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.

Untargeted NMR Metabolomics Reveals Alternative Biomarkers and Pathways in Alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare metabolic disease caused by the accumulation of homogentisic acid (HGA), an intermediate product of phenylalanine and tyrosine degradation. AKU patients carry variants within the gene coding for homogentisate-1,2-dioxygenase (HGD), which are responsible for reducing the enzyme catalytic activity and the consequent accumulation of HGA and formation of a dark pigment called the ochronotic pigment. In individuals with alkaptonuria, ochronotic pigmentation of connective tissues occurs, leading to inflammation, degeneration, and eventually osteoarthritis. The molecular mechanisms underlying the multisystemic development of the disease severity are still not fully understood and are mostly limited to the metabolic pathway segment involving HGA. In this view, untargeted metabolomics of biofluids in metabolic diseases allows the direct investigation of molecular species involved in pathways alterations and their interplay. Here, we present the untargeted metabolomics study of AKU through the nuclear magnetic resonance of urine from a cohort of Italian patients; the study aims to unravel molecular species and mechanisms underlying the AKU metabolic disorder. Dysregulation of metabolic pathways other than the HGD route and new potential biomarkers beyond homogentisate are suggested, contributing to a more comprehensive molecular signature definition for AKU and the development of future adjuvant treatment.

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.

Homogentisic acid induces cytoskeleton and extracellular matrix alteration in alkaptonuric cartilage.

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.

Padina pavonica Extract Promotes In Vitro Differentiation and Functionality of Human Primary Osteoblasts.

Marine algae have gained much importance in the development of nutraceutical products due to their high content of bioactive compounds. In this work, we investigated the activity of Padina pavonica with the aim to demonstrate the pro-osteogenic ability of its extract on human primary osteoblast (HOb). Our data indicated that the acetonic extract of P. pavonica (EPP) is a safe product as it did not show any effect on osteoblast viability. At the same time, EPP showed to possess a beneficial effect on HOb functionality, triggering their differentiation and mineralization abilities. In particular EPP enhanced the expression of the earlier differentiation stage markers: a 5.4-fold increase in collagen type I alpha 1 chain (COL1A1), and a 2.3-fold increase in alkaline phosphatase (ALPL), as well as those involved in the late differentiation stage: a 3.7-fold increase in osteocalcin (BGLAP) expression and a 2.8-fold in osteoprotegerin (TNFRSF11B). These findings were corroborated by the enhancement in ALPL enzymatic activity (1.7-fold increase) and by the reduction of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) ratio (0.6-fold decrease). Moreover, EPP demonstrated the capacity to enhance the bone nodules formation by 3.2-fold in 4 weeks treated HOb. Therefore, EPP showed a significant capability of promoting osteoblast phenotype. Given its positive effect on bone homeostasis, EPP could be used as a useful nutraceutical product that, in addition to a healthy lifestyle and diet, can be able to contrast and prevent bone diseases, especially those connected with ageing, such as osteoporosis (OP).

Novel smoothened antagonists as anti-neoplastic agents for the treatment of osteosarcoma.

Osteosarcoma (OS) is an ultra-rare highly malignant tumor of the skeletal system affecting mainly children and young adults and it is characterized by an extremely aggressive clinical course. OS patients are currently treated with chemotherapy and complete surgical resection of cancer tissue. However, resistance to chemotherapy and the recurrence of disease, as pulmonary metastasis, remain the two greatest challenges in the management, and treatment of this tumor. For these reasons, it is of primary interest to find alternative therapeutic strategies for OS. Dysregulated Hedgehog signalling is involved in the development of various types of cancers including OS. It has also been implicated in tumor/stromal interaction and cancer stem cell biology, and therefore presents a novel therapeutic strategy for cancer treatment. In our work, we tested the activity of five potent Smoothened (SMO) inhibitors, four acylguanidine and one acylthiourea derivatives, against an OS cell line. We found that almost all our compounds were able to inhibit OS cells proliferation and to reduce Gli1 protein levels. Our results also indicated that SMO inhibition in OS cells by such compounds, induces apoptosis with a nanomolar potency. These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with OS.

Differentially activated Src kinase in chemo-naïve human primary osteosarcoma cells and effects of a Src kinase inhibitor.

The therapeutic treatment of osteosarcoma (OS), a rare malignant teenage cancer of the skeletal system, still represents a great challenge as patient survival after conventional protocol chemotherapy treatment has not improved in the last four decades leaving poor patient prognoses. Therefore, many efforts have been done to find increasingly reliable OS cell models and to identify "druggable" targets in OS, in order to identify novel effective therapeutic approaches and treatment strategies. In this contest, the more successful use of patient-derived cell cultures in respect to human commercial lines and findings of Src kinase deregulation in cancer, prompted us to study for the first time the activation state of Src and the potential activity of our Src inhibitor SI-83 in a number of chemo-naïve patient-derived primary OS cells. We here demonstrate that Src is hyperactivated in OS cells in respect to the nonmalignant counterpart and that SI-83 is able to strongly decrease cell viability, proliferation, Src416 phosphorylation, and cell migration. © 2017 BioFactors, 43(6):801-811, 2017.

