Summary /

The inhibition of DNA unwinding by both truncated and full-length BLM DNA unwinding for the probe ML216 is displayed in Figure 1.

Figure 1. A) Representative gels for ML216 in the DNA unwinding gel electrophoresis assay using the truncated (left) and full-length (right) forms of BLM helicase and a fluorescent-labeled forked DNA substrate. B) Graphical representation of the percent inhibition of BLM by ML216. Each point represents the mean ± SD for three independent experiments.

Summary /

ML216 selectively inhibits cell proliferation of BLM-proficient fibroblast cells (PSNF5) over BLM-deficient cells (PSNG13) (Figure 2), whereas the initial “hit molecule” 2 shows minimal effect on either cell line. More strikingly, ML216 caused a significant increase in the frequency of sister chromatid exchanges in PSNF5 cells, a key cytogenetic marker of cells lacking BLM activity (Figure 3). Together, these results serve to validate the ontarget cellular activity of ML216, as well as highlight a major advantage of the probe molecule over the initial hit.

Figure 2. Inhibition of cell proliferation of BLM-complemented (PSNF5) fibroblast cell line, but not the BLM-deficient (PSNG13) fibroblast cell line after 72 hr treatment with ML216.

Figure 3. Increase in sister chromatid exchanges (SCEs) in the BLM-complemented (PSNF5) fibroblast cell line, but not in the BLM-deficient (PSNG13) fibroblast cell line after treatment with ML216 (50 μM).

Summary /

ML216 exhibits excellent mouse liver microsomal (MLM) stability and plasma stability (Table 2), indicating that the probe could prove to be useful in vivo. However, for this compound to have appropriate oral bioavailability, additional improvement in the aqueous solubility needs to be made.

Table 2. ADME profile of ML216. (a) In vitro ADME profile of the probe compound (ML216). Kinetic solubility, Caco-2 permeability, MLM stability and plasma stability were all carried out by Pharmaron Inc. PBS buffer (pH 7.4) stability was performed in-house using LC/MS monitoring UV @ 254 and 220 nm.

Summary /

Besides the forked DNA duplex substrate, BLM has been shown to display an ATPdependent 3’-5’ DNA helicase activity that unwinds a variety of DNA substrates arising during DNA replication and repair such as Holliday junctions, G-quadruplex DNA, and displacement loops (D-loops). The BLM-dependent unwinding of such structures is also inhibited by ML216 (Figure 4), albeit to a lesser extent than the forked DNA duplex substrate. In addition, a fluorescence polarization assay, which monitored the binding of BLM to a fluorescently-labeled singled-stranded DNA oligonucleotide, was utilized to gain insight into the mechanism of action of ML216. As Figure 5 illustrates, ML216 inhibited the DNA binding of BLM with an IC50 of 5.1 μM, while having only a minimal effect on RecQ1 (IC50 = 101.8 μM). These results suggest the DNA binding domain of BLM as the putative binding pocket for ML216.

Figure 4. Inhibition of the processing of alternative DNA structures by BLM. Representative nondenaturing polyacrylamide gels showing the inhibition of BLM processing of Holliday-like junctions (left), G-quadruplex DNA (middle), and D-loop-like structures (right) by ML216 at 10 and 50 μM. Asterisks indicate location of 32P-radiolabel.

Figure 5. Inhibition of DNA binding by fluorescence polarization. A) Schematic diagram of the fluorescence polarization-based helicase-DNA binding assay. B) Inhibition of BLM and RecQ1 DNA binding by ML216.