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Calculated values for IC 50, nH and 95% CI are given in each case. Experimental data were fitted with a four parameter, log(inhibitor) vs. ( B) Quantification of inhibitory activity by 2 in the gel-based activity assay. ( A) Titration of BLM-HD with 2 prevents the unwinding of a forked-50mer dsDNA substrate into its component strands, as judged by native gel electrophoresis. These data pave the way for the development of allosteric inhibitors of BLM helicase with the potential to generate trapped and highly cytotoxic BLM-DNA complexes. Crystallographic analysis of the BLM-DNA-2 complex identifies a novel allosteric binding site and reveals a distinctive conformational step in the helicase mechanism, that can be trapped by small-molecules. While ML216 appears to act, at least in part, through direct DNA binding and has poor specificity, we find that 2 and derivatives thereof are highly specific binders of a defined BLM-DNA complex. Here, we determine the mode of action for two reported inhibitors of BLM – ML216 ( Nguyen et al., 2013 Rosenthal, 2010) and a substituted benzamide (compound 2). Despite the therapeutic opportunities this presents, no drugs targeting RECQ helicases have yet been licensed, although potential leads have been reported ( Nguyen et al., 2013 Rosenthal, 2010 Yin et al., 2019). Loss of function of WRN underlies the complex progeria Werner Syndrome defects in BLM underlie Bloom Syndrome, which is characterised by growth retardation and immunodeficiency and defects in RECQ4 are associated with Rothmund-Thompson syndrome, which displays growth retardation, skeletal abnormalities and premature ageing.Ī number of experimental and computational studies have implicated RECQ helicases – primarily BLM and WRN - as potential targets for cancer therapy, due to the synthetic lethality of their silencing or downregulation with genetic defects inherent in a range of different cancers ( Chan et al., 2019 Lieb et al., 2019 Aggarwal and Brosh, 2009 Behan et al., 2019 Datta et al., 2021 Kategaya et al., 2019 Pearl et al., 2015 Wang et al., 2018). Defects in RECQ-family members are responsible for rare genetic diseases displaying substantial genomic instability and cancer predisposition ( Bernstein et al., 2010).
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RECQ-helicases are strongly implicated in the maintenance of genomic integrity, principally through their participation in the homologous recombination (HR) pathway for repair of DNA double-strand breaks and restart of collapsed or blocked replication forks (reviewed in Croteau et al., 2014 Urban et al., 2017), but also have roles in toleration of microsatellite instability ( Chan et al., 2019 Lieb et al., 2019) and sister chromatid decatenation ( Chan et al., 2007). As well as simple DNA duplexes, the various members of the RECQ helicase family (BLM, WRN, RECQ1, RECQ4, and RECQ5 in humans) are able to unwind DNA within a range of complex DNA structures and DNA repair intermediates, including: forks, bubbles, triple helices, displacement (D)-loops, G-quadraplexes, and three- or four-way Holliday junctions (extensively reviewed in Croteau et al., 2014 Wu, 2012).
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RECQ helicases catalyse the unwinding of duplex DNA with 3’ to 5’ directionality, driven by energy liberated by ATP-hydrolysis.