Identification of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based small molecules as selective BRD9 binders

Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for thedevelopment of new agents endowed with anticancer properties. With this aim, a set of 2,4,5 trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following acombined approach based on in silico investigations, chemical synthesis and biological evaluation of the most promising items. The protocol was based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested against the bromodomain ofBRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bindBRD9 in the high nanomolar range of concentrations (IC50 = 0.47±0.27 ?M and 0.18±0.30 ?M,respectively) showing a high selectivity profile when tested against a panel of further ninebromodomains. Taking advantage of structure-based 3D pharmacophore models, further 10derivatives were selected in silico for the synthetic step and binding assessment, disclosing sevencompounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9. The ability of the identifiedBRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very goodpassive permeability profile. Preliminary studies were carried out on a panel of healthy and cancerhuman cell lines to explore the biological behavior of the selected compounds, disclosing amoderate activity and significant selectivity profile towards leukaemia cells.These results highlighted the applicability of the reported multidisciplinary approach foraccelerating the selection of promising items and for driving the chemical synthesis of novelselective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for furtherexploring the chemical space in order to obtain new analogues with improved potency, with aparticular interest to design of related proteolysis targeting chimera (PROTAC) derivatives.