Identification
and validation of BCL2 i-motif–interactive
compounds. (A) Principle of the FRET high-throughput screening assay
used to identify compound IMC-76 (right) that destabilizes or unfolds
the i-motif and IMC-48 (left) that stabilizes or facilitates the folding.
(B) Representative results of high-throughput screening with the NCI
Diversity Library. The BCL2 i-motif (1 μM)
labeled with FAM and BHQ1 at the 5′- and 3′-ends, respectively,
was used for compound screening (5 μM) at pH 5.8. (C) Selectivity
of IMC-48 (pH 6.6, left) and IMC-76 (pH 5.9, right) for the BCL2 i-motif (wild-type) over the i-motif mutant, c-MYC i-motif, VEGF i-motif, BCL2 duplex, and BCL2 G-quadruplex probes. Probes were
labeled with FAM at the 5′-end and TAMRA at the 3′-end.
Fluorescence intensity at 520 nm was normalized to the DMSO (0 equiv)
to obtain the relative fluorescence. (D) Comparison of Kd values of IMC-48 (left) and IMC-76 (right) for the BCL2 and VEGF i-motifs, showing that both compounds have
higher affinity for the BCL2 i-motif than for the
VEGF i-motif.