Vasorelaxant Activity of (2S)-Sakuranetin and Other Flavonoids Isolated from the Green Propolis of the Caatinga Mimosa tenuiflora.

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 CaV1.2 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 KCa1.1 channels both in single myocytes and vascular rings. In silico analysis provided interesting insights into the mode of action of (2S)-sakuranetin within both CaV1.2 and KCa1.1 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.

A Drug Discovery Approach to a Reveal Novel Antioxidant Natural Source: The Case of Chestnut Burr Biomass.

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.

Alkaptonuria.

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.

3,3'-O-dimethylquercetin: A bi-functional vasodilator isolated from green propolis of the Caatinga Mimosa tenuiflora.

In the search for novel, bi-functional compounds acting as CaV1.2 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 Ba2+ currents through CaV1.2 channels (IBa1.2) 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 KCa1.1 channel currents (IKCa1.1). In silico simulations provided additional evidence for the binding of 3,3'-O-dimethylquercetin to KCa1.1 and CaV1.2 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.

Electrophysiology, molecular modelling, and functional analysis of the effects of dietary quercetin and flavonoid analogues on Kir6.1 channels in rat stomach fundus smooth muscle.

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+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) 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 IKir6.1 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 Kir6.1 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, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.

Supporting Machine Learning Model in the Treatment of Chronic Pain.

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.

Dynamin-independent CaV1.2 and KCa1.1 channels regulation and vascular tone modulation by the mitochondrial fission inhibitors dynasore and dyngo-4a.

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 Ba2+ currents through CaV1.2 channels (IBa1.2), stimulate KCa1.1 channel currents (IKCa1.1), 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 IBa1.2 and stimulating IKCa1.1 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 IKCa1.1, did not affect IBa1.2 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 CaV1.2 and KCa1.1 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.

The Impact of Artificial Intelligence in the Odyssey of Rare Diseases.

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.

Structure-Function Relationship of Homogentisate 1,2-dioxygenase: Understanding the Genotype-Phenotype Correlations in the Rare Genetic Disease Alkaptonuria.

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.

Bioinformatics Approaches to Predict Mutation Effects in the Binding Site of the Proangiogenic Molecule CD93.

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.

Effects of Nitisinone on Oxidative and Inflammatory Markers in Alkaptonuria: Results from SONIA1 and SONIA2 Studies.

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.

Artificial Intelligence Approaches in Drug Discovery: Towards the Laboratory of the Future.

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.

The Pharmacogenetics of Cannabis in the Treatment of Chronic Pain.

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.

Targeting neuronal calcium channels and GSK3ß for Alzheimer's disease with naturally-inspired Diels-Alder adducts.

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-3ß), 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 Cav modulator, inhibiting both L- and N-calcium channels, likely responsible for the Ca2+ overload observed in Alzheimer's disease. In a multitarget perspective, compound 11 appeared as an effective prototype, endowed with improved Cav inhibitory activity, with respect to the reference drug nifedipine, and encouraging modulating activity on GSK-3ß.

HGDiscovery: An online tool providing functional and phenotypic information on novel variants of homogentisate 1,2- dioxigenase.

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/).

Pharmacological and In Silico Analysis of Oat Avenanthramides as EGFR Inhibitors: Effects on EGF-Induced Lung Cancer Cell Growth and Migration.

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.

The drp-1-mediated mitochondrial fission inhibitor mdivi-1 impacts the function of ion channels and pathways underpinning vascular smooth muscle tone.

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 CaV1.2 and KCa1.1 channels, both in vitro and in silico as well as on the mechanical activity of rat aorta rings. Mdivi-1 was an effective CaV1.2 channel blocker, docking in a CaV1.2 channel antagonist binding region, stimulated KCa1.1 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 Ca2+ 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.

Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators.

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 KCa1.1 and CaV1.2 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. Ba2+ currents through CaV1.2 channels (IBa1.2) 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-[ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside], ambocin, butin, and biochanin A increased IKCa1.1. 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.

In Silico Multi-Target Approach Revealed Potential Lead Compounds as Scaffold for the Synthesis of Chemical Analogues Targeting SARS-CoV-2.

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.

Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria.

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.