Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates

Abstract

Protein synthesis inhibitors are commonly used for measuring protein degradation rates, but may cause cytotoxicity via direct or indirect mechanisms. This study aimed to identify concentrations providing optimal inhibition in the absence of overt cytotoxicity. Actinomycin D, cycloheximide, emetine, and puromycin were assessed individually, and in two‐, three‐, and four‐drug combinations for protein synthesis inhibition (IC 50) and cytotoxicity (CC 50) over 72 h. Experiments were conducted in HepG2 cells and primary rat hepatocytes (PRH). IC 50 for actinomycin D, cycloheximide, emetine, and puromycin were 39 ± 7.4, 6600 ± 2500, 2200 ± 1400, and 1600 ± 1200 nmol/L; with corresponding CC 50 values of 6.2 ± 7.3, 570 ± 510, 81 ± 9, and 1300 ± 64 nmol/L, respectively, in HepG2 cells. The IC 50 were 1.7 ± 1.8, 290 ± 90, 620 ± 920, and 2000 ± 2000 nmol/L, with corresponding CC 50 values of 0.98 ± 1.8, 680 ± 1300, 180 ± 700, and 1600 ± 1000 (SD) nmol/L, respectively, in PRH. CC 50 were also lower than the IC 50 for all drug combinations in HepG2 cells. These data indicate that using pharmacological interference is inappropriate for measuring protein degradation over a protracted period, because inhibitory effects cannot be extricated from cytotoxicity.

A Lipophilic 4‐Phenylbutyric Acid Derivative That Prevents Aggregation and Retention of Misfolded Proteins

“…Therefore, we tested the effect of compound 5 on the protein production using the total protein synthesis assay kit, which exploits a fluorescence‐activated cell sorting (FACS) analysis. Figure shows that the known total protein synthesis inhibitor cycloheximide blocks production of proteins in PC12 cells. On the other hand, in the presence of 5 , the rate of total protein synthesis was not significantly affected.…”

Evaluation of the anti‐inflammatory effect of 1,4‐dihydropyridine derivatives

“…Significance was set at P < 0.05. The values of IC50 for NOx were calculated by nonlinear regression analysis using the logarithm of concentration versus normalized response (percentage NOx inhibition), with the aid of the software program GraphPad Prism ® , version 9.1.1 (San Diego, CA, USA) [23].…”

Synapse integrity and function: Dependence on protein synthesis and identification of potential failure points

“…In this study we used protein synthesis inhibitors to block the supply of new proteins in neurons. Translational inhibitors, however, have been reported to have widespread effects on neuronal function, some of which might be unrelated to their capacity to suppress protein synthesis: Superinduction of immediate early genes ( Mahadevan and Edwards, 1991 ; Hazzalin et al, 1998 ; Radulovic and Tronson, 2008 ; Santos et al, 2019 ); increased synthesis /hyperproduction of specific proteins ( Törocsik and Szeberényi, 2000a ; Radulovic and Tronson, 2008 ; Kenney et al, 2016 ); activation of signaling pathways ( Cano et al, 1994 ; Kardalinou et al, 1994 ; Zinck et al, 1995 ; Iordanov et al, 1997 ; Hazzalin et al, 1998 ; Iordanov and Magun, 1998 ; Törocsik and Szeberényi, 2000a ; Monaghan et al, 2014 ; Tyssowski et al, 2018 ); apoptosis/cytotoxicity (in certain cell types; Törocsik and Szeberényi, 2000b ; Monaghan et al, 2014 ; Chan et al, 2017 ); effects on protein degradation ( Franklin and Johnson, 1998 ; Dai et al, 2013 ; see also Ding et al, 2007 ; Kaang and Choi, 2012 ; Jarome and Helmstetter, 2014 ); and altered axonal transport ( Levy et al, 1990 ). Despite these reports, the similarity of the findings in experiments based on two different inhibitors (CHX, ANI) suggest that these are unlikely to stem primarily from off-target effects of these inhibitors.…”