Design and SAR Analysis of Covalent Inhibitors Driven by Hybrid QM/MM Simulations

Quantum mechanics/molecular mechanics (QM/MM) hybrid technique is emerging as a reliable computational method to investigate and characterize chemical reactions occurring in enzymes. From a drug discovery perspective, a thorough understanding of enzyme catalysis appears pivotal to assist the design of inhibitors able to covalently bind one of the residues belonging to the enzyme catalytic machinery. Thanks to the current advances in computer power, and the availability of more efficient algorithms for QM-based simulations, the use of QM/MM methodology is becoming a viable option in the field of covalent inhibitor design. In the present review, we summarized our experience in the field of QM/MM simulations applied to drug design problems which involved the optimization of agents working on two well-known drug targets, namely fatty acid amide hydrolase (FAAH) and epidermal growth factor receptor (EGFR). In this context, QM/MM simulations gave valuable information in terms of geometry (i.e., of transition states and metastable intermediates) and reaction energetics that allowed to correctly predict inhibitor binding orientation and substituent effect on enzyme inhibition. What is more, enzyme reaction modelling with QM/MM provided insights that were translated into the synthesis of new covalent inhibitor featured by a unique combination of intrinsic reactivity, on-target activity, and selectivity

Anisakis spp. larvae in different kinds of ready to eat products made of anchovies (Engraulis encrasicolus) sold in Italian supermarkets

In this study the occurrence of visible anisakid larvae in semi-preserved anchovy products sold on the Italian market was investigated. Totally, 107 ready to eat products (33 salted-ripened, 49 in oil and 25 marinated) were sampled. Each sample was digested, then the digested material was observed under natural and UV light. Parasites were counted, collected and microscopically identified to genus level. A representative subset was molecularly identified using the cox2 gene. At least one visible Anisakis sp. larva was found in 54.2% of the total 107 products analysed and totally 1283 dead larvae were collected. Anisakis sp. larvae were found in all the 33 salted products and 1139 (88.8%) larvae were collected, with a range of 1â105 parasites per product. Larval density per gram was 0.13. Anisakis sp. larvae were found in 49.0% of the products in oil and 143 (11.1%) larvae were isolated, with a range of 0â28 and a density of 0.03. Only 1 larva was found in the 25 marinated products (4.0%, density 0.00). A highly significant difference between all the product categories in respect of number of larvae per product, frequency of products contaminated by at least one larva and larval density per gram was found. Within the subset of larvae molecularly analysed (n = 122), 92 (75.4%) were identified as A. pegreffii and 30 (24.6%) as A. simplex. This study showed that semi-preserved anchovy products heavily contaminated with Anisakis spp. larvae reach the market. Beyond the negligible risk for anisakidosis, the presence of dead visible parasites may cause immediate rejection in consumers. In addition, the potential risk related to allergic reactions in sensitized individuals needs to be further assessed. In order to avoid commercialization of obviously contaminated products, fresh anchoviesâ batches intended for the production of such products should be accurately selected by the processing industry applying inspection methods

Different roles for the acyl chain and the amine leaving group in the substrate selectivity of N-Acylethanolamine acid amidase

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations

Protein-Protein Interaction Inhibitors Targeting the Eph-Ephrin System with a Focus on Amino Acid Conjugates of Bile Acids

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings

N-Acylethanolamine Acid Amidase (NAAA): Mechanism of Palmitoylethanolamide Hydrolysis Revealed by Mechanistic Simulations

The N-terminal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) catalyzes the hydrolytic deactivation of the lipid messenger palmitoylethanolamide (PEA), with optimal activity at acidic pH. Using the crystal structure of human NAAA as a starting point, we investigated the catalytic mechanism of PEA hydrolysis with a multiscale approach based on classic molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) simulations coupled with enhanced sampling and path-collective variables (PCVs). The proton configuration of the catalytic nucleophile, Cys126, and of the surrounding carboxylates was critical to preserve the active site architecture. A stable Michaelis complex was then used to reconstruct the free-energy surfaces of NAAA acylation and deacylation during PEA hydrolysis. Acylation emerged as the critical step, with Cys126 acting both as an acid, to protonate the ethanolamine leaving group, and as a nucleophile, to attack the PEA carbonyl carbon. The ethanol fragment of PEA did not appear to play an indispensable role in acylation, a result further supported by kinetic experiments showing that NAAA hydrolyzes palmitoyl methyl amide (PMA) with high catalytic efficiency. Our multiscale approach identified a distinctive protonation state and catalytic mechanism for NAAA which accounts for pH-dependent activity, mutagenesis data, and mechanism of covalent inhibitors

N-Acylethanolamine Acid Amidase (NAAA): Structure, Function, and Inhibition

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation and to NAAA itself as a target for anti-inflammatory medicines. The present Perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and noncovalent agents; and finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders

Phenotype Screening of an Azole-bisindole Chemical Library Identifies URB1483 as a New Antileishmanial Agent Devoid of Toxicity on Human Cells

We report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC50 values ranging from 4 to 10 μM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells

UniPR1331: small Eph/ephrin antagonist beneficial in intestinal inflammation by interfering with type-B signaling

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn’s disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses

Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene

G.M.P. and E.C. contributed equally to this work. G.M.P. acknowledges the financial support from Fondazione Cariplo, grant no. 2018-0979. The authors thank the financial support from the EU Horizon 2020 Research and Innovation Programme under Grant Agreement No. 643238 (SYNCHRONICS). The authors also thank Dr. Daniele Viola for helping with the analysis of the TA data.The non‐covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo‐stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans→cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane.Fondazione Cariplo. Grant Number: 2018‐0979EU Horizon 2020 Research and Innovation Programme. Grant Number: 64323

Examining the effects of sodium ions on the binding of antagonists to dopamine D2 and D3 receptors

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo