Design, synthesis, and biological evaluation of dual targeting inhibitors of histone deacetylase 6/8 and bromodomain BRPF1

Abstract

Histone modifying proteins, specifically histone deacetylases (HDACs) and bromodomains, have emerged as novel promising targets for anticancer therapy. In the current work, based on available crystal structures and docking studies, we designed dual inhibitors of both HDAC6/8 and the bromodomain and PHD finger containing protein 1 (BRPF1). Biochemical and biophysical tests showed that compounds 23a,b and 37 are nanomolar inhibitors of both target proteins. Detailed structure-activity relationships were deduced for the synthesized inhibitors which were supported by extensive docking and molecular dynamics studies. Cellular testing in acute myeloid leukemia (AML) cells showed only a weak effect, most probably because of the poor permeability of the inhibitors. We also aimed to analyse the target engagement and the cellular activity of the novel inhibitors by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines.

Synthesis, structure-activity relationships, cocrystallization and cellular characterization of novel smHDAC8 inhibitors for the treatment of schistosomiasis

“…The HDAC8 activity assays were performed according to the commercial HDAC8 Fluorometric Drug Discovery Kit [Fluor de Lys(R)-HDAC8, BML-KI178] corresponding to the manufacturer´s instructions as described earlier [32]. As substrate a tetrapeptide connected to aminomethylcoumarin (AMC) H 2 N-Arg-His-Lys(Ac)-Lys(Ac)-AMC was synthesized as previously described [40]. The enzyme was incubated for 90 min at 37 °C, with a substrate concentration of 50 μM and increasing concentrations of inhibitors.…”

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity

“…The in vitro testing on recombinant HDACs were performed as previously described [62] Recombinant human HDAC1 and -6 were purchased from BPS Biosciences. The enzyme inhibition was determined by using a reported homogenous fluorescence assay.…”

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity

“…The in vitro testing on recombinant HDACs were performed as previously described [63] Recombinant human HDAC1 and -6 were purchased from BPS Biosciences. The enzyme inhibition was determined by using a reported homogenous fluorescence assay.…”

“…As substrate a tetrapeptide connected to aminomethylcoumarin (AMC) H2N-Arg-His-Lys(Ac)-Lys(Ac)-AMC was synthesized as previously described. [63] The enzyme was incubated for 90 min at 37 °C, with a substrate concentration of 50 M and increasing concentrations of inhibitors. The stop-solution containing inhibitor, to stop the hHDAC8 activity, and Trypsin, to release the AMC, was added.…”