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The flavonoid chrysin is an effective vascular Ca V 1.2 channel blocker. The aim of this study was to explore the chemical space around chrysin to identify the structural features that can be modified to develop novel and more effective blockers. Four derivatives (Chrysin 1-4) were synthesised and a functional, electrophysiology and molecular docking approach was pursued to assess their binding mode to Ca V 1.2 channels and their activity in vascular preparations. Methylation of the 5- and 7-OH of the chrysin backbone caused a marked reduction of the Ca 2+ antagonistic potency and efficacy. However, C-8 derivatives showed biophysical features similar to those of the parent compound and, like nicardipine, bound with high affinity to and stabilised the Ca V 1.2 channel in its inactivated state. The vasorelaxant effects of the four derivatives appeared vessel-specific, addressing the molecules' derivatization towards different targets. In conclusion, the scaffold of chrysin may be considered a valuable starting point for the development of innovative vascular Ca V 1.2 channel blockers., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Repurposing saffron ( Crocus sativus ) waste presents a sustainable strategy for generating high-value products within the bioeconomy framework. Typically, flower components are discarded after stigma harvest, resulting in significant waste-350 kg of tepals per kilogram of stigmas. This research employed a comprehensive approach, integrating bioactivity studies (in vitro and in silico) with Life Cycle Assessment (LCA) evaluations, to extract and assess bioactive compounds from C. sativus tepals sourced in Tuscany, Italy. Phytochemical characterization using UPLC-MS/MS revealed a high abundance and variety of flavonoids in the hydro-ethanolic extract (CST). The antioxidant capacity was validated through various assays, and the ability to mitigate H 2 O 2 -induced oxidative stress and enhance fermentation was demonstrated in Saccharomyces cerevisiae . This study reports that C. sativus tepals extract reduces oxidative stress and boosts ethanol fermentation in yeast, paving the way for applications in the food and biofuels sectors. Further validation in RAW 264.7 macrophages confirmed CST's significant anti-inflammatory effects, indicating its potential for pharmaceutical, cosmeceutical, and nutraceutical applications. In silico studies identified potential targets involved in antioxidant and anti-inflammatory processes, shedding light on possible interaction mechanisms with Kaempferol 3-O-sophoroside (KOS-3), the predominant compound in the extract. The integration of LCA studies highlighted the environmental benefits of this approach. Overall, this research underscores the value of using waste-derived extracts through "green" methodologies, offering a model that may provide significant advantages for further evaluations compared to traditional methodologies and supporting the circular bioeconomy.

Lung cancer (LC) continues to be an important public health problem, being the most common form of cancer and a major cause of cancer deaths worldwide. Despite the great bulk of research to identify genetic susceptibility genes by genome-wide association studies, only few loci associated to nicotine dependence have been consistently replicated. Our previously published study in few phenotypically discordant sib-pairs identified a combination of germline truncating mutations in known cancer susceptibility genes in never-smoker early-onset LC patients, which does not present in their healthy sib. These results firstly demonstrated the presence of an oligogenic combination of disrupted cancer-predisposing genes in non-smokers patients, giving experimental support to a model of a "private genetic epidemiology". Here, we used a combination of whole-exome and RNA sequencing coupled with a discordant sib's model in a novel cohort of pairs of never-smokers early-onset LC patients and in their healthy sibs used as controls. We selected rare germline variants predicted as deleterious by CADD and SVM bioinformatics tools and absent in the healthy sib. Overall, we identified an average of 200 variants per patient, about 10 of which in cancer-predisposing genes. In most of them, RNA sequencing data reinforced the pathogenic role of the identified variants showing: (i) downregulation in LC tissue (indicating a "second hit" in tumor suppressor genes); (ii) upregulation in cancer tissue (likely oncogene); and (iii) downregulation in both normal and cancer tissue (indicating transcript instability). The combination of the two techniques demonstrates that each patient has an average of six (with a range from four to eight) private mutations with a functional effect in tumor-predisposing genes. The presence of a unique combination of disrupting events in the affected subjects may explain the absence of the familial clustering of non-small-cell lung cancer. In conclusion, these findings indicate that each patient has his/her own "predisposing signature" to cancer development and suggest the use of personalized therapeutic strategies in lung cancer.

Transient receptor potential vanilloid 1 (TRPV1) was reported to be a putative target for recovery from chronic pain, producing analgesic effects after its inhibition. A series of drug candidates were previously developed, without the ability to ameliorate the therapeutic outcome. Starting from previously designed compounds, derived from the hybridization of antagonist SB-705498 and partial agonist MDR-652, we performed a virtual screening on a pharmacophore model built by exploiting the Cryo-EM 3D structure of a nanomolar antagonist in complex with the human TRPV1 channel. The pharmacophore model was described by three pharmacophoric features, taking advantage of both the bioactive pose of the antagonist and the receptor exclusion spheres. The results of the screening were implemented inside a 3D-QSAR model, correlating with the negative decadic logarithm of the inhibition rate of the ligands. After the validation of the obtained 3D-QSAR model, we designed a new series of compounds by introducing key modifications on the original scaffold. Again, we determined the compounds' binding poses after alignment to the pharmacophoric model, and we predicted their inhibition rates with the validated 3D-QSAR model. The obtained values resulted in being even more promising than parent compounds, demonstrating that ongoing research still leaves much room for improvement.

The study of rare diseases is important not only for the individuals affected but also for the advancement of medical knowledge and a deeper understanding of human biology and genetics. The wide repertoire of structural information now available from reliable and accurate prediction methods provides the opportunity to investigate the molecular origins of most of the rare diseases reviewed in the Orpha.net database. Thus, it has been possible to analyze the topology of the pathogenic missense variants found in the 2515 proteins involved in Mendelian rare diseases (MRDs), which form the database for our structural bioinformatics study. The amino acid substitutions responsible for MRDs showed different mutation site distributions at different three-dimensional protein depths. We then highlighted the depth-dependent effects of pathogenic variants for the 20,061 pathogenic variants that are present in our database. The results of this structural bioinformatics investigation are relevant, as they provide additional clues to mitigate the damage caused by MRD.

