Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Grazia Raffaella Tundo,Massimo Coletta,Gabriele Macari,Francesco Bellia,Anna Maria Santoro,Francesco Oddone,Diego Sbardella,Danilo Milardi,Giuseppe Grasso,Mariacristina Parravano,Fabio Polticelli,Paolo Cascio,Stefano Marini,Maria Grazia Atzori,Grazia Graziani

doi:10.3390/biom12020315

Abstract

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the β5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Highlights

The proteasome is the central proteolytic assembly of the Ubiquitin Proteasome System (UPS), a main catabolic pathway which carries out the targeted proteolysis of the majority of intracellular proteins, mostly short-lived, being involved in the regulation of all cellular metabolic processes [1,2]
By combining biochemical, molecular and bioinformatic tools, we report that CFZ is an Insulin-Degrading Enzyme (IDE) ligand in vitro, exerting some inhibition of catalytic activity on synthetic and natural substrates
CFZ is well known to be an efficient and specific inhibitor of 20S proteasome ac[41,42], as it clearly emerges from the dependence of the 20S chymotrypsin-like activity tivity [41,42], as it clearly emerges from the dependence of the 20S chymotrypsin-like on CFZ concentration (Figure 1)

Summary

Introduction

The proteasome is the central proteolytic assembly of the Ubiquitin Proteasome System (UPS), a main catabolic pathway which carries out the targeted proteolysis of the majority of intracellular proteins, mostly short-lived, being involved in the regulation of all cellular metabolic processes [1,2]. The 19S couples the ATP-hydrolysis with the binding, unfolding and translocation of the substrate (e.g., poly-ubiquitinated proteins) into the 20S catalytic core.

Methods

Results

Conclusion