Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin (2020)
Figure 2

Figure 2

Figure 2. GSK716 inhibits polymerisation of Z α1-antitrypsin in cell models of disease.

(A) GSK716 was added to CHO-TET-ON-Z-A1AT cells (Ordóñez et al., 2013) with simultaneous induction of Z α1-antitrypsin expression using doxycycline, and polymer load was quantified with the 2C1 monoclonal antibody that is specific to pathological polymers of α1-antitrypsin (Miranda et al., 2010). The parent cell line that did not express Z α1-antitrypsin provided a negative control. GSK716 completely prevented intracellular polymer formation. Scale bar: 50 µm.

(B) Quantification of immunostained CHO-TET-ON-Z-A1AT cells showed that GSK716 reduced intracellular polymer formation and increased the secretion of Z α1-antitrypsin in a dose-dependent manner with similar potencies. Data were normalised to vehicle and a control compound from the GSK716 series at saturating concentration. Data presented as mean ± SD, n = 61 (secretion) and n = 67 (polymer inhibition).

(C, D) GSK716 was (C) administered to iPSC-derived-hepatocytes and (D) inhibited polymerisation and increased secretion with a similar potency. It induced an approximately 3-fold increase in secreted levels of Z α1-antitrypsin compared with vehicle control. This was apparent even after polymers had been allowed to form. Scale bar: 100 µm. Data presented as mean ± SD, n = 8 (secretion) and n = 6 (polymer inhibition).

(E) CHO tetracycline-inducible cells expressing Z α1-antitrypsin were induced with 0.5 μg/mL doxycycline for 48h and treated with 10μM GSK716. Cells are then lysed in 1% v/v NP-40 buffer at different time points (0, 12, 24, 36, 48, 60, 72, 84, 96 and 108 hours). For every time point, NP-40-soluble and insoluble fractions were separated and immunoprecipitated with the 2C1 mAb and separated by 4-12% w/v SDS-PAGE and α1-antitrypsin detected by immunoblotting. PRE indicates cells pre-treated for 48 hours with 10μM GSK716 and induced for the same time with 0.5 μg/mL doxycycline. The rate of clearance of soluble and insoluble polymer is shown.

(F) CHO inducible cells expressing either wildtype M or Z α1-antitrypsin were induced with 0.5μg/mL doxycycline and treated with 10μM GSK716 or with 0.1% DMSO vehicle (NT, not treated). After induction for 48 h, cells were treated with various doses of tunicamycin for 36 h. Cell viability was measured by Cell Counting Kit-8. The results are shown as mean±SEM, n=4.

(G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography.

(H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2).