Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation

Abstract

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

Dual blockade of misfolded alpha-sarcoglycan degradation by bortezomib and givinostat combination

“…This study was performed on immortalized fibroblasts from a LGMD R3 patient homozygous for R77C-α-SG mutation and stably transduced with a R77C-α-SGmCh reporter lentivirus. This model was previously described as a relevant cellular model to concomitantly investigate the R77C-α-SG expression and localization after compound treatments (Hoch et al, 2019). The drug screening strategy is based on simultaneous identification of positive cells for heterologous R77C-α-SGmCh protein by immunostaining and for presence of the R77C-α-SGmCh protein at the cell membrane compartment using a high-content imaging automate in 384 well plates on non-permeabilized cells (Supplementary Figure S1A ).…”

“…Informed consents were obtained from the parents of the patient included in this study, complying with the ethical guidelines of the institutions involved and with the legislation requirements of the country of origin. The R77C fibroblasts were transduced with a pBABE-puro-based retroviral vector containing sequence encoding the catalytic subunit of human telomerase reverse transcriptase (hTERT) and then selected in the presence of puromycin (1 mg/ml) for 10 days, as previously described (Hoch et al, 2019). Experimental protocols were approved by the french minister of research: AC-2013-1868.…”

“…The sarcoglycan fusion protein was designed based on the human SGCA and SGCG consensus coding sequence (CCDS) found in the NCBI portal (Gene ID: 6442, CCDS number 45729.1 and Gene ID: 6445, CCDS number 9299.1) and the mCherry sequence. The linker, in the amino-acid form of -GGGGS-, was chosen as a flexible type linker that also increased stability/folding of the fusion proteins (Hoch et al, 2019). The fusion protein nucleotide sequence was synthesized by Genecust and cloned in a vector plasmid prepared for lentivirus production.…”

“…Recently, our group developed a pharmacological model of LGMD R3 to identify drugs rescuing the proteasomal degradation of R77C-α-SG. We previously reported that among 2000 tested drugs only one drug, thiostrepton, was capable to rescue R77C-α-SG membrane localization via a direct inhibition of the proteasome (Hoch et al, 2019). Here we have extended the library to 958 other drugs and performed the screening of these drugs alone or in combination with a low dose of BTZ.…”

Introduction to Thiopeptides: Biological Activity, Biosynthesis, and Strategies for Functional Reprogramming

“…Additionally, proteasome and FOXM1 inhibition, when combined, can affect an array of cellular processes. For example, thiostrepton was reported to rescue folding of mutated a-sarcoglycan, the major cause of limb-girdle muscular dystrophy type 2D (Hoch et al, 2019); inhibit toll-like receptors 7-9, which attenuates psoriasis-like inflammation and pathogenesis of autoimmune diseases (Lai et al, 2015;Ueno et al, 2004); reactivate latent HIV-1 in CD4 + T cells through nuclear factor kB pathway mediated by heat-shock response (Peng et al, 2020); and inhibit sonic hedgehog signaling pathway (Yang et al, 2016), thus showing potential as a treatment of triple-negative breast cancer.…”