Early investigation of a novel SI306 theranostic prodrug for glioblastoma treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive malignancies with a high recurrence rate and poor prognosis. Theranostic, combining therapeutic and diagnostic approaches, arises as a successful strategy to improve patient outcomes through personalized medicine. Src is a non-receptor tyrosine kinase (nRTK) whose involvement in GBM has been extensively demonstrated. Our previous research highlighted the effectiveness of the pyrazolo[3,4-d]pyrimidine SI306 and its more soluble prodrug CMP1 as Src inhibitors both in in vitro and in vivo GBM models. In this scenario, we decided to develop a theranostic prodrug of SI306, ProSI-DOTA(68 Ga) 1, which was designed to target GBM cells after hydrolysis and follow-up on the disease's progression and improve the therapy's outcome. First, the corresponding nonradioactive prodrug 2 was tested to evaluate its ADME profile and biological activity. It showed good metabolic stability, no inhibition of CYP3A4, suboptimal aqueous solubility, and slight gastrointestinal and blood-brain barrier passive permeability. Compound 2 exhibited a drastic reduction of cell vitality after 72 h on two different GBM cell lines (GL261 and U87MG). Then, 2 was subjected to complexation with the radionuclide Gallium-68 to give ProSI-DOTA(68 Ga) 1. The cellular uptake of 1 was evaluated on GBM cells, highlighting a slight but significant time-dependent uptake. The data obtained from our preliminary studies reflect the physiochemical properties of 1. The use of an alternative route of administration, such as the intranasal route, could overcome the physiochemical limitations and enhance the pharmacokinetic properties of 1, paving the way for its future development.

Consensus screening for a challenging target: the quest for P-glycoprotein inhibitors.

ATP-binding cassette (ABC) transporters are a large family of proteins involved in membrane transport of a wide variety of substrates. Among them, ABCB1, also known as MDR-1 or P-glycoprotein (P-gp), is the most characterized. By exporting xenobiotics out of the cell, P-gp activity can affect the ADME properties of several drugs. Moreover, P-gp has been found to mediate multidrug resistance in cancer cells. Thus, the inhibition of P-gp activity may lead to increased absorption and/or intracellular accumulation of co-administered drugs, enhancing their effectiveness. Using the human-mouse chimeric cryoEM 3D structure of the P-gp in the inhibitor-bound intermediate form (PDBID: 6qee), approximately 200 000 commercially available natural compounds from the ZINC database were virtually screened. To build a model able to discriminate between substrate and inhibitors, two datasets of compounds with known activity, including P-gp inhibitors, substrates, and inactive molecules were also docked. The best docking pose of selected substrates and inhibitors were used to generate 3D common feature pharmacophoric models that were combined with the Autodock Vina binding energy values to prioritize compounds for visual inspection. With this consensus approach, 13 potential candidates were identified and then tested for their ability to inhibit P-gp, using zosuquidar, a third generation P-gp inhibitor, as a reference drug. Eight compounds were found to be active with 6 of them having an IC50 lower than 5 µM in a membrane-based ATPase activity assay. Moreover, the P-gp inhibitory activity was also confirmed by two different cell-based in vitro methods. Both retrospective and prospective results demonstrate the ability of the combined structure-based pharmacophore modeling and docking-based virtual screening approach to predict novel hit compounds with inhibitory activity toward P-gp. The resulting chemical scaffolds could serve as inspiration for the optimization of novel and more potent P-gp inhibitors.

Antiviral Activity of Cannabidiolic Acid and Its Methyl Ester against SARS-CoV-2.

In the present study, the antiviral activity of cannabinoids isolated from Cannabis sativa L. was assessed in vitro against a panel of SARS-CoV-2 variants, indicating cannabidiolic acid (CBDA) was the most active. To overcome the instability issue of CBDA, its methyl ester was synthesized and tested for the first time for its antiviral activity. CBDA methyl ester showed a neutralizing effect on all the SARS-CoV-2 variants tested with greater activity than the parent compound. Its stability in vitro was confirmed by ultra-high-performance liquid chromatography (UHPLC) analysis coupled with high-resolution mass spectrometry (HRMS). In addition, the capacity of both CBDA and its derivative to interact with the virus spike protein was assessed in silico. These results showed that CBDA methyl ester can be considered as a lead compound to be further developed as a new effective drug against COVID-19 infection.

Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol.

