Cannabidiol and positive effects on object recognition memory in an in vivo model of Fragile X Syndrome: Obligatory role of hippocampal GPR55 receptors.

Cannabidiol (CBD), a non-psychotomimetic constituent of Cannabis sativa, has been recently approved for epileptic syndromes often associated with Autism spectrum disorder (ASD). However, the putative efficacy and mechanism of action of CBD in patients suffering from ASD and related comorbidities remain debated, especially because of the complex pharmacology of CBD. We used pharmacological, immunohistochemical and biochemical approaches to investigate the effects and mechanisms of action of CBD in the recently validated Fmr1-Δexon 8 rat model of ASD, that is also a model of Fragile X Syndrome (FXS), the leading monogenic cause of autism. CBD rescued the cognitive deficits displayed by juvenile Fmr1-Δexon 8 animals, without inducing tolerance after repeated administration. Blockade of CA1 hippocampal GPR55 receptors prevented the beneficial effect of both CBD and the fatty acid amide hydrolase (FAAH) inhibitor URB597 in the short-term recognition memory deficits displayed by Fmr1-Δexon 8 rats. Thus, CBD may exert its beneficial effects through CA1 hippocampal GPR55 receptors. Docking analysis further confirmed that the mechanism of action of CBD might involve competition for brain fatty acid binding proteins (FABPs) that deliver anandamide and related bioactive lipids to their catabolic enzyme FAAH. These findings demonstrate that CBD reduced cognitive deficits in a rat model of FXS and provide initial mechanistic insights into its therapeutic potential in neurodevelopmental disorders.

Nitrobindin versus myoglobin: A comparative structural and functional study.

Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-ß-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-ß-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O2/NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-ß-barrel heme-proteins.

Membrane Transporters Involved in Iron Trafficking: Physiological and Pathological Aspects.

Iron is an essential transition metal for its involvement in several crucial biological functions, the most notable being oxygen storage and transport. Due to its high reactivity and potential toxicity, intracellular and extracellular iron levels must be tightly regulated. This is achieved through transport systems that mediate cellular uptake and efflux both at the level of the plasma membrane and on the membranes of lysosomes, endosomes and mitochondria. Among these transport systems, the key players are ferroportin, the only known transporter mediating iron efflux from cells; DMT1, ZIP8 and ZIP14, which on the contrary, mediate iron influx into the cytoplasm, acting on the plasma membrane and on the membranes of lysosomes and endosomes; and mitoferrin, involved in iron transport into the mitochondria for heme synthesis and Fe-S cluster assembly. The focus of this review is to provide an updated view of the physiological role of these membrane proteins and of the pathologies that arise from defects of these transport systems.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation.

Nitrobindins (Nbs) represent an evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum-classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

In Silico Analysis of the Structural Dynamics and Substrate Recognition Determinants of the Human Mitochondrial Carnitine/Acylcarnitine SLC25A20 Transporter.

The Carnitine-Acylcarnitine Carrier is a member of the mitochondrial Solute Carrier Family 25 (SLC25), known as SLC25A20, involved in the electroneutral exchange of acylcarnitine and carnitine across the inner mitochondrial membrane. It acts as a master regulator of fatty acids ß-oxidation and is known to be involved in neonatal pathologies and cancer. The transport mechanism, also known as "alternating access", involves a conformational transition in which the binding site is accessible from one side of the membrane or the other. In this study, through a combination of state-of-the-art modelling techniques, molecular dynamics, and molecular docking, the structural dynamics of SLC25A20 and the early substrates recognition step have been analyzed. The results obtained demonstrated a significant asymmetry in the conformational changes leading to the transition from the c- to the m-state, confirming previous observations on other homologous transporters. Moreover, analysis of the MD simulations' trajectories of the apo-protein in the two conformational states allowed for a better understanding of the role of SLC25A20 Asp231His and Ala281Val pathogenic mutations, which are at the basis of Carnitine-Acylcarnitine Translocase Deficiency. Finally, molecular docking coupled to molecular dynamics simulations lend support to the multi-step substrates recognition and translocation mechanism already hypothesized for the ADP/ATP carrier.

A computational study of the structure and function of human Zrt and Irt-like proteins metal transporters: An elevator-type transport mechanism predicted by AlphaFold2.