The concept of a "circular bioeconomy" holds great promise for the health, cosmetic, and nutrition sectors by re-using Castanea sativa (Mill.) by-products. This sustainable resource is rich in bioactive secondary metabolites with antioxidant and anti-inflammatory properties. By transforming these by-products into high-value products for human health, we can promote sustainable economic growth and reduce the environmental impact of traditional waste disposal, adding value to previously underutilized resources. In the present study, we investigated the antioxidant capacity, phytochemical composition, and in vitro antioxidant and anti-inflammatory activity of C. sativa burr (CSB) aqueous extract. The spectrophotometric study revealed high total phenolic content (TPC) values with significant antioxidant and anti-radical properties. Using UPLC-MS/MS techniques, the phytochemical investigation identified 56 metabolites, confirming the presence of phenolic compounds in CSBs. In addition, CSBs significantly downregulated pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophage cells without significant cell toxicity. Lastly, in silico studies pinpointed three kinases from RAW 264.7 cells as binding partners with ellagic acid, the predominant compound found in our extract. These findings strongly advocate for the recycling and valorization of C. sativa by-products, challenging their conventional classification as mere "waste".

Utilizing plant-based resources, particularly their by-products, aligns with sustainability principles and circular bioeconomy, contributing to environmental preservation. The therapeutic potential of plant extracts is garnering increasing interest, and this study aimed to demonstrate promising outcomes from an extract obtained from an underutilized plant waste. Chaetomorpha linum , an invasive macroalga found in the Orbetello Lagoon, thrives in eutrophic conditions, forming persistent mats covering approximately 400 hectares since 2005. The biomass of C. linum undergoes mechanical harvesting and is treated as waste, requiring significant human efforts and economic resources-A critical concern for municipalities. Despite posing challenges to local ecosystems, the study identified C. linum as a natural source of bioactive metabolites. Phytochemical characterization revealed lipids, amino acids, and other compounds with potential anti-inflammatory activity in C. linum extract. In vitro assays with LPS-stimulated RAW 264.7 and TNF-α/IFN-γ-stimulated HaCaT cells showed the extract inhibited reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2) productions, and reduced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions via NF-κB nuclear translocation, in RAW 264.7 cells. It also reduced chemokines (TARC/CCL17, RANTES/CCL5, MCP-1/CCL2, and IL-8) and the cytokine IL-1β production in HaCaT cells, suggesting potential as a therapeutic candidate for chronic diseases like atopic dermatitis. Finally, in silico studies indicated palmitic acid as a significant contributor to the observed effect. This research not only uncovered the untapped potential of C. linum but also laid the foundation for its integration into the circular bioeconomy, promoting sustainable practices, and innovative applications across various industries.

Some in vitro and in vivo evidence is consistent with the cardiovascular beneficial activity of propolis. As the single actors responsible for this effect have never been identified, an in-depth investigation of flavonoids isolated from the green propolis of the Caatinga Mimosa tenuiflora was performed and their mechanism of action was described. A comprehensive electrophysiology, functional, and molecular docking approach was applied. Most flavanones and flavones were effective Ca V 1.2 channel blockers with a potency order of (2S)-sakuranetin > eriodictyol-7,3'-methyl ether > quercetin 3-methyl ether > 5,4'-dihydroxy-6,7-dimethoxyflavanone > santin > axillarin > penduletin > kumatakenin, ermanin and viscosine being weak or modest stimulators. Except for eriodictyol 5-O-methyl ether, all the flavonoids were also effective spasmolytic agents of vascular rings, kumatakenin and viscosine also showing an endothelium-dependent activity. (2S)-Sakuranetin also stimulated K Ca 1.1 channels both in single myocytes and vascular rings. In silico analysis provided interesting insights into the mode of action of (2S)-sakuranetin within both Ca V 1.2 and K Ca 1.1 channels. The green propolis of the Caatinga Mimosa tenuiflora is a valuable source of multi-target vasoactive flavonoids: this evidence reinforces its nutraceutical value in the cardiovascular disease prevention arena., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

In the search for novel, bi-functional compounds acting as Ca V 1.2 channel blockers and K + channel stimulators, which represent an effective therapy for hypertension, 3,3'-O-dimethylquercetin was isolated for the first time from Brazilian Caatinga green propolis. Its effects were investigated through electrophysiological, functional, and computational approaches. In rat tail artery myocytes, 3,3'-O-dimethylquercetin blocked Ba 2+ currents through Ca V 1.2 channels (I Ba1.2 ) in a concentration-dependent manner, with the inhibition being reversed upon washout. The compound also shifted the voltage dependence of the steady-state inactivation curve to more negative potentials without affecting the slope of the inactivation and activation curves. Furthermore, the flavonoid stimulated K Ca 1.1 channel currents (I KCa1.1 ). In silico simulations provided additional evidence for the binding of 3,3'-O-dimethylquercetin to K Ca 1.1 and Ca V 1.2 channels and elucidated its mechanism of action. In depolarized rat tail artery rings, the flavonoid induced a concentration-dependent relaxation. Moreover, in rat aorta rings its antispasmodic effect was inversely related to the transmembrane K + gradient. In conclusion, 3,3'-O-dimethylquercetin demonstrates effective in vitro vasodilatory properties, encouraging the exploration of its scaffold to develop novel derivatives for potential use in the treatment of hypertension., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Alkaptonuria is a rare inborn error of metabolism caused by the deficiency of homogentisate 1,2-dioxygenase activity. The consequent homogentisic acid (HGA) accumulation in body fluids and tissues leads to a multisystemic and highly debilitating disease whose main features are dark urine, ochronosis (HGA-derived pigment in collagen-rich connective tissues), and a painful and severe form of osteoarthropathy. Other clinical manifestations are extremely variable and include kidney and prostate stones, aortic stenosis, bone fractures, and tendon, ligament and/or muscle ruptures. As an autosomal recessive disorder, alkaptonuria affects men and women equally. Debilitating symptoms appear around the third decade of life, but a proper and timely diagnosis is often delayed due to their non-specific nature and a lack of knowledge among physicians. In later stages, patients' quality of life might be seriously compromised and further complicated by comorbidities. Thus, appropriate management of alkaptonuria requires a multidisciplinary approach, and periodic clinical evaluation is advised to monitor disease progression, complications and/or comorbidities, and to enable prompt intervention. Treatment options are patient-tailored and include a combination of medications, physical therapy and surgery. Current basic and clinical research focuses on improving patient management and developing innovative therapies and implementing precision medicine strategies., (© 2024. Springer Nature Limited.)

