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.

BACKGROUND AND PURPOSE: Although chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is limited. ClC-1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC-1 blockers have been discovered, such as anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA), whereas no activator exists. The absence of a ClC-1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators. EXPERIMENTAL APPROACH: Here we provide an in-depth characterization of drug binding pockets in ClC-1 through an integrated in silico and experimental approach. We first searched putative cavities in a homology model of ClC-1 built upon an eukaryotic CLC crystal structure, and then validated in silico data by measuring the blocking ability of 9-AC and NFA on mutant ClC-1 channels expressed in HEK 293 cells. KEY RESULTS: We identified four putative binding cavities in ClC-1. 9-AC appears to interact with residues K231, R421 and F484 within the channel pore. We also identified one preferential binding cavity for NFA and propose R421 and F484 as critical residues. CONCLUSIONS AND IMPLICATIONS: This study represents the first effort to delineate the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins.

The 'preliminary neuropsychological battery'. An instrument to grade the cognitive level of minimally responsive patients.

An early, formalized cognitive evaluation of'minimally responsive' patients could be important in the planning of their care and rehabilitation as well as in providing a realistic prognosis and studying the modality of the cognitive recovery. A short, bedside, neuropsychologically-oriented test-battery, the Preliminary Neuropsychological Battery, a psychometric tool which enables the cognitive evaluation of these patients and of patients unable to give verbal or complex motor answers, has been devised. The BNP was administered to a sample of 40 head-injured patients and to a sample of 34 healthy subjects. The aim was (i) to evaluate its usefulness, and (ii) to study its correlation with cortical functions as assessed by a more extensive battery. The findings suggest that the BNP is useful for assessing the general cognitive level of head-injured post-comatose patients. It was able to detect patients deserving a wider, analytic, neuropsychological assessment. Attentional defects emerged as an important variable determining the BNP score.

A normative study on visual reaction times and two Stroop colour-word tests.

Two-hundred and nine normal subjects underwent a study aimed at providing norms for some attention tests currently used in neuropsychological examination. Norms were calculated taking into account the demographic variables. Our battery included single visuo-manual and go/no-go reaction times, and two versions of the Stroop Colour-Word Test. For the latter test, we employed a traditional version based on card presentation and a new version based on computer-assisted presentation. We evaluated the intercorrelation of the different tests, and studied the statistical regression models in order to adjust the original scores according to age, education and gender. Normative values were calculated following the Equivalent Scores method. The pattern of intercorrelation of our battery and the regression models of the scores are discussed with reference to the literature. The newly developed computer-assisted presentation of the Stroop test offers some advantages over the traditional version, especially with patients affected by spatial disorders.