A new and underexplored target class in cancer therapy is the family of DNA unwinding enzymes known as helicases. The BLM helicase is one of the best validated drug targets within the helicase family. Therefore, multiple research groups have been trying to identify of BLM inhibitors in the recent past. However, the published inhibitors are neither suitable as tool compounds to elucidate BLM biology in cells, nor as starting points for further optimization, due to issues such as poor solubility (ML216) or lack of specificity for BLM, and insufficient or non-specific activity in cells.

The primary aim of this project was to identify new BLM inhibitors which can be used to study BLM biology, facilitating a better understanding of its role in DDR, as well as starting points for further medicinal chemistry optimization. In order to avoid the pitfalls of academic screening campaigns which often result in non-specific and low quality hits, we carefully sought to develop a suite of advanced assays which allowed us to interrogate the specificity, mode of action, and cellular activity of hits early in the screening cascade, as shown in Figure 41. As a proof of concept, we employed this assay cascade to test, and finally devalidate published BLM inhibitors as low affinity or unspecific binders.

We first established a protocol to purify active BLM protein from baculovirus culture. This enzyme was the basis for development of a fluorescence-based helicase unwinding assay, which was used to screen a library of 330,000 small molecules. The primary hits were rigorously tested in a cascade of custom-developed biochemical and cellular assays. The biochemical validation process started with retesting using fluorescence and luminescence assays at varying dose concentrations, followed by structural filtration, hit scaffold clustering, and counter-assays for DNA binding.

Notably, we identified a series of sub-micromolar inhibitors with selectivity for BLM in a panel of five different helicases while demonstrating favorable solubility and cell (membrane) permeability. Most importantly, we were able to confirm that the frontrunner hits inhibit the activity of BLM in its native, cellular context in human cancer cell lines. Cellular target engagement assays confirmed that a series of compounds bound to and thereby stabilized a substructure of BLM containing the catalytic domain. Phenotypic assays revealed that these BLM inhibitors selectively increase DNA damage homologous recombination repair levels in a BLM specific way, depending on the presence of catalytically active BLM in cells. Ultimately, these compounds demonstrated synthetic lethality combined with ATM inhibitors and showed increased efficacy selectively in ALT (Alternative Lengthening of Telomer)-positive cancer cells.

These findings demonstrate the utility of the advanced hits as tool compounds to elucidate the biological role of BLM in a cellular context, as well as their suitability for further optimization into a targeted cancer therapy. 

In sum, we have identified the first starting points for development of BLM specific helicase inhibitors, as well as developed an assay platform for the identification and of inhibitors of high-quality inhibitors of the other members of the helicases family.