The prevalence of obesity is steadily rising, making safe and more efficient anti-obesity treatments an urgent medical need. Growing evidence correlates obesity and comorbidities, including anxiety and depression, with the development of a low-grade inflammation in peripheral and central tissues. We hypothesized that attenuating neuroinflammation might reduce weight gain and improve mood. We investigated the efficacy of a methanolic extract from Helichrysum stoechas (L.) Moench (HSE), well-known for its anti-inflammatory properties, and its main constituent arzanol (AZL). HPLC-ESI-MS2 and HPLC-UV were used to characterize the extract. HSE effects on mood and feeding behavior was assessed in mice. The mechanism of action of HSE and AZL was investigated in hippocampus samples and SH-SY5Y cells by western blotting and immunofluorescence. Oral administration of HSE for 3 weeks limited weight gain with no significant decrease in food intake. HSE produced an anxiolytic-like and antidepressant-like phenotype comparable to diazepam and amitriptyline, respectively, in the absence of locomotor and cognitive impairments and induced neuroprotective effects in glutamate-exposed SH-SY5Y cells. A dose-dependent reduction of SIRT1 expression was detected in SH-SY5Y cells and in hippocampal samples from HSE-treated mice. The inhibition of the SIRT1-FoxO1 pathway was induced in the hypothalamus. Molecular docking studies proposed a mechanism of SIRT1 inhibition by AZL, confirmed by the evaluation of inhibitory effects on SIRT1 enzymatic activity. HSE limited weight gain and comorbidities through an AZL-mediated SIRT1 inhibition. These activities indicate HSE an innovative therapeutic perspective for obesity and associated mood disorders.

CanB, a Druggable Cellular Target in Mycobacterium tuberculosis.

Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis ß-carbonic anhydrases that catalyze the reversible reaction of CO2 hydration to form HCO3- and H+. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.

Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

Effect of in vitro simulated digestion on the anti-Helicobacter Pylori activity of different Propolis extracts.

Helicobacter pylori (HP) is among the most common pathogens causing infection in humans worldwide. Oxidative stress and gastric inflammation are involved in the progression of HP-related gastric diseases, and they can be targeted by integrating conventional antibiotic treatment with polyphenol-enriched natural products. In this work, we characterised three different propolis extracts and evaluated their stability under in vitro simulated gastric digestion, compared to their main constituents alone. The extract with the highest stability to digestion (namely, the dark propolis extract, DPE) showed a minimum bactericidal concentration (MBC) lower than 1 mg/mL on HP strains with different virulence factors. Finally, since urease is one of the virulence factors contributing to the establishment of a microenvironment that promotes HP infection, we evaluated the possible inhibition of this enzyme by using molecular docking simulations and in vitro colorimetric assay, showing that galangin and pinocembrin may be involved in this activity.

Zingiberene, a non-zinc-binding class I HDAC inhibitor: A novel strategy for the management of neuropathic pain.

BACKGROUND: Even though numerous Histone deacetylase inhibitors (HDACi) have been approved for the treatment of different types of cancer, and others are in clinical trials for the treatment of neurodegenerative diseases, the main problem related to the clinical use of available HDACi is their low isoform selectivity which causes undesirable effects and inevitably limits their therapeutic application. Previously, we demonstrated that a standardized Zingiber officinalis Roscoe rhizome extract (ZOE) reduced neuroinflammation through HDAC1 inhibition in a mice model of neuropathy, and this activity was related to terpenes fraction. HYPOTHESIS/PURPOSE: The aim of this work was to identify the ZOE constituent responsible for the activity on HDAC1 and to study its possible application in trauma-induced neuropathic pain. METHODS: The ability of ZOE and its terpenes fraction (ZTE) to inhibit HDAC and SIRT isoforms activity and protein expression was assessed in vitro. Then, a structure-based virtual screening approach was applied to predict which constituent could be responsible for the activity. In the next step, the activity of selected compound was tested in an in vitro model of neuroinflammation and in an in vivo model of peripheral neuropathy (SNI). RESULTS: ZTE resulted to be more potent than ZOE on HDAC1, 2, and 6 isoforms, while ZOE was more active on HDAC8. Zingiberene (ZNG) was found to be the most promising HDAC1 inhibitor, with an IC50 of 2.3 ± 0.1 µM. A non-zinc-binding mechanism of inhibition was proposed based on molecular docking. Moreover, the oral administration of ZNG reduced thermal hyperalgesia and mechanical allodynia in animals with neuropathy after 60 min from administration, and decreased HDAC-1 levels in the spinal cord microglia. CONCLUSION: We found a new non-zinc-dependent inhibitor of HDAC class I, with a therapeutic application in trauma-related neuropathic pain forms in which microglia-spinal overexpression of HDAC1 occurs. The non-zinc-binding mechanism has the potential to reduce off target effects, leading to a higher selectivity and better safety profile, compared to other HDAC inhibitors.

Cannabidiol-rich non-psychotropic Cannabis sativa L. oils attenuate peripheral neuropathy symptoms by regulation of CB2-mediated microglial neuroinflammation.

