Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 50 (22), 5357-63

Small Molecules Block the Polymerization of Z alpha1-antitrypsin and Increase the Clearance of Intracellular Aggregates

Affiliations

Small Molecules Block the Polymerization of Z alpha1-antitrypsin and Increase the Clearance of Intracellular Aggregates

Meera Mallya et al. J Med Chem.

Abstract

The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.2 million small molecules and 6 compounds were identified that reduced polymer formation in vitro. Modeling the effects of ligand binding on the cavity and re-screening the library identified an additional 10 compounds that completely blocked polymerization. The best antagonists were effective at ratios of compound to Z alpha1-antitrypsin of 2.5:1 and reduced the intracellular accumulation of Z alpha1-antitrypsin by 70% in a cell model of disease. Identifying small molecules provides a novel therapy for the treatment of liver disease associated with the Z allele of alpha1-antitrypsin.

Figures

Figure 1
Figure 1
Rational drug design to prevent the polymerisation of α1-antitrypsin. Fig. 1a. Pathway of the polymerisation of α1-antitrypsin. The Z mutation of α1-antitrypsin (Glu342Lys at P17; arrowed) perturbs the structure of β-sheet A (green) and the mobile reactive centre loop (red) to form the intermediate M*. The patent β-sheet A can then accept the loop of another molecule (as strand 4) to form a dimer (D) which then extends into polymers (P)–. It is these polymers that accumulate within hepatocytes to cause liver disease. The position of the lateral hydrophobic pocket that is the target of rational drug design is shown with a blue arrowhead. Note the change in conformation in this region of the molecule as it forms M* and then dimers and polymers. Fig. 1b. The predicted binding pose of CG to the lateral hydrophobic cavity with Asn104 in an alternate conformation. The residues that most effect the dimensions of the pocket, Asn104 and His139, are highlighted. Fig. 1c. The parent compounds Jub, AG, PC, DV and CO and their analogues that also blocked polymerisation. Compound TT (red) was designed as a control that did not block polymer formation. Fig. 1d. The parent compound CG and its analogues. Compounds labelled in red (NY and JS) are designed not to block polymerisation.
Figure 1
Figure 1
Rational drug design to prevent the polymerisation of α1-antitrypsin. Fig. 1a. Pathway of the polymerisation of α1-antitrypsin. The Z mutation of α1-antitrypsin (Glu342Lys at P17; arrowed) perturbs the structure of β-sheet A (green) and the mobile reactive centre loop (red) to form the intermediate M*. The patent β-sheet A can then accept the loop of another molecule (as strand 4) to form a dimer (D) which then extends into polymers (P)–. It is these polymers that accumulate within hepatocytes to cause liver disease. The position of the lateral hydrophobic pocket that is the target of rational drug design is shown with a blue arrowhead. Note the change in conformation in this region of the molecule as it forms M* and then dimers and polymers. Fig. 1b. The predicted binding pose of CG to the lateral hydrophobic cavity with Asn104 in an alternate conformation. The residues that most effect the dimensions of the pocket, Asn104 and His139, are highlighted. Fig. 1c. The parent compounds Jub, AG, PC, DV and CO and their analogues that also blocked polymerisation. Compound TT (red) was designed as a control that did not block polymer formation. Fig. 1d. The parent compound CG and its analogues. Compounds labelled in red (NY and JS) are designed not to block polymerisation.
Figure 1
Figure 1
Rational drug design to prevent the polymerisation of α1-antitrypsin. Fig. 1a. Pathway of the polymerisation of α1-antitrypsin. The Z mutation of α1-antitrypsin (Glu342Lys at P17; arrowed) perturbs the structure of β-sheet A (green) and the mobile reactive centre loop (red) to form the intermediate M*. The patent β-sheet A can then accept the loop of another molecule (as strand 4) to form a dimer (D) which then extends into polymers (P)–. It is these polymers that accumulate within hepatocytes to cause liver disease. The position of the lateral hydrophobic pocket that is the target of rational drug design is shown with a blue arrowhead. Note the change in conformation in this region of the molecule as it forms M* and then dimers and polymers. Fig. 1b. The predicted binding pose of CG to the lateral hydrophobic cavity with Asn104 in an alternate conformation. The residues that most effect the dimensions of the pocket, Asn104 and His139, are highlighted. Fig. 1c. The parent compounds Jub, AG, PC, DV and CO and their analogues that also blocked polymerisation. Compound TT (red) was designed as a control that did not block polymer formation. Fig. 1d. The parent compound CG and its analogues. Compounds labelled in red (NY and JS) are designed not to block polymerisation.
Figure 1
Figure 1
