Alfonso Trezza

The surge of flavonoids as novel, fine regulators of cardiovascular Cav channels

ABSTRACT Ion channels underlie a wide variety of physiological processes that involve rapid changes in cell dynamics, such as cardiac and vascular smooth muscle contraction. Overexpression or dysfunction of these membrane proteins are the basis of many cardiovascular diseases that represent the leading cause of morbidity and mortality for human beings. In the last few years, flavonoids, widely distributed in the plant kingdom, have attracted the interest of many laboratories as an emerging class of fine ion, in particular Cav, channels modulators. Pieces of in vitro evidence for direct as well as indirect effects exerted by various flavonoids on ion channel currents are now accumulating in the scientific literature. This activity may be responsible, at least in part, for the beneficial and protective effects of dietary flavonoids toward cardiovascular diseases highlighted in several epidemiological studies. Here we examine numerous studies aimed at analysing this feature of flavonoids, focusing on the mechanisms that promote their sometimes controversial activities at cardiovascular Cav channels. New methodological approaches, such as molecular modelling and docking to Cav1.2 channel &agr;1c subunit, used to elucidate flavonoids intrinsic mechanism of action, are introduced. Moreover, flavonoid‐membrane interaction, bioavailability, and antioxidant activity are taken into account and discussed.

Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca2+ antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (ICa1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1xa0μM (FRA77, GDE6, GDE19) and >10xa0μM (verapamil) in EA.hy926 cells, or >10xa0μM (FRA77, GDE6, GDE19) and at 100xa0μM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60xa0mM K+ (K60)-induced contraction at concentrations ≥1 and ≥3xa0μM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10xa0μM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited ICa1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies

&NA; To characterize the role of cAMP‐dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H‐89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H‐89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in‐depth analysis of H‐89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole‐cell patch‐clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H‐89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6–22 amide or enhanced by the PKA stimulators 6‐Bnz‐cAMP and 8‐Br‐cAMP. Inhibition of ICa1.2 by H‐89 appeared almost irreversible upon washout, was charge carrier‐ and voltage‐dependent, and antagonised by the Cav1.2 channel agonist (S)‐(‐)‐Bay K 8644. H‐89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage‐dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration‐dependent manner. H‐89 docked at the &agr;1C subunit in a pocket region close to that of (S)‐(‐)‐Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr‐1489. Finally, both high K+‐ and (S)‐(‐)‐Bay K 8644‐induced contractions of rings were fully reverted by H‐89. In conclusion, these results indicate that H‐89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA‐independent mechanism. Therefore, caution is recommended when interpreting experiments where H‐89 is used to inhibit vascular smooth muscle PKA.

A Computational Approach From Gene to Structure Analysis of the Human ABCA4 Transporter Involved in Genetic Retinal Diseases.

PurposenThe aim of this article is to report the investigation of the structural features of ABCA4, a protein associated with a genetic retinal disease. A new database collecting knowledge of ABCA4 structure may facilitate predictions about the possible functional consequences of gene mutations observed in clinical practice.nnnMethodsnIn order to correlate structural and functional effects of the observed mutations, the structure of mouse P-glycoprotein was used as a template for homology modeling. The obtained structural information and genetic data are the basis of our relational database (ABCA4Database).nnnResultsnSequence variability among all ABCA4-deposited entries was calculated and reported as Shannon entropy score at the residue level. The three-dimensional model of ABCA4 structure was used to locate the spatial distribution of the observed variable regions. Our predictions from structural in silico tools were able to accurately link the functional effects of mutations to phenotype. The development of the ABCA4Database gathers all the available genetic and structural information, yielding a global view of the molecular basis of some retinal diseases.nnnConclusionsnABCA4 modeled structure provides a molecular basis on which to analyze protein sequence mutations related to genetic retinal disease in order to predict the risk of retinal disease across all possible ABCA4 mutations. Additionally, our ABCA4 predicted structure is a good starting point for the creation of a new data analysis model, appropriate for precision medicine, in order to develop a deeper knowledge network of the disease and to improve the management of patients.

From in silico to in vitro: a trip to reveal flavonoid binding on the Rattus norvegicus Kir6.1 ATP-sensitive inward rectifier potassium channel

Background ATP-sensitive inward rectifier potassium channels (Kir), are a potassium channel family involved in many physiological processes. KATP dysfunctions are observed in several diseases such as hypoglycaemia, hyperinsulinemia, Prinzmetal angina–like symptoms, cardiovascular diseases. Methods A broader view of the KATP mechanism is needed in order to operate on their regulation, and in this work we clarify the structure of the Rattus norvegicus ATP-sensitive inward rectifier potassium channel 8 (Kir6.1), which has been obtained through a homology modelling procedure. Due to the medical use of flavonoids, a considerable increase in studies on their influence on human health has recently been observed, therefore our aim is to study, through computational methods, the three-dimensional (3D) conformation together with mechanism of action of Kir6.1 with three flavonoids. Results Computational analysis by performing molecular dynamics (MD) and docking simulation on rat 3D modelled structure have been completed, in its closed and open conformation state and in complex with Quercetin, 5-Hydroxyflavone and Rutin flavonoids. Our study showed that only Quercetin and 5-Hydroxyflavone were responsible for a significant down-regulation of the Kir6.1 activity, stabilising it in a closed conformation. This hypothesis was supported by in vitro experiments demonstrating that Quercetin and 5-Hydroxyflavone were capable to inhibit KATP currents of rat tail main artery myocytes recorded by the patch-clamp technique. Conclusion Combined methodological approaches, such as molecular modelling, docking and MD simulations of Kir6.1 channel, used to elucidate flavonoids intrinsic mechanism of action, are introduced, revealing a new potential druggable protein site.