Neuropathic pain (NP) is a chronic disease that affects the normal quality of life of patients. To date, the therapies available are only symptomatic and they are unable to reduce the progression of the disease. Many studies reported the efficacy of Cannabis sativa L. (C. sativa) on NP, but no Δ9 -tetrahydrocannabinol (Δ9 -THC)-free extracts have been investigated in detail for this activity so far. The principal aim of this work is to investigate the potential pain-relieving effect of innovative cannabidiol-rich non-psychotropic C. sativa oils, with a high content of terpenes (K2), compared to the same extract devoid of terpenes (K1). Oral administration of K2 (25 mg kg-1 ) induced a rapid and long-lasting relief of pain hypersensitivity in a mice model of peripheral neuropathy. In spinal cord samples, K2 reduced mitogen-activated protein kinase (MAPKs) levels and neuroinflammatory factors. These effects were reverted by the administration of a CB2 antagonist (AM630), but not by a CB1 antagonist (AM251). Conversely, K1 showed a lower efficacy in the absence of CB1/CB2-mediated mechanisms. In LPS-stimulated murine microglial cells (BV2), K2 reduced microglia pro-inflammatory phenotype through the downregulation of histone deacetylase 1 (HDAC-1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IKBα) and increased interleukin-10 (IL-10) expression, an important antiinflammatory cytokine. In conclusion, these results suggested that K2 oral administration attenuated NP symptoms by reducing spinal neuroinflammation and underline the important role of the synergism between cannabinoids and terpenes.

Synthesis and biological activity evaluation of 3-(hetero) arylideneindolin-2-ones as potential c-Src inhibitors.

Inhibition of c-Src is considered one of the most studied approaches to cancer treatment, with several heterocyclic compounds approved during the last 15 years as chemotherapeutic agents. Starting from the biological evaluation of an in-house collection of small molecules, indolinone was selected as the most promising scaffold. In this work, several functionalised indolinones were synthesised and their inhibitory potency and cytotoxic activity were assayed. The pharmacological profile of the most active compounds, supported by molecular modelling studies, revealed that the presence of an amino group increased the affinity towards the ATP-binding site of c-Src. At the same time, bulkier derivatizations seemed to improve the interactions within the enzymatic pocket. Overall, these data represent an early stage towards the optimisation of new, easy-to-be functionalised indolinones as potential c-Src inhibitors.

Investigation on the neuroprotective effect of a cannabidiol-enriched non-psychotropic Cannabis sativa L. extract in an in vitro model of excitotoxicity.

The purpose of this study was to evaluate the neuroprotective effect of a cannabidiol-enriched non-psychotropic Cannabis sativa L. extract (CSE) and its main constituents, cannabidiol and ß-caryophyllene. An in vitro model of glutamate-induced neuronal excitotoxicity using SH-SY5Y cells was optimized. The impact of CSE on glutamate-impaired cell viability, brain-derived neurotrophic factor release, CB1 protein expression, and ERK levels was evaluated. The involvement of CB1 modulation was verified by the cotreatment with the CB1 antagonist AM4113. CSE was able to significantly protect SH-SY5Y from glutamate-impaired cell viability, and to counteract the changes in brain-derived neurotrophic factor levels, with a mechanism of action involving ERK modulation. Moreover, CSE completely reversed the reduction of CB1 receptor expression induced by glutamate, and the presence of the CB1 antagonist AM4113 reduced CSE effectiveness, suggesting that CBr play a role in the modulation of neuronal excitotoxicity. This work demonstrated the in vitro effectiveness of CSE as a neuroprotective agent, proposing the whole cannabis phytocomplex as a more effective strategy, compared to its main constituents alone, and suggested further investigations by using more complex cell models before moving to in vivo studies.

Quinolines and Oxazino-quinoline Derivatives as Small Molecule GLI1 Inhibitors Identified by Virtual Screening.

A virtual screening approach based on a five-feature pharmacophoric model for negative modulators of GLI1 was applied to databases of commercially available compounds. The resulting quinoline derivatives showed significant ability to reduce the GLI1 protein level and were characterized by submicromolar antiproliferative activity toward human melanoma A375 and medulloblastoma DAOY cell lines. Decoration of the quinoline ring and chemical rigidification to an oxazino-quinoline scaffold allowed us to deduce SAR considerations for future ligand optimization.

Survey on the Recent Advances in 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) Inhibition by Diketone and Triketone Derivatives and Congeneric Compounds: Structural Analysis of HPPD/Inhibitor Complexes and Structure-Activity Relationship Considerations.

The serendipitous discovery of the HPPD inhibitors from allelopathic plants opened the way for searching new and effective herbicidal agents by application of classical hit-to-lead optimization approaches. A plethora of active and selective compounds were discovered that belong to three major classes of cyclohexane-based triketones, pyrazole-based diketones, and diketonitriles. In addition, to enhance inhibitory constant and herbicidal activity, many efforts were also made to gain broader weed control, crop safety, and eventual agricultural applicability. Moreover, HPPD inhibitors emerged as therapeutic agents for inherited and metabolic human diseases as well as vector-selective insecticides in the control of hematophagous arthropods. Given the large set of experimental data available, structure-activity relationship analysis could be used to derive suggestions for next generation optimized compounds.

