Ritterostatin G_N1_N, a Cephalostatin–Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78

Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine–cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine–cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin G_N1_N, a cephalostatin–ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.