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.

ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.

KEY POINTS: Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. ABSTRACT: Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

Estimation of the binding free energy by Linear Interaction Energy models.

Since Hansch's extra thermodynamic multi-parameter approach, originally coined as Linear Free Energy Relationship, great efforts in medicinal chemistry have been made to properly estimate the binding free energy. Despite the often small amount, its value is however very critical in determining a successful binding. As a result, its correct estimation may provide a guide for a prospective rational drug design. The calculation of the absolute binding free energies is however a very challenging task as it requires a rigorous treatment of a number of physical terms that are both very time demanding and to some extent not immediately interpretable. In view of this, the introduction of some numerical approximations has permitted to develop the so called Linear Interaction Energy method that, at present, constitutes the best compromise among accuracy, speed of computation and easy interpretation. The initially developed Linear Interaction Energy method was subsequently revisited and several important improvements have been made. Significant examples are the Extended Linear Response, the surface generalized Born LIE, the molecular mechanics generalized Born surface area, the linear interaction energy in continuum electrostatics as well as its quantum mechanics variant. Principles and selected applications of these methods will be herein reviewed.

New strategies in the chemotherapy of leukemia: eradicating cancer stem cells in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia-positive chromosome deriving from a translocation between chromosomes 22 and 9. The oncogenic product of this aberrant chromosome is the constitutively active tyrosine kinase BCR-ABL that is responsible for leukemic cell growth, proliferation and survival driven by the dysregulation of a large array of signal transduction pathways. Inhibition of BCR-ABL with tyrosine kinase inhibitors proved to be an efficient therapy of CML in the chronic phase. Unfortunately, the impressive success of BCR-ABL inhibitors as front-line therapy in CML has been tempered by problems of disease persistence or relapse arising from different mechanisms, including mutations in the kinase domain of the enzyme BCRABL and mechanisms independent from BCR-ABL activity. Growing evidence has also suggested a pivotal role of persistent leukemic cancer stem cells, characterized by high self-renewal and pluripotency, in CML maintenance and/or relapse. The present review deals with the most recent advances in this challenging field and focuses on the development of new drugs and therapeutic approaches to eradicate the subtle and dangerous leukemic stem cells responsible for maintaining and sustaining tumor growth.

D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.

Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness.

Targeting monoamine oxidases with multipotent ligands: an emerging strategy in the search of new drugs against neurodegenerative diseases.

The socioeconomic burden of multi-factorial pathologies, such as neurodegenerative diseases (NDs), is enormous worldwide. Unfortunately, no proven disease-modifying therapy is available yet and in most cases (e.g., Alzheimer's and Parkinson's disease) the approved drugs exert only palliative and symptomatic effects. Nowadays, an emerging strategy for the discovery of disease-modifying drugs is based on the multi-target directed ligand (MTDL) design, an innovative shift from the traditional approach one-drug-one-target to the more ambitious one-drug-more-targets goal. Herein, we review the discovery strategy, the mechanism of action and the biopharmacological evaluation of multipotent ligands exhibiting monoamine oxidase (MAO) inhibition as the core activity with a potential for the treatment of NDs. In particular, MAO inhibitors exhibiting additional acetylcholinesterase (AChE) or nitric oxide synthase (NOS) inhibition, or ion chelation/antioxidant-radical scavenging/anti-inflammatory/A2A receptor antagonist/APP processing modulating activities have been thoroughly examined.

BCR-ABL inhibitors in chronic myeloid leukemia: process chemistry and biochemical profile.

Chronic myeloid leukemia (CML) is a myeloproliferative disease originating from a constitutively active tyrosine kinase, called BCR-ABL, expressed by an oncogene resulting from a reciprocal translocation between chromosome 9 and chromosome 22, coded as (t[9,22][q34;q11]). Inhibition of BCR-ABL with tyrosine kinase inhibitors (TKI) proved to be an efficient targeted therapy of Philadelphia-positive (Ph+) CML in the chronic phase. This review mainly addresses the synthetic pathways and process chemistry leading to the large scale preparation for pre-clinical demands and clinical supply of the three TKIs approved for Ph+ CML, i.e., imatinib, dasatinib and nilotinib and three more investigational drugs, i.e., bosutinib, ponatinib and bafetinib. Recent progress on the biochemical profiling of the six examined TKIs has been also reported.

Quinolizidinyl derivatives of bi- and tricyclic systems as potent inhibitors of acetyl- and butyrylcholinesterase with potential in Alzheimer's disease.