Currently, many environmental and energy-related problems are threatening the future of our planet. In October 2022, the Worldmeter recorded the world population as 7.9 billion people, estimating that there will be an increase of 2 billion by 2057. The rapid growth of the population and the continuous increase in needs are causing worrying conditions, such as pollution, climate change, global warming, waste disposal, and natural resource reduction. Looking for novel and innovative methods to overcome these global troubles is a must for our common welfare. The circular bioeconomy represents a promising strategy to alleviate the current conditions using biomass-like natural wastes to replace commercial products that have a negative effect on our ecological footprint. Applying the circular bioeconomy concept, we propose an integrated in silico and in vitro approach to identify antioxidant bioactive compounds extracted from chestnut burrs (an agroforest waste) and their potential biological targets. Our study provides a novel and robust strategy developed within the circular bioeconomy concept aimed at target and drug discovery for a wide range of diseases. Our study could open new frontiers in the circular bioeconomy related to target and drug discovery, offering new ideas for sustainable scientific research aimed at identifying novel therapeutical strategies.

Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K + (K ir 6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through K ir 6.1 channels (I Kir6.1 ) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive I Kir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the K ir 6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K + - and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K + , rutin being ineffective. In conclusion, I Kir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors mdivi-1 and dynasore. However, mdivi-1 is capable to inhibit Ba 2+ currents through Ca V 1.2 channels (I Ba1.2 ), stimulate K Ca 1.1 channel currents (I KCa1.1 ), and modulate pathways key to the maintenance of vessel active tone in a dynamin-independent manner. Using a multidisciplinary approach, the present study demonstrates that dynasore, like mdivi-1, is a bi-functional vasodilator, blocking I Ba1.2 and stimulating I KCa1.1 in rat tail artery myocytes, as well as promoting relaxation of rat aorta rings pre-contracted by either high K + or phenylephrine. Conversely, its analogue dyngo-4a, though inhibiting mitochondrial fission triggered by phenylephrine and stimulating I KCa1.1 , did not affect I Ba1.2 but potentiated both high K + - and phenylephrine-induced contractions. Docking and molecular dynamics simulations identified the molecular basis supporting the different activity of dynasore and dyngo-4a at Ca V 1.2 and K Ca 1.1 channels. Mito-tempol only partially counteracted the effects of dynasore and dyngo-4a on phenylephrine-induced tone. In conclusion, the present data, along with previous observations (Ahmed et al., 2022) rise caution for the use of dynasore, mdivi-1, and dyngo-4a as tools to investigate the role of mitochondrial fission in vascular contraction: to this end, a selective dynamin inhibitor and/or a different experimental approach are needed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Conventional therapy options for chronic pain are still insufficient and patients most frequently request alternative medical treatments, such as medical cannabis. Although clinical evidence supports the use of cannabis for pain, very little is known about the efficacy, dosage, administration methods, or side effects of widely used and accessible cannabis products. A possible solution could be given by pharmacogenetics, with the identification of several polymorphic genes that may play a role in the pharmacodynamics and pharmacokinetics of cannabis. Based on these findings, data from patients treated with cannabis and genotyped for several candidate polymorphic genes (single-nucleotide polymorphism: SNP) were collected, integrated, and analyzed through a machine learning (ML) model to demonstrate that the reduction in pain intensity is closely related to gene polymorphisms. Starting from the patient's data collected, the method supports the therapeutic process, avoiding ineffective results or the occurrence of side effects. Our findings suggest that ML prediction has the potential to positively influence clinical pharmacogenomics and facilitate the translation of a patient's genomic profile into useful therapeutic knowledge.

Emerging machine learning (ML) technologies have the potential to significantly improve the research and treatment of rare diseases, which constitute a vast set of diseases that affect a small proportion of the total population. Artificial Intelligence (AI) algorithms can help to quickly identify patterns and associations that would be difficult or impossible for human analysts to detect. Predictive modeling techniques, such as deep learning, have been used to forecast the progression of rare diseases, enabling the development of more targeted treatments. Moreover, AI has also shown promise in the field of drug development for rare diseases with the identification of subpopulations of patients who may be most likely to respond to a particular drug. This review aims to highlight the achievements of AI algorithms in the study of rare diseases in the past decade and advise researchers on which methods have proven to be most effective. The review will focus on specific rare diseases, as defined by a prevalence rate that does not exceed 1-9/100,000 on Orphanet, and will examine which AI methods have been most successful in their study. We believe this review can guide clinicians and researchers in the successful application of ML in rare diseases., Competing Interests: The authors declare no conflicts of interest.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs, which occurs because the homogentisate 1,2-dioxygenase (HGD) enzyme is not functional due to gene variants. Over time, HGA oxidation and accumulation cause the formation of the ochronotic pigment, a deposit that provokes tissue degeneration and organ malfunction. Here, we report a comprehensive review of the variants so far reported, the structural studies on the molecular consequences of protein stability and interaction, and molecular simulations for pharmacological chaperones as protein rescuers. Moreover, evidence accumulated so far in alkaptonuria research will be re-proposed as the bases for a precision medicine approach in a rare disease., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The complexity of neurodegenerative diseases, among which Alzheimer's disease plays a pivotal role, poses one of the tough therapeutic challenges of present time. In this perspective, a multitarget approach appears as a promising strategy to simultaneously interfere with different defective pathways. In this paper, a structural simplification plan was performed on our previously reported multipotent polycyclic compounds, in order to obtain a simpler pharmacophoric central core with improved pharmacokinetic properties, while maintaining the modulating activity on neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3β), as validated targets to combat Alzheimer's disease. The molecular pruning approach applied here led to tetrahydroisoindole-dione (1), tetrahydromethanoisoindole-dione (2) and tetrahydroepoxyisoindole-dione (3) structures, easily affordable by Diels-Alder cycloaddition. Preliminary data indicated structure 3 as the most appropriate, thus a SAR study was performed by introducing different substituents, selected on the basis of the commercial availability of the furan derivatives required for the synthetic procedure. The results indicated compound 10 as a promising, structurally atypical, safe and BBB-penetrating Ca v modulator, inhibiting both L- and N-calcium channels, likely responsible for the Ca 2+ overload observed in Alzheimer's disease. In a multitarget perspective, compound 11 appeared as an effective prototype, endowed with improved Ca v inhibitory activity, with respect to the reference drug nifedipine, and encouraging modulating activity on GSK-3β., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nitisinone (NTBC) was recently approved to treat alkaptonuria (AKU), but there is no information on its impact on oxidative stress and inflammation, which are observed in AKU. Therefore, serum samples collected during the clinical studies SONIA1 (40 AKU patients) and SONIA2 (138 AKU patients) were tested for Serum Amyloid A (SAA), CRP and IL-8 by ELISA; Advanced Oxidation Protein Products (AOPP) by spectrophotometry; and protein carbonyls by Western blot. Our results show that NTBC had no significant effects on the tested markers except for a slight but statistically significant effect for NTBC, but not for the combination of time and NTBC, on SAA levels in SONIA2 patients. Notably, the majority of SONIA2 patients presented with SAA > 10 mg/L, and 30 patients in the control group (43.5%) and 40 patients (58.0%) in the NTBC-treated group showed persistently elevated SAA > 10 mg/L at each visit during SONIA2. Higher serum SAA correlated with lower quality of life and higher morbidity. Despite no quantitative differences in AOPP, the preliminary analysis of protein carbonyls highlighted patterns that deserve further investigation. Overall, our results suggest that NTBC cannot control the sub-clinical inflammation due to increased SAA observed in AKU, which is also a risk factor for developing secondary amyloidosis., Competing Interests: The authors declare that they have no conflicts of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.

