Discovering inhibitors of human Bloom syndrome protein (BLM)

Human Bloom Syndrome protein (BLM) is a member of the RecQ helicase family that catalyses the unwinding of DNA duplexes. BLM plays a key role in genome maintenance and stability, with mutations in BLM being associated with the rare genetic disease ‘Bloom Syndrome’. BLM has recently been identified as an important anti-cancer drug target, particularly in ALT-proficient cells as it facilitates recombination-driven amplification of telomeres, independently of telomerase. The X-ray crystal structure for the helicase domain of BLM has recently been published, along with some promising small-molecule leads. However, the mode of action of these compounds was unclear. The aim of this programme of research was to discover novel small-molecule inhibitors of human BLM helicase and to validate and understand their mode of action. The first part of this project was to obtain sufficient recombinant protein for assay development and for crystallisation studies; resulting in two helicase domain constructs that could be readily expressed and purified. Ten separate in vivo and in vitro assays were then developed to identify small molecular inhibitors of BLM and to determine mode of action. Analysis of 10 series of compounds identified by an NIH high-throughput screen led to two series of compounds being explored further: the first based on ML216, a previously reported inhibitor of BLM; and the second on a separate unrelated series (Series C). Our data suggest that ML216 is an indirect inhibitor of BLM, inhibiting activity by binding to the DNA rather than protein. In contrast, biophysical data demonstrate that Series C compounds are direct allosteric inhibitors of BLM, binding to the protein only in the presence of a DNA substrate; supported by a series of X-ray crystal structure. In this early stage drug discovery project, we have identified a novel and selective series of compounds that interact and allosterically inhibit the helicase activity of human BLM in vitro. Clearly additional experimental work will be required in order to take this project forward; initially to produce a highly cell penetrant compound with demonstrated ontarget effect in cellular and ultimately in vivo.