A new integrated and interactive tool applicable to inborn errors of metabolism: Application to alkaptonuria

This paper describes our experience with the development and implementation of a database for the rare disease Alkaptonuria (AKU, OMIM: 203500). AKU is an autosomal recessive disorder caused by a gene mutation leading to the accumulation of homogentisic acid (HGA). Analogously to other rare conditions, currently there are no approved biomarkers to monitor AKU progression or severity. Although some biomarkers are under evaluation, an extensive biomarker analysis has not been undertaken in AKU yet. In order to fill this gap, we gained access to AKU-related data that we carefully processed, documented and stored in a database, which we named ApreciseKUre. We undertook a suitable statistical analysis by associating every couple of potential biomarkers to highlight significant correlations. Our database is continuously updated allowing us to find novel unpredicted correlations between AKU biomarkers and to confirm system reliability. ApreciseKUre includes data on potential biomarkers, patients quality of life and clinical outcomes facilitating their integration and possibly allowing a Precision Medicine approach in AKU. This framework may represent an online tool that can be turned into a best practice model for other rare diseases.

Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak

Graphical abstract Figure. No caption available. &NA; 3,5‐Di‐t‐butyl‐4‐hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+‐uptake, endowed with antioxidant and L‐type Ca2+ channel blocking activities. In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico. In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration‐dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time‐constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 &mgr;M), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration‐dependent manner. DTBHA, at concentrations higher than 10 &mgr;M, increased Ca2+ uptake in cardiac SR without affecting Ca2+‐dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin‐stimulated Ca2+ leak in cardiac SR and reduced caffeine‐induced, RyR2‐activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein. In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

In silico screening of anthraquinones from Prismatomeris memecyloides as novel phosphodiesterase type-5 inhibitors (PDE-5Is)

OBJECTIVEnPrismatomeris memecyloides Craib (Rubiaceae) is a medicinal plant traditionally used by ethnic minorities in Vietnam for the treatment of erectile dysfunction (ED). The aim of this study was to investigate the chemical compositions and screen in silico its possible inhibitory effect against PDE-5 which reduced cyclic guanosine-3,5-monophosphate (cGMP) levels and indirectly caused the male ED.nnnMETHODSnSeparation of natural compounds were carried out on chromatographic column with silica gel or reversed phase materials, eluting with different solvent gradients. The structures of all isolated compounds were elucidated on the basis of spectroscopic data (HR-MS, 1D/2D-NMR). Docking simulation study of compound (1-7) was performed by using flexible side chains protocol based on Iterated Local Search Global Optimizer Algorithm of AutoDock/Vina v.1.1.2. Pharmacokinetic parameters and toxicity prediction were also calculated by appropriate softwares.nnnRESULTSnFrom the methanol extract of roots of P. memecyloides collected in Vietnam, seven compounds including four anthraquinone/one anthraquinone glycoside namely damnacanthal (1), lucidin-ω-methyl ether (2), 3-methylalizarin (3), rubiadin-3-methyl ether (4), and 1-O-methylrubiadin 3-O-primeveroside (5) along with two iridoid glucosides, asperulosidic acid (6) and aitchisonide A (7) were isolated. The molecular modeling results showed that 5 anthraquinone compounds possess the lowest binding energies to PDE-5. The anthraquinone glucoside 1-O-methylrubiadin 3-O-primeveroside (5) potentially inhibited PDE-5 similarly to commercial PDE-5Is sildenafil (SLD) and tadalafil (TLD). Calculated pharmacokinetic results like pIC50,pred; miLogP, TPSA, enzyme inhibitory of anthraquinone glucoside (5) were similar and even higher to those of the commercial PDE-5 inhibitors. Especially the predictive toxicity of 1-O-methylrubiadin 3-O-primeveroside (5) was even lower than those of SLD and TLD.nnnCONCLUSIONnThis is the first study to find a scientific-based evidence for the ethnic use of P. memecyloides as medicinal plant for the treatment of ED. The result indicates that the anthraquinones (damnacanthal (1), lucidin-ω-methyl ether (2), 3-methylalizarin (3) and rubiadin-3-methyl ether (4)), especially anthraquinone glycoside (1-O-methylrubiadin 3-O-primeveroside (5)) are compounds of potential novel drug class for the ED treatment.