The ZIP (Zrt and Irt-like proteins) protein family includes transporters responsible for the translocation of zinc and other transition metals, such as iron and cadmium, between the extracellular space (or the lumen of organelles) and the cytoplasm. This protein family is present at all the phylogenetic levels, including bacteria, fungi, plants, insects, and mammals. ZIP proteins are responsible for the homeostasis of metals essential for the cell physiology. The human ZIP family consists of fourteen members (hZIP1-hZIP14), divided into four subfamilies: LIV-1, containing nine hZIPs, the subfamily I, with only one member, the subfamily II, which includes three members and the subfamily gufA, which has only one member. Apart from the extracellular domain, typical of the LIV-1 subfamily, the highly conserved transmembrane domain, containing the binuclear metal center (BMC), and the histidine-rich intracellular loop are the common features characterizing the ZIP family. Here is presented a computational study of the structure and function of human ZIP family members. Multiple sequence alignment and structural models were obtained for the 14 hZIP members. Moreover, a full-length three-dimensional model of the hZIP4-homodimer complex was also produced. Different conformations of the representative hZIP transporters were obtained through a modified version of the AlphaFold2 algorithm. The inward and outward-facing conformations obtained suggest that the hZIP proteins function with an "elevator-type" mechanism.

Haptoglobin and the related haptoglobin protein: the N-terminus makes the difference.

Haptoglobin related protein (Hpr) is a component of the trypanosome lytic factor (TLF), a complex acting in the innate immune response against African trypanosomes. Like haptoglobin (Hp), Hpr binds hemoglobin (Hb) in the blood, but unlike Hp, Hpr does not bind the CD163 receptor. Moreover, unlike Hp, Hpr retains the N-terminal signal peptide that is required for the association with Apolipoprotein L-1 (ApoL-1), a component of the TLF complex. Here, the molecular model of human Hpr has been built based on the high sequence identity with human Hp (91%). The structural bases of Hpr:Hpr dimerization and Hpr recognition by Hb and Trypanosoma brucei brucei Hp receptor (TbHpHbR) have been analyzed in parallel with those of Hp:Hp, Hp:Hb, and TbHpHbR:Hp:Hb complexes. We show that the Cys33-Cys33 intermolecular disulfide bridge that stabilizes the Hp1:Hp1 complex is replaced by the Phe33, Pro34, and Phe48 hydrophobic core in the Hpr:Hpr dimer. Moreover, we show that the N-terminal peptide of Hpr participates in the stabilization of the Hpr:Hpr dimer. Thus, the N-terminal peptide seems to have been retained in Hpr to mediate its critical role in the human innate immunity towards Trypanosoma brucei brucei infection.Communicated by Ramaswamy H. Sarma.

Structural determinants of ligands recognition by the human mitochondrial basic amino acids transporter SLC25A29. Insights from molecular dynamics simulations of the c-state.

In mitochondria, metabolic processes require the trafficking of solutes and organic molecules, such as amino acids. This task is accomplished by the Mitochondrial Carrier Family members (also known as SLC25), among which the SLC25A29 is responsible for the translocation of basic amino acids. In this regard, nitric oxide levels originated by the arginine mitochondrial catabolism have been shown to strongly affect cancer cells' metabolic status. Furthermore, the metabolic disease saccharopinuria has been linked to a mitochondrial dysregulation caused by a toxic intermediate of the lysine catabolism. In both cases, a reduction of the activity of SLC25A29 has been shown to ameliorate these pathological conditions. However, no detailed structural data are available on SLC25A29. In the present work, molecular modelling, docking and dynamics simulations have been employed to analyse the structural determinants of ligands recognition by SLC25A29 in the c-state. Results confirm and reinforce earlier predictions that Asn73, Arg160 and Glu161, and Arg257 represent the ligand contact points I, II, and III, respectively, and that Arg160, Trp204 and Arg257 form a stable interaction, likely critical for ligand binding and translocation. These results are discussed in view of the experimental data available for SLC25A29 and other homologous carriers of the same family.

Binding of direct oral anticoagulants to the FA1 site of human serum albumin.