Tapping into the antitubercular potential of 2,5-dimethylpyrroles: A structure-activity relationship interrogation.

An exploration of the chemical space around a 2,5-dimethylpyrrole scaffold of antitubercular hit compound 1 has led to the identification of new derivatives active against Mycobacterium tuberculosis and multidrug-resistant clinical isolates. Analogues incorporating a cyclohexanemethyl group on the methyleneamine side chain at C3 of the pyrrole core, including 5n and 5q, exhibited potent inhibitory effects against the M. tuberculosis strains, substantiating the essentiality of the moiety to their antimycobacterial activity. In addition, selected derivatives showed promising cytotoxicity profiles against human pulmonary fibroblasts and/or murine macrophages, proved to be effective in inhibiting the growth of intracellular mycobacteria, and elicited either bactericidal effects, or bacteriostatic activity comparable to 1. Computational studies revealed that the new compounds bind to the putative target, MmpL3, in a manner similar to that of known inhibitors BM212 and SQ109.

Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling.

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and ß-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

Recent research results have converted gp120 binders to a therapeutic option for the treatment of HIV-1 infection. A medicinal chemistry point of view.

Current therapeutic armamentarium for treatment of HIV-1 infection is based on the use of highly active antiretroviral therapy that, unfortunately, does not act as a curative remedy. Moreover, duration of the therapy often results in lack of compliance with the consequent emergence of multidrug resistance. Finally, drug toxicity issues also arise during treatments. In the attempt to achieve a curative effect, in addition to invest substantial resources in finding new anti-HIV-1 agents and in optimizing antiviral lead compounds and drugs currently available, additional efforts should be done to deplete viral reservoir located within host CD4+ T cells. Gp120 binders represent a class of compounds able to affect the interactions between viral envelope proteins and host CD4, thus avoiding virus-to-cell attachment and fusion, and the consequent viral entry into host cells. This review summarizes the efforts done in the last five years to design new gp120 binders, that finally culminated in the approval of fostemsavir as an anti-HIV-1 drug.

Structural Rigidification of N-Aryl-pyrroles into Indoles Active against Intracellular and Drug-Resistant Mycobacteria.

A series of indolyl-3-methyleneamines incorporating lipophilic side chains were designed through a structural rigidification approach and synthesized for investigation as new chemical entities against Mycobacterium tuberculosis (Mtb). The screening led to the identification of a 6-chloroindole analogue 7j bearing an N-octyl chain and a cycloheptyl moiety, which displayed potent in vitro activity against laboratory and clinical Mtb strains, including a pre-extensively drug-resistant (pre-XDR) isolate. 7j also demonstrated a marked ability to restrict the intracellular growth of Mtb in murine macrophages. Further assays geared toward mechanism of action elucidation have thus far ruled out the involvement of various known promiscuous targets, thereby suggesting that the new indole 7j may inhibit Mtb via a unique mechanism.

The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme.

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

Conformational insights into the C-terminal mutations of human rhodopsin in retinitispigmentosa.

Rhodopsin is a light-sensitive transmembrane receptor involved in the visual transduction cascade. Among the several rhodopsin mutations related to retinitis pigmentosa (RP), those affecting the C-terminal VAPA-COOH motif that is implicated in rhodopsin trafficking from the Golgi to the rod outer segment are notably associated with more aggressive RP forms. However, molecular reasons for defective rhodopsin signaling due to VAPA-COOH mutations, which might include steric hindrance, physicochemical features and structural determinants, are yet unknown, thus limiting further drug design approaches. In this work, clinically relevant rhodopsin mutations at the P347 site within the VAPA-COOH motif were investigated by molecular dynamics (MD) simulations and compared to the wild-type (WT) system. In agreement with experimental evidence, conformational fluctuations of the intrinsically disordered C-terminal tail of WT and mutant rhodopsin were found not to affect the overall structure of the transmembrane domain, including binding to the retinal cofactor. The WT VAPA-COOH motif adopts a unique conformation that is not found in pathological mutants, suggesting that structural features could better explain the pathogenicity of P347 rhodopsin mutants than physicochemical or steric determinants. These results were confirmed by MD simulations in both membrane-embedded full-length opsin and membrane-free C-terminal deca-peptides, these latter becoming very useful and small-size model systems for further investigations of rhodopsin C-terminal mutations. Structural details elucidated in this work might facilitate the understanding of the pathological mechanisms of this class of rhodopsin mutants, which will be instrumental to the development of new therapeutic strategies.

A molecular spectroscopy approach for the investigation of early phase ochronotic pigment development in Alkaptonuria.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.