Rational drug design to prevent the polymerisation of α1-antitrypsin. Fig. 1a. Pathway of the polymerisation of α1-antitrypsin. The Z mutation of α1-antitrypsin (Glu342Lys at P17; arrowed) perturbs the structure of β-sheet A (green) and the mobile reactive centre loop (red) to form the intermediate M*. The patent β-sheet A can then accept the loop of another molecule (as strand 4) to form a dimer (D) which then extends into polymers (P)–. It is these polymers that accumulate within hepatocytes to cause liver disease. The position of the lateral hydrophobic pocket that is the target of rational drug design is shown with a blue arrowhead. Note the change in conformation in this region of the molecule as it forms M* and then dimers and polymers. Fig. 1b. The predicted binding pose of CG to the lateral hydrophobic cavity with Asn104 in an alternate conformation. The residues that most effect the dimensions of the pocket, Asn104 and His139, are highlighted. Fig. 1c. The parent compounds Jub, AG, PC, DV and CO and their analogues that also blocked polymerisation. Compound TT (red) was designed as a control that did not block polymer formation. Fig. 1d. The parent compound CG and its analogues. Compounds labelled in red (NY and JS) are designed not to block polymerisation.
Figure 2
Figure 2
7.5 % (w/v) non-denaturing PAGE to assess the effect of compounds identified in silico on the polymerisation of Z α1-antitrypsin. Each lane contains 2μg of protein. Fig. 2a (top left). Lane 1, monomeric Z α1-antitrypsin; lane 2, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days; lane 3, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days in the presence of 4.75% (v/v) DMSO; lanes 4–9, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days with 100- fold molar excess of AG, BS, CG, EC, FP and GP respectively, all with a final concentration of 4.75% (v/v) DMSO. Fig. 2b (top right). Lane 1, monomeric Z α1-antitrypsin; lane 2, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days; lane 3, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days in the presence of 4.75% (v/v) DMSO; lanes 4–9, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 7 days with 100-fold molar excess of OC, PC, RS, SK, TCR and UX respectively, all with a final concentration of 4.75% (v/v) DMSO. Fig. 2c (bottom left). 7.5 % (w/v) non-denaturing PAGE to assess the effect of decreasing concentrations of CG on the polymerisation of Z α1-antitrypsin. Lane 1, monomeric Z α1-antitrypsin; lane 2, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days; lane 3, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days in the presence of 4.75% (v/v) DMSO; lanes 4–8, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days with 200μM, 100μM, 50μM, 20μM and 10μM CG respectively, all with a final concentration of 4.75% (v/v) DMSO. Fig. 2d (bottom right). 7.5 % (w/v) non-denaturing PAGE to assess the effect of decreasing concentrations of PC on the polymerisation of Z α1-antitrypsin. Lane 1, monomeric Z α1-antitrypsin; lane 2, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days; lane 3, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days in the presence of 4.75% (v/v) DMSO; lanes 4–8, Z α1-antitrypsin heated at 0.1mg/ml and 41°C for 3 days with 200μM, 100μM, 50μM, 20μM and 10μM PC respectively, all with a final concentration of 4.75% (v/v) DMSO.
Figure 3
Figure 3
The binding of CG and its analogues to Z α1-antitrypsin and other serpins. Fig. 3a (top left). 7.5 % (w/v) non-denaturing PAGE with each lane containing 2μg protein. The interaction between Z α1-antitrypsin and CG leads to an irreversible transition. Lane 1, Z α 1-antitrypsin; lane 2, Z α1-antitrypsin incubated with CG at 37°C for 3 days; lane 3, Z α1-antitrypsin incubated with CG at 37°C for 3 days and then dialysed in 3x1l PBS; lane 4, Z α 1-antitrypsin heated at 0.1mg/ml and 60°C for 3 hours; lane 5, Z α1-antitrypsin incubated with CG at 37°C for 3 days, dialysed in 3x1l PBS and then heated at 0.1mg/ml and 60°C for 3 hours. Fig. 3b (top right). CG does not inhibit the polymerisation of α1-antichymotrypsin. 7.5 % (w/v) non-denaturing PAGE with each lane containing 2μg protein. Lane 1, α1-antichymotrypsin; lane 2, α1-antichymotrypsin heated at 0.1mg/ml and 45°C for 3 days in 3.7% (v/v) ethanol; lane 3, α1-antichymotrypsin incubated at 0.1mg/ml and 45°C for 3 days with CG in 3.7% (v/v) ethanol. Fig. 3c (bottom left). Effect of the analogues of CG on the polymerisation of Z α1-antitrypsin. Lane 1, Z α1-antitrypsin; lane 2, Z α1-antitrypsin heated at 0.1mg/ml and 37°C for 3 days in 3.7% (v/v) ethanol; lanes 3–9, Z α1-antitrypsin heated at 0.1mg/ml and 37°C for 3 days in 100- fold molar excess of CG, LA, ENO, MS, SD, TR and WH respectively, all with a final concentration of 3.7% (v/v) ethanol. Fig. 3d (bottom right). Effect of the LTM on the polymerisation of Z α1-antitrypsin. Lane 1, Z α1-antitrypsin; lane 2 Z α1-antitrypsin heated at 0.1mg/ml and 37°C for 3 days in 3.7% (v/v) ethanol; lanes 3–7, Z α1-antitrypsin heated at 0.1mg/ml and 37°C for 3 days in 10μM, 7.5μM, 5μM, 2.5μM and 1μM of LTM respectively, all with a final concentration of 3.7% (v/v) ethanol.
Figure 4
Figure 4
The effect of CG on the secretion of Z α1-antitrypsin. Hepa1a cells were transiently transfected with Z α1-antitrypsin in the presence or absence of CG. Lanes 1–4, Z α1-antitrypsin radiolabeled with [35S]methionine and chased up to 5 hours. Immunoprecipitation experiments demonstrate that the half-life of the Z variant is between 3–5 hours. Lanes 5–8, transfected Hepa1a cells were treated with 100μM CG for 16h prior to the pulse-chase experiment. Z α1-antitrypsin was radiolabeled with [35S]methionine in the presence of CG and chased up to 5 hours. Glycan trimming of Z α1-antitrypsin was not detected in association with an increased rate of intracellular clearance.

Similar articles

See all similar articles

Cited by 30 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback