Alternative strategies for testing the proteotoxic crisis hypothesis through proteasome inhibition. (A) Bortezomib (BTZ; red asterisk) has a very slow off-rate from 26S proteasome (26S; gray cylinders). Coupled with the high concentrations of proteasomes in red blood cells (RBCs), this results in sequestration of most BTZ in the RBC compartment following intravenous injection. (B) MLN9708 (purple asterisk) dissociates from proteasome six-fold faster than BTZ, enabling better equilibration throughout the body and stronger inhibition of proteasome in tumors. (C) Hypothetical pharmacodynamic response of β5 activity (red trace) in a patient repeatedly dosed (blue arrows) with an oral proteasome inhibitor during the course of a single day. Repeat dosing may suffice to keep β5 activity in the `kill zone’ (in this example, >60% inhibition) for a sufficiently long time interval (denoted by dark gray bar) to kill solid tumor cells. (D) Alternative drug targets in the proteasome: the Rpt1-6 ATPase, Rpn11, and the pockets in 29S outer rings that serve as docking sites for Rpt ATPases in 19S regulatory particle.