Abstract
The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib.
We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib.
In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity.
Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors.
Disclosures:
No relevant conflicts of interest to declare.
Nucleotide sequences of highly repeated DNAs; compilation and comments