Background: The increase in the medical use of cannabis has revealed a number of beneficial effects, a variety of adverse side effects and great inter-individual variability. Association studies connecting consumption, addiction and side effects related to recreational cannabis use have led to the identification of several polymorphic genes that may play a role in the pharmacodynamics and pharmacokinetics of cannabis., Method: In total, 600 patients treated with cannabis were genotyped for several candidate polymorphic genes (single-nucleotide polymorphism; SNP), encoding receptors CNR1 and TRPV1; for the ABCB1 transporter; for biotransformation, bioactivation and biosynthesis; and CYP3A4, COMT and UGT2B7 conjugation., Results: Three polymorphic genes (ABCB1, TRPV1 and UGT2B7) were identified as being significantly associated with decline in pain after treatment with cannabis. Patients simultaneously carrying the most favourable allele combinations showed a greater reduction (polygenic effect) in pain compared to those with a less favourable combination. Considering genotype combinations, we could group patients into good responders, intermediate responders and poor or non-responders. Results suggest that genetic makeup is, at the moment, a significant predictive factor of the variability in response to cannabis., Conclusions: This study proves, for the first time, that certain polymorphic candidate genes may be associated with cannabis effects, both in terms of pain management and side effects, including therapy dropout., Significance: Our attention to pharmacogenetics began in 2008, with the publication of a first study on the association between genetic polymorphisms and morphine action in pain relief. The study we are presenting is the first observational study conducted on a large number of patients involving several polymorphic candidate genes. The data obtained suggest that genetic makeup can be a predictive factor in the response to cannabis therapy and that more extensive and planned studies are needed for the opening of new scenarios for the personalization of cannabis therapy.

Mdivi-1 is widely used as a pharmacological tool to inhibit dynamin-related protein-1-mediated mitochondrial fission. Whether this compound may interact directly or indirectly with ion channels or cellular pathways fundamental for the regulation of vascular smooth muscle tone remains unknown. The present study aimed to assess the effect of mdivi-1 on Ca V 1.2 and K Ca 1.1 channels, both in vitro and in silico as well as on the mechanical activity of rat aorta rings. Mdivi-1 was an effective Ca V 1.2 channel blocker, docking in a Ca V 1.2 channel antagonist binding region, stimulated K Ca 1.1 channel current, binding to a sensing region common to other stimulators, and possibly inhibited the Rho-kinase pathway. These effects contributed to its vasorelaxant activity observed in rings stimulated with high KCl, phenylephrine, or NaF. Neither structurally different dynamin inhibitors nor a stimulator affected the Ca 2+ antagonistic and vasorelaxant activities of the compound. However, mito-tempol reduced its vasorelaxant potency towards phenylephrine. Finally, mdivi-1 antagonized mitochondrial fission triggered by phenylephrine. In conclusion, mdivi-1 is an effective in vitro vasorelaxant agent at concentrations routinely employed to block dynamin-related protein-1. Ion channels and pathways key to the maintenance of vessel active tone are involved in this mechanism. These yet undiscovered off-target effects raise caution for the interpretation of mitochondrial fission signalling., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in the body. Affected individuals lack functional levels of an enzyme required to breakdown HGA. Mutations in the homogentisate 1,2-dioxygenase (HGD) gene cause AKU and they are responsible for deficient levels of functional HGD, which, in turn, leads to excess levels of HGA. Although HGA is rapidly cleared from the body by the kidneys, in the long term it starts accumulating in various tissues, especially cartilage. Over time (rarely before adulthood), it eventually changes the color of affected tissue to slate blue or black. Here we report a comprehensive mutation analysis of 111 pathogenic and 190 non-pathogenic HGD missense mutations using protein structural information. Using our comprehensive suite of graph-based signature methods, mCSM complemented with sequence-based tools, we studied the functional and molecular consequences of each mutation on protein stability, interaction and evolutionary conservation. The scores generated from the structure and sequence-based tools were used to train a supervised machine learning algorithm with 89% accuracy. The empirical classifier was used to generate the variant phenotype for novel HGD missense mutations. All this information is deployed as a user friendly freely available web server called HGDiscovery (https://biosig.lab.uq.edu.au/hgdiscovery/)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.