The anticoagulant therapy is widely used to prevent and treat thromboembolic events. Until the last decade, vitamin K antagonists were the only available oral anticoagulants; recently, direct oral anticoagulants (DOACs) have been developed. Since 55% to 95% of DOACs are bound to plasma proteins, the in silico docking and ligand-binding properties of drugs apixaban, betrixaban, dabigatran, edoxaban, and rivaroxaban and of the prodrug dabigatran etexilate to human serum albumin (HSA), the most abundant plasma protein, have been investigated. DOACs bind to the fatty acid (FA) site 1 (FA1) of ligand-free HSA, whereas they bind to the FA8 and FA9 sites of heme-Fe(III)- and myristic acid-bound HSA. DOACs binding to the FA1 site of ligand-free HSA has been validated by competitive inhibition of heme-Fe(III) recognition. Values of the dissociation equilibrium constant for DOACs binding to the FA1 site (ie, calc KDOAC ) derived from in silico docking simulations (ranging between 1.2 × 10-8 M and 1.4 × 10-6 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (ie, exp KDOAC ; ranging between 2.5 × 10-7 M and 2.2 × 10-6 M). In addition, this study highlights the inequivalence of rivaroxaban binding to mammalian serum albumin. Given the HSA concentration in vivo (~7.5 × 10-4 M), values of KDOAC here determined indicate that the formation of the HSA:DOACs complexes in the absence and presence of FAs and heme-Fe(III) may occur in vivo. Therefore, HSA appears to be an important determinant for DOACs transport.

Kinetic inequivalence between α and ß subunits of ligand dissociation from ferrous nitrosylated human haptoglobin:hemoglobin complexes. A comparison with O2 and CO dissociation.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αß dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of koff(NO)1 and koff(NO)2 (i.e., (6.4 ± 0.8) × 10-5 s-1 and (3.6 ± 0.6) × 10-5 s-1 for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10-5 s-1 and (3.1 ± 0.6) × 10-5 s-1 for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and ß subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and ß(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α1ß1 and α2ß2 dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and ß chains is closely similar for the O2, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.

DockingApp RF: A State-of-the-Art Novel Scoring Function for Molecular Docking in a User-Friendly Interface to AutoDock Vina.

MOTIVATION: Bringing a new drug to the market is expensive and time-consuming. To cut the costs and time, computer-aided drug design (CADD) approaches have been increasingly included in the drug discovery pipeline. However, despite traditional docking tools show a good conformational space sampling ability, they are still unable to produce accurate binding affinity predictions. This work presents a novel scoring function for molecular docking seamlessly integrated into DockingApp, a user-friendly graphical interface for AutoDock Vina. The proposed function is based on a random forest model and a selection of specific features to overcome the existing limits of Vina's original scoring mechanism. A novel version of DockingApp, named DockingApp RF, has been developed to host the proposed scoring function and to automatize the rescoring procedure of the output of AutoDock Vina, even to nonexpert users. RESULTS: By coupling intermolecular interaction, solvent accessible surface area features and Vina's energy terms, DockingApp RF's new scoring function is able to improve the binding affinity prediction of AutoDock Vina. Furthermore, comparison tests carried out on the CASF-2013 and CASF-2016 datasets demonstrate that DockingApp RF's performance is comparable to other state-of-the-art machine-learning- and deep-learning-based scoring functions. The new scoring function thus represents a significant advancement in terms of the reliability and effectiveness of docking compared to AutoDock Vina's scoring function. At the same time, the characteristics that made DockingApp appealing to a wide range of users are retained in this new version and have been complemented with additional features.

Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism.

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein's dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.

Computational Methods for the Identification of Molecular Targets of Toxic Food Additives. Butylated Hydroxytoluene as a Case Study.

Butylated hydroxytoluene (BHT) is one of the most commonly used synthetic antioxidants in food, cosmetic, pharmaceutical and petrochemical products. BHT is considered safe for human health; however, its widespread use together with the potential toxicological effects have increased consumers concern about the use of this synthetic food additive. In addition, the estimated daily intake of BHT has been demonstrated to exceed the recommended acceptable threshold. In the present work, using BHT as a case study, the usefulness of computational techniques, such as reverse screening and molecular docking, in identifying protein-ligand interactions of food additives at the bases of their toxicological effects has been probed. The computational methods here employed have been useful for the identification of several potential unknown targets of BHT, suggesting a possible explanation for its toxic effects. In silico analyses can be employed to identify new macromolecular targets of synthetic food additives and to explore their functional mechanisms or side effects. Noteworthy, this could be important for the cases in which there is an evident lack of experimental studies, as is the case for BHT.

Uncovering the structure and function of Pseudomonas aeruginosa periplasmic proteins by an in silico approach.