Histological and Ultrastructural Characterization of Alkaptonuric Tissues.

Alkaptonuria (AKU) is a hereditary disorder that results from altered structure and function of homogentisate 1,2 dioxygenase (HGD). This enzyme, predominantly produced by liver and kidney, is responsible for the breakdown of homogentisic acid (HGA), an intermediate in the tyrosine degradation pathway. A deficient HGD activity causes HGA levels to rise systemically. The disease is clinically characterized by homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and joint arthropathy. Additional manifestations are cardiovascular abnormalities, renal, urethral and prostate calculi and scleral and ear involvement. While the radiological aspect of ochronotic spondyloarthropathy is known, there are only few data regarding an exhaustive ultrastructural and histologic study of different tissues in AKU. Moreover, an in-depth analysis of tissues from patients of different ages, having varied symptoms, is currently lacking. A complete microscopic and ultrastructural analysis of different AKU tissues, coming from six differently aged patients, is here presented thus significantly contributing to a more comprehensive knowledge of this ultra-rare pathology.

Smoothened-antagonists reverse homogentisic acid-induced alterations of Hedgehog signaling and primary cilium length in alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway.

Cytoskeleton Aberrations in Alkaptonuric Chondrocytes.

Alkaptonuria (AKU) is an ultra-rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2-dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called "ochronosis." The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co-localization of SAA with three cytoskeletal proteins: actin, vimentin, and ß-tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4-HNE presence has been evaluated by means of immunofluorescence. J. Cell. Physiol. 232: 1728-1738, 2017. © 2016 Wiley Periodicals, Inc.

Angiogenesis in alkaptonuria.

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of AKU treatment is palliative and little is known about its physiopathology. Neovascularization is involved in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes AKU. Here, we investigated the presence of neoangiogenesis in AKU synovium and healthy controls. Synovium from AKU patients, who had undergone total joint replacement or arthroscopy, or from healthy patients without any history of rheumatic diseases, who underwent surgical operation following sport trauma was subjected to hematoxylin and eosin staining. Histologic grades were assigned for clinical disease activity and synovitis based on cellular content of the synovium. By immunofluorescence microscopy, using different endothelial cell markers, we observed large vascularization in AKU but not in healthy synovium. Moreover, Western blotting and quantification analyses confirmed strong expression of endothelial cell markers in AKU synovial tissues. Importantly, AKU synovium vascular endothelium expressed high levels of ß-dystroglycan, a protein previously involved in the regulation of angiogenesis in osteoarthritic synovium. This is the first report providing experimental evidences that new blood vessels are formed in AKU synovial tissues, opening new perspectives for AKU therapy.

Comparative proteomics in alkaptonuria provides insights into inflammation and oxidative stress.

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism associated with a defective catabolism of phenylalanine and tyrosine leading to increased systemic levels of homogentisic acid (HGA). Excess HGA is partly excreted in the urine, partly accumulated within the body and deposited onto connective tissues under the form of an ochronotic pigment, leading to a range of clinical manifestations. No clear genotype/phenotype correlation was found in AKU, and today there is the urgent need to identify biomarkers able to monitor AKU progression and evaluate response to treatment. With this aim, we provided the first proteomic study on serum and plasma samples from alkaptonuric individuals showing pathological SAA, CRP and Advanced Oxidation Protein Products (AOPP) levels. Interesting similarities with proteomic studies on other rheumatic diseases were highlighted together with proteome alterations supporting the existence of oxidative stress and inflammation in AKU. Potential candidate biomarkers to assess disease severity, monitor disease progression and evaluate response to treatment were identified as well.

Inhibition of para-Hydroxyphenylpyruvate Dioxygenase by Analogues of the Herbicide Nitisinone As a Strategy to Decrease Homogentisic Acid Levels, the Causative Agent of Alkaptonuria.

Alkaptonuria (AKU) is a rare multisystem metabolic disease caused by deficient activity of homogentisate 1,2-dioxygenase (HGD), which leads to the accumulation of homogentisic acid (HGA). Currently, there is no treatment for AKU. The sole drug with some beneficial effects is the herbicide nitisinone (1), an inhibitor of p-hydroxyphenylpyruvate dioxygenase (4-HPPD). 1 has been used as a life-saving drug in infants with type I tyrosinemia despite severe side effects due to the buildup of tyrosine. Four clinical trials of nitisinone to treat AKU have shown that 1 consistently decreases HGA levels, but also caused the accumulation of tyrosine in blood serum. Moreover, the human preclinical toxicological data for 1 are incomplete. In this work, we performed pharmacodynamics and toxicological evaluations of 1, providing the first report of LD50 values in human cells. Intracellular tyrosinemia was also evaluated. Three additional 4-HPPD inhibitors with a more favorable profile than that of 1 in terms of IC50, LD50, and tyrosine accumulation were also identified among commercially available compounds. These may be promising starting points for the development of new therapeutic strategies for the treatment of AKU.

