophoresis and electrospray ionization mass spectrometry (ESI-MS), Sun et al have studied the G-quadruplex–rutin interaction by 1 H NMR spectroscopy . To the best of our knowledge, there are only two literature reports that contain a comparison between quercetin and its glycoside rutin binding with G-quadruplexes.…”