Hypertension is a risk factor for cardiovascular diseases, which are the main cause of morbidity and mortality in the world. In the search for new molecules capable of targeting K Ca 1.1 and Ca V 1.2 channels, the expression of which is altered in hypertension, the in vitro vascular effects of a series of flavonoids extracted from the heartwoods, roots, and leaves of Dalbergia tonkinensis Prain, widely used in traditional medicine, were assessed. Rat aorta rings, tail artery myocytes, and docking and molecular dynamics simulations were used to analyse their effect on these channels. Formononetin, orobol, pinocembrin, and biochanin A showed a marked myorelaxant activity, particularly in rings stimulated by moderate rather than high KCl concentrations. Ba 2+ currents through Ca V 1.2 channels (I Ba1.2 ) were blocked in a concentration-dependent manner by sativanone, 3'- O -methylviolanone, pinocembrin, and biochanin A, while it was stimulated by ambocin. Sativanone, dalsissooside, and eriodictyol inhibited, while tectorigenin 7- O -[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside], ambocin, butin, and biochanin A increased I KCa1.1 . In silico analyses showed that biochanin A, sativanone, and pinocembrin bound with high affinity in target-sensing regions of both channels, providing insight into their potential mechanism of action. In conclusion, Dalbergia tonkinensis is a valuable source of mono- and bifunctional, vasoactive scaffolds for the development of novel antihypertensive drugs.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3 L412F -encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

The transmembrane glycoprotein CD93 has been identified as a potential new target to inhibit tumor angiogenesis. Recently, Multimerin-2 (MMRN2), a pan-endothelial extracellular matrix protein, has been identified as a ligand for CD93, but the interaction mechanism between these two proteins is yet to be studied. In this article, we aim to investigate the structural and functional effects of induced mutations on the binding domain of CD93 to MMRN2. Starting from experimental data, we assessed how specific mutations in the C-type lectin-like domain (CTLD) affect the binding interaction profile. We described a four-step workflow in order to predict the effects of variations on the inter-residue interaction network at the PPI, based on evolutionary information, complex network metrics, and energetic affinity. We showed that the application of computational approaches, combined with experimental data, allowed us to gain more in-depth molecular insights into the CD93-MMRN2 interaction, offering a platform for developing innovative therapeutics able to target these molecules and block their interaction. This comprehensive molecular insight might prove useful in drug design in cancer therapy., Competing Interests: VC was employed by the company Toscana Life Sciences Foundation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cicaloni, Karmakar, Frusciante, Pettini, Visibelli, Orlandini, Galvagni, Mongiat, Silk, Nardi, Ascher, Santucci and Spiga.)

With a structural bioinformatic approach, we have explored amino acid compositions at PISA defined interfaces between small molecules and proteins that are contained in an optimized subset of 11,351 PDB files. The use of a series of restrictions, to prevent redundancy and biases from interactions between amino acids with charged side chains and ions, yielded a final data set of 45,230 protein-small molecule interfaces. We have compared occurrences of natural amino acids in surface exposed regions and binding sites for all the proteins of our data set. From our structural bioinformatic survey, the most relevant signal arose from the unexpected Gly abundance at enzyme catalytic sites. This finding suggested that Gly must have a fundamental role in stabilizing concave protein surface moieties. Subsequently, we have tried to predict the effect of in silico Gly mutations in hen egg white lysozyme to optimize those conditions that can reshape the protein surface with the appearance of new pockets. Replacing amino acids having bulky side chains with Gly in specific protein regions seems a feasible way for designing proteins with additional surface pockets, which can alter protein surface dynamics, therefore, representing controllable switches for protein activity.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease that spreads rapidly in humans. In March 2020, the World Health Organization (WHO) declared a COVID-19 pandemic. Identifying a multi-target-directed ligand approach would open up new opportunities for drug discovery to combat COVID-19. The aim of this work was to perform a virtual screening of an exclusive chemical library of about 1700 molecules containing both pharmacologically active compounds and synthetic intermediates to propose potential protein inhibitors for use against SARS-CoV-2. In silico analysis showed that our compounds triggered an interaction network with key residues of the SARS-CoV-2 spike protein (S-protein), blocking trimer formation and interaction with the human receptor hACE2, as well as with the main 3C-like protease (3CLpro), inhibiting their biological function. Our data may represent a step forward in the search for potential new chemotherapeutic agents for the treatment of COVID-19.

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Quercetin, a flavonoid abundantly present in the Mediterranean diet, is considered a vasodilator despite its recognized capability to stimulate vascular Ca V 1.2 channel current (I Ca1.2 ). The present study was undertaken to assess its possible vasocontractile activity. Functional and electrophysiology experiments were performed in vitro on rat aorta rings and tail artery myocytes along with an in-depth molecular modelling analysis. The Ca V 1.2 channel stimulator (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (Bay K 8644) was used as reference compound. Quercetin and Bay K 8644 caused a significant leftward shift of KCl concentration-response curve. Neither agent affected basal muscle tone, though in rings pre-treated with thapsigargin or 15 mM KCl they caused a strong, concentration-dependent contraction. Both quercetin and Bay K 8644 potentiated the response to Ca 2+ in weakly depolarised rings. At high KCl concentrations, however, quercetin caused vasorelaxation. While Bay K 8644 stimulated I Ca1.2 , this effect being sustained with time, quercetin-induced stimulation was transient, although the molecule in solution underwent only marginal oxidation. Quercetin transient stimulation was not affected by pre-treatment with isoprenaline, sodium nitroprusside, or dephostatin; however, it converted to a sustained one in myocytes pre-incubated with Gö6976. Classical molecular dynamics simulations revealed that quercetin and Bay K 8644 formed hydrogen bonds with target sensing residues of Ca V 1.2 channel favouring the inactivated conformation. In conclusion, quercetin-induced stimulation of I Ca1.2 promoted vasocontraction when Ca 2+ buffering function of sarcoplasmic reticulum was impaired and/or smooth muscle cell membrane was moderately depolarised, as it may occur under certain pathological conditions., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease caused by a mutation in the homogentisate 1,2-dioxygenase gene. One of the main obstacles in studying AKU and other ultra-rare diseases, is the lack of a standardized methodology to assess disease severity or response to treatment. Based on that, a multi-purpose digital platform, called ApreciseKUre, was implemented to facilitate data collection, integration and analysis for patients affected by AKU. It includes genetic, biochemical, histopathological, clinical, therapeutic resources and Quality of Life (QoL) scores that can be shared among registered researchers and clinicians to create a Precision Medicine Ecosystem. The combination of machine learning applications to analyse and re-interpret data available in the ApreciseKUre clearly indicated the potential direct benefits to achieve patients' stratification and the consequent tailoring of care and treatments to a specific subgroup of patients. In order to generate a comprehensive patient profile, computational modeling and database construction support the identification of potential new biomarkers, paving the way for more personalized therapy to maximize the benefit-risk ratio. In this work, different Machine Learning implemented approaches were described., Competing Interests: Authors OS and AS were employed by company Competence Center ARTES 4.0 and SienabioACTIVE—SbA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Visibelli, Cicaloni, Spiga and Santucci.)

