Discovery of an inhibitor of Z-alpha1 antitrypsin polymerization

Abstract

Polymerization of the Z variant alpha-1-antitrypsin (Z-α1AT) results in the most common and severe form of α1AT deficiency (α1ATD), a debilitating genetic disorder whose clinical manifestations range from asymptomatic to fatal liver and/or lung disease. As the altered conformation of Z-α1AT and its attendant aggregation are responsible for pathogenesis, the polymerization process per se has become a major target for the development of therapeutics. Based on the ability of Z-α1AT to aggregate by recruiting the reactive center loop (RCL) of another Z-α1AT into its s4A cavity, we developed a high-throughput screening assay that uses a modified 6-mer peptide mimicking the RCL to screen for inhibitors of Z-α1AT polymer growth. A subset of compounds from the Library of Pharmacologically Active Compounds (LOPAC) with molecular weights ranging from 300 to 700 Da, was used to evaluate the assay's capabilities. The inhibitor S-(4-nitrobenzyl)-6-thioguanosine was identified as a lead compound and its ability to prevent Z-α1AT polymerization confirmed by secondary assays. To further investigate the binding location of S-(4-nitrobenzyl)-6-thioguanosine, an in silico strategy was pursued and the intermediate α1AT M* state modeled to allow molecular docking simulations and explore various potential binding sites. Docking results predict that S-(4-nitrobenzyl)-6-thioguanosine can bind at the s4A cavity and at the edge of β-sheet A. The former binding site would directly block RCL insertion whereas the latter site would prevent β-sheet A from expanding between s3A/s5A, and thus indirectly impede RCL insertion. Altogether, our investigations have revealed a novel compound that inhibits the formation of Z-α1AT polymers, as well as in vitro and in silico strategies for identifying and characterizing additional blocking molecules of Z-α1AT polymerization.

Fig 1. Kinetic diagram of bPEG-peptide binding to α1AT.

(A) Four micrograms per well of attached (■) Z-α1AT or (●) M-α1AT were incubated for various times in presence of 38.4 μM bPEG-peptide. (B) Z-α1AT was incubated in presence of 5% DMSO and bPEG-peptide for 16 h. Errors bars reflect the standard deviation of three replicates.

Fig 2. Pattern of inhibition resulting from the screening of 80 unknown LOPAC compounds.

A 96-well plate was coated with 4 μg/well of Z-α1AT and incubated for 16 h with 100 μM of various compounds and 38.4 μM of bPEG-peptide. The black arrow indicates the compound that corresponds to S-(4-nitrobenzyl)-6-thioguanosine and gives an inhibition effect of 67 ± 2% and. The error bars are the standard deviation of three individual experiments.

Fig 3. S-(4-nitrobenzyl)-6-thioguanosine inhibits bPEG-peptide binding to Z-α1AT.

(A) Dose-response curves were assayed for various concentrations of (●) S-(4-nitrobenzyl)-6-thioguanosine and (■) its homologue S-(4-nitrobenzyl)-6-thioinosine. (B) Chemical structures of (left) S-(4-nitrobenzyl)-6-thioguanosine and (right) S-(4-nitrobenzyl)-6-thioinosine. The errors bars are the standard deviation of an experiment conducted in triplicate.

Fig 4. Effect of S-(4-nitrobenzyl)-6-thioguanosine on Z-α1AT polymerization.

The protein was incubated with (●) or without (■) 100 μM of S-(4-nitrobenzyl)-6-thioguanosine for various time at 37°C. The error bars are the standard deviation of three separate experiments.

Fig 5. The three models of α1AT protein.

(Top left) Structure of wild type (PDB: 1QLP) (Top right) Structure of Z-mutant (PDB: 3T1P) (Bottom middle) Intermediate M* model with an expanded β-sheet A (retained from structure 3T1P), RCL not inserted into the RCL cavity (retained from structure 1QLP), and C_term loop inserted into β-sheet B (retained from structure 1QLP). β-sheet A is colored red and β-sheet B is colored green. Shades of green and red distinguish discontinuous fragments from the same initial crystal structure (light/dark green for 1QLP fragments and light/dark red for 3T1P fragments) used to generate the M* model. Grey colored regions represent residues that were not used to generate the M* model.

Fig 6. The fragments of structures 1QLP (green) and 3T1P (red) used to homology model the M* intermediate state of α1AT.

β-sheet A is the red beta sheet across the top half of the model and β-sheet B is the green beta sheet across the bottom of the model. Residue numbers at the start and end of each fragment transition are labeled with an arrow in the N_term to C_term direction. Shades of green and red distinguish discontinuous fragments from the same initial crystal structure (light/dark green for 1QLP fragments and light/dark red for 3T1P fragments).

Fig 7. Binding Sites for S-(4-nitrobenzyl)-6-thioguanosine.

Two protein ribbon models are shown for each structure: (A) 3CWM, (B) 3T1P and (C) M* Model. The left model and right representations in each panel are rotated 90° with respect to one another. The best binding poses for S-(4-nitrobenzyl)-6-thioguanosine at each available binding site are shown with space filling atoms with the carbon atoms colored green. (Purple) Strands 3 and 5 from β-sheet A. (Dark blue) C_term loop within β-sheet B. (Light blue) RCL. (Orange) Residues of the RCL corresponding to the analogous 6-mer peptide.