Oxidative switch as a means of regulating catalytic activity of proteasomes

Using a novel cell-based assay we identified a new cupper based class of proteasome inhibitors. Of these PI-6 was chosen as a lead compound that inhibit the mammalian proteasome (initial affinity measured at sub-micromolar range).

Here we present the whole medicinal-chemistry process, starting from a lead compound in a screening assay, through iterations of in-silico design and synthesis, followed by biochemical and cell culture based assays. Using this rational we have successfully synthesized reversible and irreversible inhibitors, with optimized physicochemical properties and improved affinity that may be used as drug candidates.

In-silico calculations predicted a possible binding site for PI-6 and its derivatives, identifying a bis-Cysteine tweezers, revealing a previously unknown oxidative switch within the 20S proteasome. This implies an inter-subunit disulfide bond formation upon binding of these inhibitors. In fact, such disulfide was verified in nonreducing SDS-PAGE, only upon incubation with the nonreversible version of the inhibitor.

We present advanced versions of reversible and nonreversible derivatives of our inhibitors and their ability to eradicate multiple myeloma cells that are velcade resistant. We propose a mechanism of action based on biochemical and cryoEM structure of 20S proteasomes treated with these inhibitors.