SOMETHING NEW ABOUT BORTEZOMIB NEUROTOXICITY

Bortezomib (BTZ) and Carfilzomib (CFZ) are proteasome inhibitors that represent the gold standard in the treatment of multiple myeloma. While both are very effective, they have different side effect profiles. In particular, BTZ induces peripheral neuropathy as a major side effect in a high percentage of patients, while this is only rarely observed with CFZ. In this study, we investigated possible BTZ and CFZ off-targets able to explain the difference in their neurotoxicity profiles. Off-targets were identified using SPILLO-PBSS, a software that perform a 3D in silico screening on a proteome-wide-scale. The hypothesis was biologically validated in vitro in adult mice dorsal root ganglia primary sensory neuron cultures and in a cell free model of tubulin polymerization and depolymerization. NMR binding studies were performed to demonstrate the interaction with the identified off-target. Using an innovative in silico approach, we demonstrated that tubulin is a potential off-target of BTZ. A direct BTZ-microtubules interaction could inhibit the GTPase activity of tubulin, thus reducing microtubule catastrophe and increasing tubulin polymerization. In neuron cultures, BTZ, but not CFZ, induced neurotoxicity and increased the percentage of polymerized tubulin. Moreover, in a cell-free model of tubulin polymerization and depolymerization only BTZ slowed down the depolymerization of microtubules and reduced the free phosphate concentration released during GTP hydrolysis. Lastly, NMR binding studies clearly demonstrated that only BTZ is able to directly interact with both tubulin dimers and polymerized form. In conclusion, our data for the first time gives evidence of a differential molecular mechanism of action of BTZ and CFZ that would explain their different side effect profiles: BTZ neurotoxicity is not related to its well-known proteasome inhibition, but to its ability to directly bind to tubulin, reducing microtubule catastrophe and consequently increasing the rate of polymerized tubulin.