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BRD4-targeted proteolysis targeting chimera (PROTAC) have exhibited promising in vitro and in vivo anticancer activity in a number of cancer models. However, the clinical development of current reported BRD4-PROTACs have stagnated, largely due to the safety risks caused by their poor degradation selectivity. In this study, we designed and synthesized a series of PROTACs based on our recently reported dual BET/PLK1 inhibitor WNY0824 , which led to the discovery of an isoform-selective and potent BRD4-PROTAC 12a (WWL0245). WWL0245 exhibited excellent selective cytotoxicity in the BETi sensitive cancer cell lines, including AR-positive prostate cancer cell lines. It could also efficiently induce ubiquitin-proteasomal degradation of BRD4 in AR-positive prostate cancer cell lines, with sub-nanomolar half-maximal degrading concentration (DC 50) and maximum degradation (D max) > 99%. Moreover, WWL0245 induced cell cycle arrest at the G0/G1 phase and apoptosis in AR-positive prostate cancer by downregulation of the protein levels of AR, PSA and c-Myc as well as transcriptionally suppressed AR-regulated genes. WWL0245 was thus expected to be developed as a promising drug candidate for AR-positive prostate cancer and a valuable tool compound to study the biological function of BRD4. [Display omitted] • A series of PROTACs were designed and synthesized based on the dual BET/PLK1 inhibitor WNY0824. • 12a (WWL0245) is a potent and highly selective BRD4-PROTAC. • 12a exhibited better antiproliferative activity in AR-positive prostate cancer cells than WNY0824. [ABSTRACT FROM AUTHOR]

Abstract: Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 μm and a viable cell density greater than 5×106 cells·ml−1 were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1×107 cells·ml−1 resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7×1011 GTU ml−1 at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect. [Copyright &y& Elsevier]