On the pattern of the potent and selective butyrylcholinesterase (BChE) inhibitors ethopropazine and Astra1397, sets of quinolizidinyl derivatives of bi- and tricyclic (hetero)aromatic systems were studied as dual, or BChE-selective inhibitors. All compounds exhibited activity against both cholinesterases, but inhibition of BChE was generally stronger, with submicromolar IC50 values for most of them (e.g. 15: IC50 versus BChE=0.15 µM; SI=47). However, in a subset of quinolizidinyl derivatives of 6-hydroxycoumarin an inverted selectivity for acetylcholinesterase (AChE) was observed (e.g. 46: IC50 versus AChE=0.35 µM; SI=0.06). Docking studies furnished a sound interpretation of the observed different enzyme activity. Several of the studied compounds have shown, in the past, additional pharmacological properties (as antagonism on presynaptic muscarinic autoreceptor; inhibition of enkephaline aminopeptidase and antipsychotic activity) of some relevance in Alzheimer's disease, and may, therefore, represent hits for the development of interesting single-entity multi-target drugs.

Cytisine derivatives as high affinity nAChR ligands: synthesis and comparative molecular field analysis.

A number of new N-substituted cytisine derivatives were prepared and tested, along with similar compounds already described by us and others, as high affinity neuronal acetylcholine receptor ligands. Structure-affinity relationships were discussed in the light of our recently proposed pharmacophore model for nicotinic receptor agonists. The most significant physicochemical interactions modulating the receptor-ligand binding were detected at the three dimensional (3D) level by means of comparative molecular field analysis (CoMFA). The best predictive PLS model was a single-field steric model showing good statistical figures: n = 17, Q2 = 0.717, s(ev) = 0.566, r2 = 0.942, s = 0.275.

Ligands of neuronal nicotinic acetylcholine receptor (nAChR): inferences from the Hansch and 3-D quantitative structure-activity relationship (QSAR) Models.

Neuronal acetylcholine ion channel receptors (nAChRs), that exist in several subtypes resulting from a different organisation of various subunits around the central ion channel, are involved in a variety of functions and disorders of the central nervous system. There is evidence to implicate a deficit of nAChRs in the symptomatology of severe neurologic pathologies, such as Alzheimer s and Parkinson s diseases. Reliable three-dimensional structures of nAChRs are not available yet, and this hampers adopting structure-based approaches in designing new ligands. Also pharmacophore models are not reliable enough to be used in ligand-based approaches to drug design and little structure-activity work has been reported so far. This paper deals with structure-activity relationships of a wide series of nicotinic ligands. It provides results from a study of the quantitative structure activity relationships (QSARs) based on literature data of about 270 nicotinic agonists, belonging to various chemical classes. The QSAR study was carried out by using either a classical Hansch approach or a Comparative Molecular Field Analysis (CoMFA). Within each congeneric series, Hansch-type equations revealed detrimental steric effects as the factors mainly modulating the receptor affinity, whereas CoMFA allowed us to merge progressively models obtained for each class of congeners into a more general one that showed good cross-validation statistics. The CoMFA coefficient isocontour maps illustrated, at the 3-D level, the most relevant interactions responsible for a high receptor affinity, whereas the robustness of the global three-dimensional QSAR/CoMFA (n = 206, q(2) = 0.749, r(2) = 0.847, s= 0.600) model was supported by the high value of the prediction statistics (r(2)pred = 0.961) and confirmed by the satisfactory predictions of the affinity data of an external set of 18 recently published ligands with chemical structures even quite diverse from those included in the training set.

Neuronal nicotinic receptor agonists: a multi-approach development of the pharmacophore.

Based on the results obtained with different automated computational approaches as applied to the study of eleven high-affinity agonists of the neuronal nicotine acetylcholine receptor (nAChR), belonging to different chemical classes, new relevant features were detected which complement the existing pharmacophores. Convergent results from DISCO (Distance Comparison), QXP (Quick Explore), Catalyst/HipHop, and MIPSIM (Molecular Interaction Potential Similarity) allowed us to identify and locate, in a well defined spatial arrangement, three geometrically independent key structural features: (i) a positively charged nitrogen atom for ionic or hydrogen bond interactions, (ii) a lone pair of the pyridine nitrogen or a specific lone pair of a carbonyl oxygen, as a hydrogen bond acceptor, and (iii) a centre of a hydrophobic area generally occupied by aliphatic cycles. The pharmacophore presented herein, along with predictive 2D and 3D QSAR models recently developed in our group, could represent valuable computational tools for the design of new nAChR agonists having therapeutical potential.