Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

Figures

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  Fig. 1. 3D pocket localization of the predicted Sites 1–3 and blind-docking/screening and selection of potential hit compounds. [A] Blind docking results showing all 1615 FDA compounds binding across various CLIC4 cavities, including the predicted Site 1 (magenta), Site 2 (red) and Site 3 (yellow) all shown in surface representation [B] Energy filtering results of the top 50 hit compounds binding to their preferred target sites on CLIC4. [C] Plot showing the interaction energies of the respective compounds in the top 50 set.

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  Fig. 2. Binding modes and target sites of top 10 potential inhibitors of CLIC4. 0. IAA94 1. Digoxin 2. AMPhB 3. Ergotamine 4. Demeclocycline 5. RAPA6. Natamycin 7. Ponatinib 8. Telithromycin 9. Ciclesonide 10. Mepron.

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  Fig. 3. 2D structures of potential CLIC4 inhibitors. A. IAA94 (control) B. AMPhB and C. Rapamycin.

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  Fig. 4. Binding of AMPhB and RAPA to CLIC4. 1H–15N TROSY HSQC spectra of CLIC4 in the absence (black) and presence of two molar excess of IAA94 (A, cyan), RAPA (B, magenta) and AMPhB (C, red). Regions showing significant chemical shift changes upon addition of RAPA and AMPhB are expanded and compared to similar regions in the IAA94-bound spectrum.

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  Fig. 5. AMPhB and RAPA inhibits CLIC4 membrane translocation and endothelial cell migration. Top Panel: (A) HUVECs (Control) immunostained for CLIC4 (red), VE-cadherin (cell junctions, green) and DAPI (nucleus, blue) under confluent conditions show the presence of CLIC4 within cytosol and nucleus with the maintenance of endothelial cell barrier indicated by the tight cell-cell junctions. (B) HUVECs treated with 0.003% hydrogen peroxide (H2O2) significantly increased the expression levels of CLIC4, including at the plasma membrane with disruption to the barrier functions. These effects were partially reversed when treated with (C)10 μM AMPhB or (D) 10 μM RAPA as indicated by decreased CLIC4 staining in the cells, especially at the plasma membrane. Scale bar = 200 µM. Bottom Panel Representative images of HUVECs “scratch-wounded” using a universal 10 μl pipette tip (scratched area indicated by the broken lines) that are treated with (E) control adenovirus (AdControl) or (F) adenovirus to overexpress CLIC4 (AdCLIC4) alone or with (G) 10 μM AMPhB or (F) 10 μM RAPA. Migration pattern of cells into the ‘scratch-wounded’ area indicate that both drugs attenuated AdCLIC4- induced cell migration.…

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  Fig. 6. RMSD plots for unbound and ligand-bound CLIC4. [A] Whole time-frame (500 ns) RMSD plot showing high deviations in unbound CLIC4 relative to stable ligand-bound systems. [B] RMSD distribution density plots for whole time RMSD. [C] Final equilibrated RMSD plots obtained from the terminal 50 ns [D] RMSD distribution density plots for final equilibrated RMSD.

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  Fig. 7. RoG and RMSF plots measuring variations in structural compactness and residual fluctuations [A] Density distribution plots of equilibrated RoGs [B] Heatmap showing fluctuations among of constituent residues [C] equilibrated RoG line plots [D] RMSF line plot mapping out corresponding residues and their degree of fluctuations as indicated in B.

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  Fig. 8. Conformational RMSD, RoG and RMSF analyses for important subsites on CLCI4 with notable ligand effects [A] proximal catalytic (GSH) loop [B] connecting loop [C] extended catalytic loop [D] N-to-C terminal connecting loop [E] flexible foot loop.

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  Fig. 9. 2D mapping of residue interactions. Residue mapping for [A] CLIC4 and AMPhB [B] CLIC4 and RAPA[C] CLIC4 and IAA94. 3D superposition of unbound and [D] AMPhB [B] RAPA[E] IAA94-bound CLIC4 showing alterations at key regions.