Homogentisate 1,2 dioxygenase is expressed in brain: implications in alkaptonuria.

Alkaptonuria is an ultra-rare autosomal recessive disease developed from the lack of homogentisate 1,2-dioxygenase (HGD) activity, causing an accumulation in connective tissues of homogentisic acid (HGA) and its oxidized derivatives in polymerized form. The deposition of ochronotic pigment has been so far attributed to homogentisic acid produced by the liver, circulating in the blood, and accumulating locally. In the present paper, we report the expression of HGD in the brain. Mouse and human brain tissues were positively tested for HGD gene expression by western blotting. Furthermore, HGD expression was confirmed in human neuronal cells that also revealed the presence of six HGD molecular species. Moreover, once cultured in HGA excess, human neuronal cells produced ochronotic pigment and amyloid. Our findings indicate that alkaptonuric brain cells produce the ochronotic pigment in loco and this may contribute to induction of neurological complications.

Establishment of Four New Human Primary Cell Cultures from Chemo-Naïve Italian Osteosarcoma Patients.

Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones.

Amyloidosis, inflammation, and oxidative stress in the heart of an alkaptonuric patient.

BACKGROUND: Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient. RESULTS: Congo Red birefringence, Th-T fluorescence, and biochemical assays demonstrated the presence of SAA-amyloid deposits in AKU stenotic aortic valve. Light and electron microscopy assessed the colocalization of ochronotic pigment and SAA-amyloid, the presence of calcified areas in the valve. Immunofluorescence detected lipid peroxidation of the tissue and lymphocyte/macrophage infiltration causing inflammation. High SAA plasma levels and proinflammatory cytokines levels comparable to those from rheumatoid arthritis patients were found in AKU patient. CONCLUSIONS: SAA-amyloidosis was present in the aortic valve from an AKU patient and colocalized with ochronotic pigment as well as with tissue calcification, lipid oxidation, macrophages infiltration, cell death, and tissue degeneration. A local HGD expression in human cardiac tissue has also been ascertained suggesting a consequent local production of ochronotic pigment in AKU heart.

Proteomics and phosphoproteomics provide insights into the mechanism of action of a novel pyrazolo[3,4-d]pyrimidine Src inhibitor in human osteosarcoma.

Osteosarcoma (OS) is a highly malignant bone tumour, affecting mainly children and young adults between 10 and 20 years of age. It represents the most frequent primitive malignant tumour of the skeletal system and is characterized by an extremely aggressive clinical course, with rapid development of lung metastases. In the last few years, targeting Src in the treatment of OS has become one of the major challenges in the development of new drugs, since an elevated Src kinase activity has been associated with the development and the maintenance of the OS malignant phenotype. Recently, SI-83, a novel pyrazolo[3,4-d]pyrimidine derivate Src inhibitor, was selected as a promising OS therapeutic drug because of its elevated anti-tumour effects toward human OS. In the present study, gel-based proteomics and phosphoproteomics revealed significant changes in proteins involved in many cancer related processes. We got insight into SI-83 proapoptotic and antiproliferative properties (overrepresentation of GRIA1, GRP78, and CALR and underrepresentation of NPM1, RCN, and P4HB). Nevertheless, the most significant findings of our work are the SI-83 induced dephosphorylation of ARPC5L, a subunit of the actin related Arp2/3 complex, and the decrease of other cytoskeleton proteins. These data, together with a dramatic impairment of SaOS-2 cell migration and adhesion, suggest that SI-83 may have antimetastatic features that enhance its use as a potent OS chemotherapeutic drug.

Proteomics of osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant tumor of bone and the third most common cancer in childhood and adolescence. Nowadays, early diagnosis, drug resistance and recurrence of the disease represent the major challenges in OS treatment. Post-genomics, and in particular proteomic technologies, offer an invaluable opportunity to address the level of biological complexity expressed by OS. Although the main goal of OS oncoproteomics is focused on diagnostic and prognostic biomarker discovery, in this review we describe and discuss global protein profiling approaches to other aspects of OS biology and pathophysiology, or to investigate the mechanism of action of chemotherapeutics. In addition, we present proteomic analyses carried out on OS cell lines as in vitro models for studying osteoblastic cell biology and the attractive opportunity offered by proteomics of OS cancer stem cells.