The role of computational tools in the drug discovery and development process is becoming central, thanks to the possibility to analyze large amounts of data. The high throughput and affordability of current omics technologies, allowing quantitative measurements of many putative targets, has exponentially increased the volume of scientific data available. The quality of the data and the speed with which in silico predictions can be validated in vitro is instrumental in accelerating clinical laboratory medicine, significantly and substantially impacting Precision Medicine (PM). PM affords the basis to develop new drugs by providing a wide knowledge of the patient as an essential step towards individualized medicine. It is, therefore, essential to collect as much information and data as possible on each patient to identify the causes of the different responses to drugs from a pharmacogenomics perspective and to identify biological biomarkers capable of accurately describing the risk signals to develop specific diseases. Furthermore, the role of biomarkers in early drug discovery is increasing, as they can significantly reduce the time it takes to develop new drugs. This review article will discuss how Artificial Intelligence fits in the drug discovery pipeline, covering the benefits of an automated, integrated laboratory framework where the application of Machine Learning methodologies to interpret omics-based data can avail the future perspective of Translational Precision Medicine., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Alkaptonuria (AKU, OMIM: 203500) is an autosomal recessive disorder caused by mutations in the Homogentisate 1,2-dioxygenase (HGD) gene. A lack of standardized data, information and methodologies to assess disease severity and progression represents a common complication in ultra-rare disorders like AKU. This is the reason why we developed a comprehensive tool, called ApreciseKUre, able to collect AKU patients deriving data, to analyse the complex network among genotypic and phenotypic information and to get new insight in such multi-systemic disease. By taking advantage of the dataset, containing the highest number of AKU patient ever considered, it is possible to apply more sophisticated computational methods (such as machine learning) to achieve a first AKU patient stratification based on phenotypic and genotypic data in a typical precision medicine perspective. Thanks to our sufficiently populated and organized dataset, it is possible, for the first time, to extensively explore the phenotype-genotype relationships unknown so far. This proof of principle study for rare diseases confirms the importance of a dedicated database, allowing data management and analysis and can be used to tailor treatments for every patient in a more effective way., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease., (© 2021 Wiley Periodicals LLC.)

Understanding the molecular mechanisms that correlate pathologies with missense mutations is of critical importance for disease risk estimations and for devising personalized therapies. Thus, we have performed a bioinformatic survey of ClinVar, a database of human genomic variations, to find signals that can account for missense mutation pathogenicity. Arginine resulted as the most frequently replaced amino acid both in benign and pathogenic mutations. By adding the structural dimension to this investigation to increase its resolution, we found that arginine mutations occurring at the protein-DNA interface increase pathogenicity 6.5 times with respect to benign variants. Glycine is the second amino acid among all the pathological missense mutations. Necessarily replaced by larger amino acids, glycine substitutions perturb the structural stability of proteins and, therefore, their functions, being mostly located in buried protein moieties. Arginine and glycine appear as representative of missense mutations causing respective changes in interaction processes and protein structural features, the two main molecular mechanisms of genome-induced pathologies.

In this review, we focus on bioinformatic oncology as an integrative discipline that incorporates knowledge from the mathematical, physical, and computational fields to further the biomedical understanding of cancer. Before providing a deeper insight into the bioinformatics approach and utilities involved in oncology, we must understand what is a system biology framework and the genetic connection, because of the high heterogenicity of the backgrounds of people approaching precision medicine. In fact, it is essential to providing general theoretical information on genomics, epigenomics, and transcriptomics to understand the phases of multi-omics approach. We consider how to create a multi-omics model. In the last section, we describe the new frontiers and future perspectives of this field.

The cardiac action potential is regulated by several ion channels. Drugs capable to block these channels, in particular the human ether-à-go-go-related gene (hERG) channel, also known as K V 11.1 channel, may lead to a potentially lethal ventricular tachyarrhythmia called "Torsades de Pointes". Thus, evaluation of the hERG channel off-target activity of novel chemical entities is nowadays required to safeguard patients as well as to avoid attrition in drug development. Flavonoids, a large class of natural compounds abundantly present in food, beverages, herbal medicines, and dietary food supplements, generally escape this assessment, though consumed in consistent amounts. Continuously growing evidence indicates that these compounds may interact with the hERG channel and block it. The present review, by examining numerous studies, summarizes the state-of-the-art in this field, describing the most significant examples of direct and indirect inhibition of the hERG channel current operated by flavonoids. A description of the molecular interactions between a few of these natural molecules and the Rattus norvegicus channel protein, achieved by an in silico approach, is also presented., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction : The term 'orphan diseases' includes conditions meeting prevalence-based or commercial viability criteria: they affect a small number of individuals and are considered an unviable market for drug development. Proteomics is an important technology to study them, providing information on mechanisms and evolution, biomarkers, and effects of therapeutic interventions. Areas covered : Herein, we review how proteomics and bioinformatic tools could be applied to the study of rare diseases and discuss pitfalls and potential. Expert opinion : Research in the field of rare diseases has to face many challenges, and implementation plans should foresee highly specialized collaborative consortia to create multidisciplinary frameworks for data sharing, advancing research, supporting clinical studies, and accelerating drug development. The integration of different technologies will allow better knowledge of disease pathophysiology, and the inclusion of proteomics and other omics technologies in this context will be pivotal to this aim.Several aspects of rare diseases, often perceived as limiting factors, might actually be advantages for a precision medicine approach: the limited number of patients, the collaboration with patient societies, and the availability of curated clinical registries could allow the development of homogeneous clinical databases and ultimately a better control over the data to be analyzed.

