HSP72 is the stress-induced isoform of the human HSP70 family; a family of 70 kDa molecular chaperones, responsible for the stabilisation and refolding of a host of native cellular proteins. The nucleotide bound state of HSP72 controls the binding and release of client proteins and subsequent ATP hydrolysis is allosterically coupled to protein refolding. HSP72 is overexpressed in numerous cancers, including breast, colon and head and neck, and its expression correlates with poor prognosis. To date no HSP70 inhibitors have been developed into clinical candidates, thus there is an unmet clinical need for HSP72 inhibitors. Numerous efforts have focussed on the development of ATP-competitive inhibitors, however HSP72’s high affinity for ATP and the high intra-cellular concentration has often resulted in poor efficacy. In an effort to circumvent this, the aim of this work is to identify novel HSP72 inhibitors that bind to an alternative site to the ATP-binding site. Here, we describe the establishment of a novel high throughput HSP72 refolding assay, which utilises chemically denatured luciferase as a model substrate. Using this assay we screened a library of approximately 20,000 small molecules for inhibition of HSP72’s refolding capability. We present the hit matter identified in this HTS and the subsequent validation of hits, using a suite of biophysical and biochemical assay technologies. Confirmed hits were selected for solubility experiments and the 8 most soluble scaffolds were taken forward for further mechanistic study. We subsequently aimed to determine whether these compounds were ATP-competitive, and thus employed a fluorescence polarisation assay utilising a fluorophore-labelled ATP probe, which determined each of the hit compounds to be non-competitive with ATP. These data indicate that hit compounds inhibit HSP72 through an alternative allosteric mechanism, and may bind to an unexplored allosteric site that would provide an alternative strategy for drug development. Further structural studies using X-ray crystallography were employed in order to determine the binding site of these novel scaffolds and further elucidate the binding mode at the molecular level. Due to the extreme flexibility of the HSP72 protein, NMR using 15N-labelled protein was employed in tandem to investigate compound binding in solution and here we present the results of this work.

Identification of novel allosteric HSP72 inhibitors

253rd National Meeting (2017)

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