Domenico Alberga

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations.

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373-385], our analysis on 200ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.

Challenging AQP4 druggability for NMO-IgG antibody binding using molecular dynamics and molecular interaction fields.

Neuromyelitis optica (NMO) is a multiple sclerosis-like immunopathology disease affecting optic nerves and the spinal cord. Its pathological hallmark is the deposition of a typical immunoglobulin, called NMO-IgG, against the water channel Aquaporin-4 (AQP4). Preventing NMO-IgG binding would represent a valuable molecular strategy for a focused NMO therapy. The recent observation that aspartate in position 69 (D69) is determinant for the formation of NMO-IgG epitopes prompted us to carry out intensive Molecular Dynamics (MD) studies on a number of single-point AQP4 mutants. Here, we report a domino effect originating from the point mutation at position 69: we find that the side chain of T62 is reoriented far from its expected position leaning on the lumen of the pore. More importantly, the strength of the H-bond interaction between L53 and T56, at the basis of the loop A, is substantially weakened. These events represent important pieces of a clear-cut mechanistic rationale behind the failure of the NMO-IgG binding, while the water channel function as well as the propensity to aggregate into OAPs remains unaltered. The molecular interaction fields (MIF)-based analysis of cavities complemented MD findings indicating a putative binding site comprising the same residues determining epitope reorganization. In this respect, docking studies unveiled an intriguing perspective to address the future design of small drug-like compounds against NMO. In agreement with recent experimental observations, the present study is the first computational attempt to elucidate NMO-IgG binding at the molecular level, as well as a first effort toward a less elusive AQP4 druggability.

Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

Background: Neuromyelitis optica autoantibodies target the aquaporin-4 (AQP4) aggregate named orthogonal arrays of particles (OAP). Results: Mutation of AQP4 aspartate 69 (Asp69) impairs NMO-IgG binding leaving the water channel function unaltered as well as its aggregation into OAPs. Conclusion: Asp69 is the key determinant for the formation of NMO-IgG epitopes. Significance: Such evidence provides additional clues on NMO pathogenesis. Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp69) of TM2 for NMO-IgG epitope assembly. Mutation of Asp69 to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp69 mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D69H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp69 is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

Docking-based classification models for exploratory toxicology studies on high-quality estrogenic experimental data

BACKGROUND The ethical and practical limitation of animal testing has recently promoted computational methods for the fast screening of huge collections of chemicals. RESULTS The authors derived 24 reliable docking-based classification models able to predict the estrogenic potential of a large collection of chemicals provided by the US Environmental Protection Agency. Model performances were challenged by considering AUC, EF1% (EFmax = 7.1), -LR (at sensitivity = 0.75); +LR (at sensitivity = 0.25) and 37 reference compounds comprised within the training set. Moreover, external predictions were made successfully on ten representative known estrogenic chemicals and on a set consisting of >32,000 chemicals. CONCLUSION The authors demonstrate that structure-based methods, widely applied to drug discovery programs, can be fairly adapted to exploratory toxicology studies.

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

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.

Trimethoxybenzanilide-based P-glycoprotein modulators: An interesting case of lipophilicity tuning by intramolecular hydrogen bonding

One of the principal reasons for the chemotherapy failure is the overexpression of drug efflux pumps, ABCB1 (also known as MDR1 or P-gp) and ABCC1 (also known as MRP1), whose inhibition remains a priority to circumvent drug resistance. We have recently shown a clear trend between lipophilicity and P-glycoprotein inhibitory activity for a class of galloyl-based modulators targeting P-glycoprotein and MRP1. Herein we report a new series of polymethoxy benzamides, whose lipophilicity was modulated through the establishment of an intramolecular hydrogen bond (IMHB) which allows reaching of P-gp inhibitory activity at the submicromolar IC50 level. The present study provides a strong rationale for candidates in the presence of IMHB as a key element for a high P-gp inhibitory activity.

Novel chemotypes targeting tubulin at the colchicine binding site and unbiasing P-glycoprotein

Retrospective validation studies carried out on three benchmark databases containing a small fraction (that is 2.80%) of known tubulin binders permitted us to develop a computational platform very effective in selecting easier manageable subsets showing by far higher percentages of actives (about 25%). These studies relied on the hierarchical application of multilayer in silico screenings employing filters implying molecular shape similarity; a structure-based pharmacophore model and molecular docking campaigns. Building on this validated approach, we performed intensive prospective studies to screen a large chemical collection, including up to 3.7 millions of commercial compounds, to across an unexplored and patent space in the search of novel colchicine binding site inhibitors. Our investigation was successful in identifying a pool of 31 initial hits showing new molecular scaffolds (such as 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one and pyrazolo[1,5-a]pyrimidine). This panel of new hits resulted antiproliferative activity in the low μM range towards MCF-7 human breast cancer, HepG2 human liver cancer, HeLa human ovarian cancer and SHSY5Y human glioblastoma cell lines as well as interesting concentration-dependent inhibition of tubulin polymerization assessed through fluorescence polymerization assays. Unlike typical tubulin inhibitors, a satisfactorily low sensitivity towards P-gp was also measured in bi-directional transport studies across MDCKII-MDR1 cells for a selected subset of seven compounds.

Organic bioelectronics probing conformational changes in surface confined proteins

The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results.

Strategies of Virtual Screening in Medicinal Chemistry

Virtualscreeningrepresentsaneffectivecomputationalstrategytorise-upthechancesoffindingnew bioactivecompoundsbyacceleratingthetimeneededtomovefromaninitialintuitiontomarket. Classically, themostpursuedapproaches relyon ligand-andstructure-based studies, the former employedwhenstructuraldatainformationaboutthetargetismissingwhilethelatteremployedwhen X-ray/NMRsolvedorhomologymodelsareinsteadavailableforthetarget.Theauthorswillfocus onthemostadvancedtechniquesappliedinthisarea.Inparticular,theywillsurveythekeyconcepts ofvirtualscreeningbydiscussinghowtoproperlyselectchemicallibraries,howtomakedatabase curation,howtoapplyingand-andstructure-basedtechniques,howtowiselyusepost-processing methods.EmphasiswillbealsogiventothemostmeaningfuldatabasesusedinVSprotocols.For theeaseofdiscussionseveralexampleswillbepresented. KeywoRdS Drug Discovery, Ligandand Structure-based Approaches, Molecular Database, Virtual Screening

Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies

Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss‐of‐function mutations in the skeletal muscle ClC‐1 channel. We report a novel ClC‐1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I‐J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch‐clamp electrophysiology on T335N and ClC‐1 wild‐type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC‐1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I‐J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K+ channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.—Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj, K., Caloiero, R., Alberga, D., Marsano, R.M., Ricci, G., Siciliano, G., Nicolotti, O., Mora, M., Bernasconi, P., Desaphy, J.‐F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new CIC‐1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies. FASEB J. 30, 3285–3295 (2016). www.fasebj.org