Nonproteasomal Targets of the Proteasome Inhibitors Bortezomib and Carfilzomib: a Link to Clinical Adverse Events

Abstract

Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR).Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors.Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure.Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734-43. Ó2011 AACR.

Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay

“…Levels of MECL1 and LMP2 inhibition have a statistically significant linear dose effect in which higher doses of carfilzomib lead to greater inhibition. While inhibition of LMP2 in samples from bortezomib‐treated patients has previously been reported (Kraus et al , 2007; Arastu‐Kapur et al , 2011), this is the first known report of MECL1 inhibition by a proteasome inhibitor in patients. The mechanism of selective LMP2 and MECL1 inhibition by carfilzomib relative to β1 and β2 inhibition remains to be defined.…”

Dual Carfilzomib and Doxorubicin–Loaded Liposomal Nanoparticles for Synergistic Efficacy in Multiple Myeloma

“…Specifically, doxorubicin and bortezomib have been shown to be synergistic and are FDA approved in combination for the treatment of multiple myeloma (15,18,19). Carfilzomib, a secondgeneration proteasome inhibitor, has shown to have reduced offtarget activity compared with bortezomib, which can eliminate the dose-limiting side-effects seen with bortezomib, such as peripheral neuropathy (20)(21)(22). Although numerous studies have been performed with bortezomib and doxorubicin, combination studies involving carfilzomib and anthracyclines have not been pursued to date (23)(24)(25).…”

Structural and Functional Characterization of the Immunoproteasome

“…Similarly to bortezomib, carfilzomib potently blocks the β5c and β5i active sites of cCP and iCP with IC 50 values of 6 nM and 33 nM, respectively, but unlike bortezomib, carfilzomib does not affect β1c [74] . Although carfilzomib also unintentionally induces neutropenia and thrombocytopenia, it does not cause peripheral neurotoxicity, as observed for the boronic acid bortezomib [68,75] .…”

“…Boronic acids such as bortezomib form a reversible tetrahedral transition state with Thr1O γ that is stabilized by hydrogen bonds with Thr1N and the oxyanion hole Gly47NH of active proteasome subunits [67] . Although these interactions promote a higher affinity of bortezomib for Ntn hydrolases, bortezomib was shown to also considerably inhibit serine proteases, including cathepsin G, cathepsin A, chymase, dipeptidyl peptidase II and HtrA2/omi, involved in neuronal survival [68] . These off-target activities cause severe neurotoxicity leading to tremor, reduced nerve conduction velocity and nerve degeneration, which affect about 30 % of all patients treated with bortezomib [68] .…”

“…Although these interactions promote a higher affinity of bortezomib for Ntn hydrolases, bortezomib was shown to also considerably inhibit serine proteases, including cathepsin G, cathepsin A, chymase, dipeptidyl peptidase II and HtrA2/omi, involved in neuronal survival [68] . These off-target activities cause severe neurotoxicity leading to tremor, reduced nerve conduction velocity and nerve degeneration, which affect about 30 % of all patients treated with bortezomib [68] . Additional drawbacks of bortezomib include thrombocytopenia, neutropenia and gastrointestinal disorders as well as its poor bioavailability that necessitates intravenous administration [69] .…”