Background: Recently, loss-of-function variants in TLR7 were identified in two families in which COVID-19 segregates like an X-linked recessive disorder environmentally conditioned by SARS-CoV-2. We investigated whether the two families represent the tip of the iceberg of a subset of COVID-19 male patients., Methods: This is a nested case-control study in which we compared male participants with extreme phenotype selected from the Italian GEN-COVID cohort of SARS-CoV-2-infected participants (<60 y, 79 severe cases versus 77 control cases). We applied the LASSO Logistic Regression analysis, considering only rare variants on young male subsets with extreme phenotype, picking up TLR7 as the most important susceptibility gene., Results: Overall, we found TLR7 deleterious variants in 2.1% of severely affected males and in none of the asymptomatic participants. The functional gene expression profile analysis demonstrated a reduction in TLR7-related gene expression in patients compared with controls demonstrating an impairment in type I and II IFN responses., Conclusions: Young males with TLR7 loss-of-function variants and severe COVID-19 represent a subset of male patients contributing to disease susceptibility in up to 2% of severe COVID-19., Funding: Funded by private donors for the Host Genetics Research Project, the Intesa San Paolo for 2020 charity fund, and the Host Genetics Initiative., Clinical Trial Number: NCT04549831., Competing Interests: CF, SD, SM, EB, NP, DF, FP, ES, MB, FF, MP, SL, FC, EQ, MV, SR, MS, MB, OS, KC, SF, FM, AR, MM, EF No competing interests declared, (© 2021, Fallerini et al.)

Ca V 1.2 channels play a fundamental role in the regulation of vascular smooth muscle tone. The aim of the present study was to synthesize morin derivatives bearing the nitrophenyl moiety of dihydropyridine Ca 2+ antagonists to increase the flavonoid vasorelaxant activity. The effects of morin and its derivatives were assessed on Ca V 1.2 and K Ca 1.1 channels, both in vitro and in silico, as well as on the contractile responses of rat aorta rings. All compounds were effective Ca V 1.2 channel blockers, positioning in the α 1C subunit region where standard blockers bind. Among the four newly synthesized morin derivatives, the penta-acetylated morin-1 was the most efficacious Ca 2+ antagonist, presenting a vasorelaxant profile superior to that of the parent compound and, contrary to morin, antagonized also the release of Ca 2+ from the sarcoplasmic reticulum; surprisingly, it also stimulated K Ca 1.1 channel current. Computational analysis demonstrated that morin-1 bound close to the K Ca 1.1 channel S6 segment. In conclusion, these findings open a new avenue for the synthesis of valuable multi-functional, vasorelaxant morin derivatives capable to target several pathways underpinning the pathogenesis of hypertension., (Copyright © 2021 Elsevier Inc. All rights reserved.)

ApreciseKUre is a multi-purpose digital platform facilitating data collection, integration and analysis for patients affected by Alkaptonuria (AKU), an ultra-rare autosomal recessive genetic disease. It includes genetic, biochemical, histopathological, clinical, therapeutic resources and quality of life scores that can be shared among registered researchers and clinicians in order to create a Precision Medicine Ecosystem (PME). The combination of machine learning application to analyse and re-interpret data available in the ApreciseKUre shows the potential direct benefits to achieve patient stratification and the consequent tailoring of care and treatments to a specific subgroup of patients. In this study, we have developed a tool able to investigate the most suitable treatment for AKU patients in accordance with their Quality of Life scores, which indicates changes in health status before/after the assumption of a specific class of drugs. This fact highlights the necessity of development of patient databases for rare diseases, like ApreciseKUre. We believe this is not limited to the study of AKU, but it represents a proof of principle study that could be applied to other rare diseases, allowing data management, analysis, and interpretation.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Quercetin represents one of the most studied dietary flavonoids; it exerts a panel of pharmacological activities particularly on the cardiovascular system. Stimulation of vascular K Ca 1.1 channels contributes to its vasorelaxant activity, which is, however, counteracted in part by its concomitant stimulation of Ca V 1.2 channels. Therefore, several quercetin hybrid derivatives were designed and synthesized to produce a more selective K Ca 1.1 channel stimulator, then assessed both in silico and in vitro. All the derivatives interacted with the K Ca 1.1 channel with similar binding energy values. Among the selected derivatives, 1E was a weak vasodilator, though displaying an interesting Ca V 1.2 channel blocking activity. The lipoyl derivatives 1F and 3F, though showing pharmacological and electrophysiological features similar to those of quercetin, seemed to be more effective as K Ca 1.1 channel stimulators as compared to the parent compound. The strategy pursued demonstrated how different chemical substituents on the quercetin core can change/invert its effect on Ca V 1.2 channels or enhance its K Ca 1.1 channel stimulatory activity, thus opening new avenues for the synthesis of efficacious vasorelaxant quercetin hybrids., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In December 2019, an initial cluster of interstitial bilateral pneumonia emerged in Wuhan, China. A human-to-human transmission was assumed and a previously unrecognized entity, termed coronavirus disease-19 (COVID-19) due to a novel coronavirus (SARS-CoV-2) was described. The infection has rapidly spread out all over the world and Italy has been the first European country experiencing the endemic wave with unexpected clinical severity in comparison with Asian countries. It has been shown that SARS-CoV-2 utilizes angiotensin converting enzyme 2 (ACE2) as host receptor and host proteases for cell surface binding and internalization. Thus, a predisposing genetic background can give reason for interindividual disease susceptibility and/or severity. Taking advantage of the Network of Italian Genomes (NIG), here we mined whole-exome sequencing data of 6930 Italian control individuals from five different centers looking for ACE2 variants. A number of variants with a potential impact on protein stability were identified. Among these, three more common missense changes, p.(Asn720Asp), p.(Lys26Arg), and p.(Gly211Arg) were predicted to interfere with protein structure and stabilization. Rare variants likely interfering with the internalization process, namely p.(Leu351Val) and p.(Pro389His), predicted to interfere with SARS-CoV-2 spike protein binding, were also observed. Comparison of ACE2 WES data between a cohort of 131 patients and 258 controls allowed identifying a statistically significant (P value < 0.029) higher allelic variability in controls compared with patients. These findings suggest that a predisposing genetic background may contribute to the observed interindividual clinical variability associated with COVID-19, allowing an evidence-based risk assessment leading to personalized preventive measures and therapeutic options.

