In silico model of the human ClC-Kb chloride channel: pore mapping, biostructural pathology and drug screening

Abstract

The human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target.

Mapping ligand binding pockets in chloride ClC‐1 channels through an integrated in silico and experimental approach using anthracene‐9‐carboxylic acid and niflumic acid

“…Moreover, the sequence alignment of ClC proteins (Supporting Information Figure S4) shows that at least four of the putative binding residues identified in this manuscript (G190, K231, F484 and F488) are conserved in other ClC proteins. These observations may help in the prediction of binding cavities in different channels, although they do not necessarily correlate with sensitivity to the same drug (Feng et al ., ; Zifarelli et al ., ; Louet et al ., ). Similarities exist for instance between ClC‐K and ClC‐1 binding pockets, thus supporting our conclusions.…”

“…Similarities exist for instance between ClC‐K and ClC‐1 binding pockets, thus supporting our conclusions. In particular, the recently published 3D model of the human channels highlighted the importance of residues D68 (D136 in ClC‐1), K165 (K231 in ClC‐1), F426 (F484 in ClC‐1) and M427 (M485 in ClC‐1) for ClC‐K function and revealed the existence of an extracellular binding cleft of the protein close to the chloride S ext position similar to pocket P1–P4 in ClC‐1 (Louet et al ., ). Remarkably, residue K165 in ClC‐Ks was found to be crucial for the binding of benzofuran derivatives and sartans to these channels (Liantonio et al ., ; Imbrici et al ., ) that are, conversely, not sensitive to 9‐AC.…”

“…Several in silico functional studies performed using different ClC homology models (from bacterial and eukaryotic transporters EcCLC, CmCLC and bovine ClC‐K channel structures) support the idea that channels belonging to the share similar, albeit not identical, structural features (Feng et al ., ; Zifarelli et al ., ; Louet et al ., ; Park et al ., ). Moreover, the sequence alignment of ClC proteins (Supporting Information Figure S4) shows that at least four of the putative binding residues identified in this manuscript (G190, K231, F484 and F488) are conserved in other ClC proteins.…”

“…G270 is replaced by an A and R421 is replaced by a K at the corresponding positions in ClC‐2, whereas L198 is replaced by an I at the corresponding position in ClC‐0. D136, K231, F484, M485 and F488 in ClC‐1 cavity P1 are equivalent to residues belonging to the extracellular binding site identified in ClC‐Kb channels (highlighted in green; Louet et al ., 2017). Identical residues are shown in red.…”

Functional Study of Novel Bartter’s Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine

“…Confocal microscopy and biochemical data support this idea, although we cannot exclude the occurrence of a minor biophysical defect. In particular, MD simulations using a model of human ClC-Kb build upon the 3D structure of the bovine channel, suggest that the rotation of V166 side chain together with the slight tilting of helix F are mainly responsible for the opening of the pore and release (or binding) of chloride ion at S ext position (Louet et al, 2017).…”