Pseudomonas aeruginosa is an opportunistic human pathogen highly relevant from a biomedical viewpoint. It is one of the main causes of infection in hospitalized patients and a major cause of mortality of cystic fibrosis patients. This is also due to its ability to develop resistance to antibiotics by various mechanisms. Therefore, it is urgent and desirable to identify novel targets for the development of new antibacterial drugs against Pseudomonas aeruginosa. In this work this problem was tackled by an in silico approach aimed at providing a reliable structural model and functional annotation for the Pseudomonas aeruginosa periplasmic proteins for which these data are not available yet. A total of 83 protein sequences were analyzed, and the corresponding structural models were built, leading to the identification of 32 periplasmic 'substrate-binding proteins', 14 enzymes and 4 proteins with different functions, including lipids and metals binding. The most interesting cases were found within the 'enzymes' group with the identification of a lipase, which can be regarded as a virulence factor, a protease involved in the assembly of ß-barrel membrane proteins and a l,d-transpeptidase, which could contribute to confer resistance to ß-lactam antibiotics to the bacterium.Communicated by Ramaswamy H. Sarma.

Neonicotinoid trapping by the FA1 site of human serum albumin.

Neonicotinoids are a widely used class of insecticides that target the acetylcholine recognition site of the nicotinic acetylcholine receptors in the central nervous system of insects. Although neonicotinoids display a high specificity for insects, their use has been recently debated since several studies led to the hypothesis that they may have adverse ecological effects and potential risks to mammals and even humans. Due to their hydrophobic nature, neonicotinoids need specific carriers to allow their distribution in body fluids. Human serum albumin (HSA), the most abundant plasma protein, is a key carrier of endogenous and exogenous compounds. The in silico docking and ligand binding properties of acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam to HSA are here reported. Neonicotinoids bind to multiple fatty acid (FA) binding sites, preferentially to the FA1 pocket, with high affinity. Values of the dissociation equilibrium constant for neonicotinoid binding FA1 of HSA (i.e., calc Kn ) derived from in silico docking simulations (ranging between 3.9 × 10-5 and 6.3 × 10-4 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (i.e., exp Kn ; ranging between 2.1 × 10-5 and 6.9 × 10-5 M). Accounting for the HSA concentration in vivo (~7.5 10-4 M), values of Kn here determined suggest that the formation of the HSA:neonicotinoid complexes may occur in vivo. Therefore, HSA appears to be an important determinant for neonicotinoid transport and distribution to tissues and organs, particularly to the liver where they are metabolized.

Contaminations in (meta)genome data: An open issue for the scientific community.

In recent years, the high throughput and the low cost of next-generation sequencing (NGS) technologies have led to an increase of the amount of (meta)genomic data, revolutionizing genomic research studies. However, the quality of sequencing data could be affected by experimental errors derived from defective methods and protocols. This represents a serious problem for the scientific community with a negative impact on the correctness of studies that involve genomic sequence analysis. As a countermeasure, several alignment and taxonomic classification tools have been developed to uncover and correct errors. In this critical review some of these integrated software tools and pipelines used to detect contaminations in reference genome databases and sequenced samples are reported. In particular, case studies of bacterial contaminations, contaminations of human origin, mitochondrial contaminations of ancient DNA, and cross contaminations are examined.

Anti-haemostatic compounds from the vampire snail Cumia reticulata: Molecular cloning and in-silico structure-function analysis.

Blood-feeding animals are known for their ability to produce bioactive compounds to impair haemostasis and suppress pain perception in the host. These compounds are extremely appealing for pharmacological development since they are generally very effective and specific for their molecular target. A preliminary RNA-Seq based characterization of the secretion from salivary and mid-oesophageal tissues of the vampire snail Cumia reticulata, revealed a complex mixture of feeding-related transcripts with potential anaesthetic and anticoagulant action. Based on the cloned full-length mRNAs, it was possible to verify the sequence of five genes encoding haematophagy-related products. The in silico modelled three-dimensional structure of each translational product was analysed to gain information on their potential biochemical activity. We have hereby validated and further investigated the assembled transcripts presumably involved in the antihaemostatic action, to improve our comprehensive understanding of this subset of the feeding secretion. The studied proteins included both inhibitors of primary haemostasis such as the vWFA domain-containing proteins, and compounds targeting different steps of the coagulation cascade, as e.g. the Turripeptide-like/protease inhibitor, the TFPI-like multiple Kunitz-type protease inhibitors, the Meprin-like metalloproteases and the Astacin/ShKT-like domain-containing proteins. All these molecules showed promising potential for pharmacological development.