Alkaptonuria (AKU) is an ultra-rare disease caused by mutations in homogentisate 1,2-dioxygenase (HGD) enzyme, characterized by the loss of enzymatic activity and the accumulation of its substrate, homogentisic acid (HGA) in different tissues, leading to ochronosis and organ degeneration. Although the pathological effects of HGD mutations are largely studied, less is known about the structure of the enzyme, in particular the pathways for dioxygen diffusion to the active site, required for the enzymatic reaction, are still uninvestigated. In the present project, the combination of two in silico techniques, Molecular Dynamics (MD) simulation and Implicit Ligand Sampling (ILS), was used to delineate gas diffusion routes in HGD enzyme. A route from the central opening of the hexameric structure of the enzyme to the back of the active site trough the protein moiety was identified as the path for dioxygen diffusion, also overlapping with a transient pocket, which then assumes an important role in dioxygen diffusion. Along the route the sequence location of the missense variant E401Q, responsible for AKU development, was also found, suggesting such mutation to be conducive of enzymatic activity loss by altering the flow dynamics of dioxygen. Our in silico approach allowed also to delineate the route of HGA substrate to the active site, until now only supposed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ca V 1.2 channel blockers or 5-HT 2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT 2 receptors and Ca V 1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT 2 receptor antagonist, on vascular Ca V 1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked Ca V 1.2 channel currents (I Ca1.2 ) in a concentration-dependent manner (K r  = 3.61 µM); I Ca1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited I Ca1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (K i  = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased I Ca1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the Ca V 1.2 α 1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of Ca V 1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

The Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The virus has rapidly spread in humans, causing the ongoing Coronavirus pandemic. Recent studies have shown that, similarly to SARS-CoV, SARS-CoV-2 utilises the Spike glycoprotein on the envelope to recognise and bind the human receptor ACE2. This event initiates the fusion of viral and host cell membranes and then the viral entry into the host cell. Despite several ongoing clinical studies, there are currently no approved vaccines or drugs that specifically target SARS-CoV-2. Until an effective vaccine is available, repurposing FDA approved drugs could significantly shorten the time and reduce the cost compared to de novo drug discovery. In this study we attempted to overcome the limitation of in silico virtual screening by applying a robust in silico drug repurposing strategy. We combined and integrated docking simulations, with molecular dynamics (MD), Supervised MD (SuMD) and Steered MD (SMD) simulations to identify a Spike protein - ACE2 interaction inhibitor. Our data showed that Simeprevir and Lumacaftor bind the receptor-binding domain of the Spike protein with high affinity and prevent ACE2 interaction.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder associated with mutation in MECP2 gene. Despite a well-defined genetic cause, there is a growing consensus that a metabolic component could play a pivotal role in RTT pathophysiology. Indeed, perturbed redox homeostasis and inflammation, i.e. oxinflammation, with mitochondria dysfunction as the central hub between the two phenomena, appear as possible key contributing factors to RTT pathogenesis and its clinical features. While these RTT-related changes have been widely documented by transcriptomic profiling, proteomics studies supporting these evidences are still limited. Here, using primary dermal fibroblasts from control and patients, we perform a large-scale proteomic analysis that, together with data mining approaches, allow us to carry out the first comprehensive characterization of RTT cellular proteome, showing mainly changes in expression of proteins involved in the mitochondrial network. These findings parallel with an altered expression of key mediators of mitochondrial dynamics and mitophagy associated with abnormal mitochondrial morphology. In conclusion, our proteomic analysis confirms the pathological relevance of mitochondrial dysfunction in RTT pathogenesis and progression., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The recent release of COVID-19 spike glycoprotein allows detailed analysis of the structural features that are required for stabilizing the infective form of its quaternary assembly. Trying to disassemble the trimeric structure of COVID-19 spike glycoprotein, we analyzed single protomer surfaces searching for concave moieties that are located at the three protomer-protomer interfaces. The presence of some druggable pockets at these interfaces suggested that some of the available drugs in Drug Bank could destabilize the quaternary spike glycoprotein formation by binding to these pockets, therefore interfering with COVID-19 life cycle. The approach we propose here can be an additional strategy to fight against the deadly virus. Ligands of COVID-19 spike glycoprotein that we have predicted in the present computational investigation, might be the basis for new experimental studies in vitro and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

ApreciseKUre is a multi-purpose digital platform facilitating data collection, integration and analysis for patients affected by Alkaptonuria (AKU), an ultra-rare autosomal recessive genetic disease. We present an ApreciseKUre plugin, called AKUImg, dedicated to the storage and analysis of AKU histopathological slides, in order to create a Precision Medicine Ecosystem (PME), where images can be shared among registered researchers and clinicians to extend the AKU knowledge network. AKUImg includes a new set of AKU images taken from cartilage tissues acquired by means of a microscopic technique. The repository, in accordance to ethical policies, is publicly available after a registration request, to give to scientists the opportunity to study, investigate and compare such precious resources. AKUImg is also integrated with a preliminary but accurate predictive system able to discriminate the presence/absence of AKU by comparing histopatological affected/control images. The algorithm is based on a standard image processing approach, namely histogram comparison, resulting to be particularly effective in performing image classification, and constitutes a useful guide for non-AKU researchers and clinicians., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R -(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R -(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba 2+ currents through Ca V 1.2 channels (I Ba1.2 ) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R -(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor N ω -nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R -(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(-)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R -(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited I Ba1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R -(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the Ca V 1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)