Development of a small molecule that corrects misfolding and increases secretion of Z α₁-antitrypsin

Abstract

Severe α1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z α1-antitrypsin. The lead compound blocks Z α1-antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that ‘mutation ameliorating’ small molecules are a viable approach to treat protein conformational diseases.

High-resolution ex vivo NMR spectroscopy of human Z α1-antitrypsin

“…7e-f). This description is consistent with the observation that relative affinities for 716 are determined primarily by different rates of association, reflecting in turn the different intermediate state populations in each variant 50 . The increased intermediate population we find for Z AAT also accounts for the observation that this variant incorporates a peptide mimetic of the RCL into β-sheet A more rapidly than M AAT 17 .…”

“…Nevertheless, the observation that the intermediate is directly stabilised by binding of the small-molecule inhibitor of polymerisation 716 provides a direct connection between our experiments and a molecular change that underpins AAT deficiency in situ: as the compound is an active inhibitor of polymerisation both in vivo and in vitro 50 , the intermediate conformation we identify must also be present and directly involved in pathological processes in the liver. This observation also suggests a paradoxical approach to the targeted treatment of protein misfolding disorders: by stabilisation of the pathological intermediate state, rather than the native state, selectivity for disease-associated variants may be achieved while nevertheless thermodynamically and kinetically inhibiting transitions out of this state along the disease pathway.…”

“…6c) revealed that 716 binds to a hydrophobic pocket, which displays altered sidechain packing and distortion at the top of strand 5 A (Fig. 1b) 50 . Our data demonstrate that this interaction is the product of conformational selection rather than an induced fit.…”

“…This molecule binds close to the site of the Z mutation, E342K, with a significantly higher affinity for Z AAT than for M or S AAT (Fig. 6b-c) 50 .…”

“…We next used ex vivo NMR to investigate the interaction of AAT variants with a small molecule, GSK716 (716), that is a potent inhibitor of AAT polymerisation in vitro, in cells, and in a mouse model of AAT deficiency (Fig. 6a) 50 . This molecule binds close to the site of the Z mutation, E342K, with a significantly higher affinity for Z AAT than for M or S AAT (Fig.…”