Bisindolylmaleimide Ligands Stabilize c-MYC G-Quadruplex DNA Structure and Downregulate Gene Expression

Abstract

G-Quadruplex (G4) structures play a pivotal role in diverse biological functions, including essential processes, such as telomere maintenance and gene regulation. G4 structures formed in functional regions of genomes are actively pursued toward therapeutics and are targeted by small-molecule ligands that alter their structure and/or stability. Herein, we report the synthesis of bisindolylmaleimide-based (BIM) ligands, which preferentially stabilize parallel G4 structures of c-MYC and c-KIT oncogenes over the telomeric h-RAS1 G4 and duplex DNAs. The preferential stabilization of parallel G4s with BIM ligands is further validated by the DNA polymerase stop assay, where stop products were only observed for templates containing the c-MYC G4 sequence. Nuclear magnetic resonance (NMR) titration studies indicate that the lead ligand BIM-Pr1 forms a 2:1 complex with c-MYC G4 DNA with a K D of 38 ± 5 μM. The BIM ligand stacks at the 5′ and 3′ quartets, with molecular modeling and dynamics studies supporting the proposed binding mode. The ligand is cytotoxic to HeLa cells and downregulates c-MYC gene expression. Collectively, the results present bisindolylmaleimide scaffolds as novel and powerful G4 targeting agents.

Two Binding Sites of SARS-CoV-2 Macrodomain 3 Probed by Oxaprozin and Meclomen

“…To rule out the potential binding of these two inhibitors to DNA-G4s, oxaprozin and meclomen were titrated to bbc3 -(dG) 14 and p53 -(dG) 14 , respectively. No significant changes were observed in the CD spectra (Figure S11a,b), indicating these drugs did not alter the parallel G4 structures. − Furthermore, CD melting − experiments exhibited no observable binding of oxaprozin/meclomen to either bbc3 -(dG) 14 or p53 -(dG) 14 , as even at a high concentration, these two inhibitors induced little change of the thermal stability of the DNA-G4s (Figure S11c,d). To evaluate a potential synergistic effect of the two compounds, we carried out the competitive FP experiment using a combination of oxaprozin and meclomen that reduced the binding between SARS-CoV-2 Mac3 and G4s more effectively than that achieved via a single inhibitor (Figure d).…”

“…The melting temperature ( T m ) was determined by fitting to a Boltzmann function from the sigmoidal curve in Origin Pro 2020 , θ = θ min + ( θ max − θ min ) / ( 1 + exp false( ( T m − T ) / s false) ) where θ represents the ellipticity, θ min and θ max refer to the minimum and maximum ellipticity, respectively, T is temperature, T m is the melting temperature, and s is a fitting parameter.…”

Targeting Parallel Topology of G-Quadruplex Structures by Indole- Fused Quindoline Scaffolds

“…The luminescence of a cell is directly related to the amount of ATP and thereby the number of viable cells present in the culture. The percentage cell viability was plotted against the logarithmic concentration of ligand, and the IC 50 value was calculated by fitting the curve using the dose response equation in Graph pad Prism 8.3.0 using a previously reported procedure …”

“…The percentage cell viability was plotted against the logarithmic concentration of ligand, and the IC 50 value was calculated by fitting the curve using the dose response equation in Graph pad Prism 8.3.0 using a previously reported procedure. 8 RNA Isolation and Purification. HeLa cells were seeded at a density of 2 × 10 5 cells per well in 6-well plates and allowed to adhere for 24 h at 5% CO 2 and a 37 °C atmosphere.…”

“…Although targeting a particular G4 topology is quite challenging, varying dimensions of different G-quartets and the unique sizes of their loops can render specificity toward a specific topology. Though several ligands that stabilize G4 structures have been reported over the years, only a few show preferential binding to promoter G4 structures. ,− Therefore, designing new molecular scaffolds, selectively binding to the promoter G4s with parallel topology, provides promising avenues for anticancer drug discovery.…”