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. 2014 Feb 21;136(11):4161–4171. doi: 10.1021/ja410934b

Figure 2.

Figure 2

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.