ABSTRACT
Proteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.
Subject(s)
Antineoplastic Agents/toxicity , Bortezomib/toxicity , Oligopeptides/toxicity , Peripheral Nervous System Diseases/chemically induced , Proteasome Inhibitors/toxicity , Tubulin/metabolism , Animals , Biopolymers/metabolism , Cell Line , Computer Simulation , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Mice , Neurons/drug effects , Protein BindingABSTRACT
[Cu(thp)4]PF6, [Cu(PTA)4]PF6, [Au(thp)4]PF6 and [Au(PTA)4]PF6 are phosphane (thp = tris(hydroxymethyl)phosphane; PTA = 1,3,5-triaza-7-phosphaadamantane) copper(I) and gold(I) water-soluble complexes characterized by high anticancer activity in a wide range of solid tumors, often able to overcome drug resistance of platinum-based compounds. For these reasons, they have been proposed as a valid alternative to platinum-based chemotherapeutic drugs (e.g., cisplatin and oxaliplatin). In vitro experiments performed on organotypic cultures of dorsal root ganglia (DRG) from 15-day-old rat embryos revealed that copper-based compounds were not neurotoxic even at concentrations higher than the IC50 obtained in human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines. The ability of these compounds to hinder the proteasome machinery in DRG neurons was tested by fluorimetric assay showing that the non-neurotoxic copper-based complexes do not inhibit proteasome activity in DRG primary neuron cultures. On the contrary, the neurotoxic complex [Au(PTA)4]PF6, induced a significant inhibition of proteasome activity even at concentrations lower than the IC50 in cancer cells. The proteasome inhibition induced by [Au(PTA)4]PF6 was associated with a significant increase in α-tubulin polymerization that was not observed following the treatment with copper-based compounds. Uptake experiments performed by atomic absorption spectrometry showed that both copper-based complexes and [Au(PTA)4]PF6 are internalized in neuron cultures. In vitro and in vivo preliminary data confirmed copper-based complexes as the most promising compounds, not only for their anticancer activity but also concerning the peripheral neurotoxicity profile.
