Insight into Iron, Oxidative Stress and Ferroptosis: Therapy Targets for Approaching Anticancer Strategies.

Based on the multifaceted molecular machinery that tightly controls iron cellular homeostasis, this review delves into its paradoxical, potentially dangerous role in biological systems, with a special focus on double-edged sword correlations with cancer. Indeed, though iron is a vital micronutrient and a required cofactor participating in several essential cell functions, its tendency to cause oxidative stress can be related both to cancer risk and to the activation of cancer cell death pathways. In this scenario, ferroptosis refers to an iron-dependent form of regulated cell death (RCD) powered by an overload of lethal peroxides sharing distinctive oxidized phospholipid profiles. As a unique cell death pathway, ferroptosis is both morphologically and mechanistically different from other types of programmed cell death involving executioner family proteins. The accumulation of cytotoxic lipid peroxides encompasses a cellular antagonism between ferroptosis execution and defense systems, with iron-dependent death occurring when ferroptosis-promoting activities significantly exceed the cellular antioxidant defenses. The most recent molecular breakthroughs in the execution of ferroptosis have aroused great consideration in tumor biology, as targeting ferroptosis can provide new tools for exploring therapeutic strategies for tumor suppression. Mutations and death/survival pathway alterations, as well as distinctive metabolic regulations of cancer cells, including the propensity to generate ROS, are seen as features that can render cancer cells unprotected to ferroptosis, thereby exposing vulnerabilities which deserve further attention to be regarded as targetable for cancers with limited therapeutic options.

Role of airway epithelium in viral respiratory infections: Can carbocysteine prevent or mitigate them?

Alterations in airway epithelial homeostasis increase viral respiratory infections risk. Viral infections frequently are associated with chronic obstructive pulmonary disease (COPD) exacerbations, events that dramatically promote disease progression. Mechanism promoting the main respiratory viruses entry and virus-evocated innate and adaptive immune responses have now been elucidated, and an oxidative stress central role in these pathogenic processes has been recognized. Presence of reactive oxygen species in macrophages and other cells allows them to eliminate virus, but its excess alters the balance between innate and adaptive immune responses and proteases/anti-proteases and leads to uncontrolled inflammation, tissue damage, and hypercoagulability. Different upper and lower airway cell types also play a role in viral entry and infection. Carbocysteine is a muco-active drug with anti-oxidant and anti-inflammatory properties used for the management of several chronic respiratory diseases. Although the use of anti-oxidants has been proposed as an effective strategy in COPD exacerbations management, the molecular mechanisms that explain carbocysteine efficacy have not yet been fully clarified. The present review describes the most relevant features of the common respiratory virus pathophysiology with a focus on epithelial cells and oxidative stress role and reports data supporting a putative role of carbocysteine in viral respiratory infections.

Gold nanowires-based sensor for quantification of H2O2 released by human airway epithelial cells.

Hydrogen peroxide (H2O2) is a biomarker relevant for oxidative stress monitoring. Most chronic airway diseases are characterized by increased oxidative stress. To date, the main methods for the detection of this analyte are expensive and time-consuming laboratory techniques such as fluorometric and colorimetric assays. There is a growing interest in the development of electrochemical sensors for H2O2 detection due to their low cost, ease of use, sensitivity and rapid response. In this work, an electrochemical sensor based on gold nanowire arrays has been developed. Thanks to the catalytic activity of gold against hydrogen peroxide reduction and the high surface area of nanowires, this sensor allows the quantification of this analyte in a fast, efficient and selective way. The sensor was obtained by template electrodeposition and consists of gold nanowires about 5 µm high and with an average diameter of about 200 nm. The high active surface area of this electrode, about 7 times larger than a planar gold electrode, ensured a high sensitivity of the sensor (0.98 µA µM-1cm-2). The sensor allows the quantification of hydrogen peroxide in the range from 10 µM to 10 mM with a limit of detection of 3.2 µM. The sensor has excellent properties in terms of reproducibility, repeatability and selectivity. The sensor was validated by quantifying the hydrogen peroxide released by human airways A549 cells exposed or not to the pro-oxidant compound rotenone. The obtained results were validated by comparing them with those obtained by flow cytometry after staining the cells with the fluorescent superoxide-sensitive Mitosox Red probe giving a very good concordance.

Formoterol Exerts Anti-Cancer Effects Modulating Oxidative Stress and Epithelial-Mesenchymal Transition Processes in Cigarette Smoke Extract Exposed Lung Adenocarcinoma Cells.

Lung cancer frequently affects patients with Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS) fosters cancer progression by increasing oxidative stress and by modulating epithelial-mesenchymal transition (EMT) processes in cancer cells. Formoterol (FO), a long-acting ß2-agonist widely used for the treatment of COPD, exerts antioxidant activities. This study explored in a lung adenocarcinoma cell line (A549) whether FO counteracted the effects of cigarette smoke extract (CSE) relative to oxidative stress, inflammation, EMT processes, and cell migration and proliferation. A549 was stimulated with CSE and FO, ROS were evaluated by flow-cytometry and by nanostructured electrochemical sensor, EMT markers were evaluated by flow-cytometry and Real-Time PCR, IL-8 was evaluated by ELISA, cell migration was assessed by scratch and phalloidin test, and cell proliferation was assessed by clonogenic assay. CSE significantly increased the production of ROS, IL-8 release, cell migration and proliferation, and SNAIL1 expression but significantly decreased E-cadherin expression. FO reverted all these phenomena in CSE-stimulated A549 cells. The present study provides intriguing evidence that FO may exert anti-cancer effects by reverting oxidative stress, inflammation, and EMT markers induced by CS. These findings must be validated in future clinical studies to support FO as a valuable add-on treatment for lung cancer management.

Antiviral activity of Taurisolo® during bovine alphaherpesvirus 1 infection.

Bovine alphaherpesvirus 1 (BoAHV-1), the pathogen causing Infectious Bovine Rhinotracheitis (IBR) and predisposing to polymicrobial infections in cattle, provokes farm economic losses and trading restrictions in the world. However, nontoxic antiviral agents for BoAHV-1 infection are still unavailable, but plant extracts, such as flavonoid derivatives possess activity against BoAHV-1. Taurisolo®, a nutraceutical produced by Aglianico grape pomace, has recently shown promising antiviral activity. Herein, the potential activity of Taurisolo® during BoAHV-1 infection in Madin Darby bovine kidney (MDBK) cells was tested. Taurisolo® enhanced cell viability and reduced morphological death signs in BoAHV-1-infected cells. Moreover, Taurisolo® influenced the expression of bICP0, the key regulatory protein of BoAHV-1, and it strongly diminished virus yield. These effects were associated with an up-regulation of aryl hydrocarbon receptor (AhR), a transcription factor involved in microbial metabolism and immune response. In conclusion, our findings indicate that Taurisolo® may represent a potential antiviral agent against BoAHV-1 infection. Noteworthy, AhR could be involved in the observed effects and become a new target in antiviral therapy.

New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function.

OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

Maternal and Neonatal Outcomes in Intrahepatic Cholestasis of Pregnancy.

The aims of our study were to evaluate the maternal and fetal outcomes of intrahepatic cholestasis of pregnancy (ICP). In this observational, retrospective case-control study, we included all pregnant women who gave birth with a diagnosis of ICP between January 2010 and December 2020 at the Unit of Obstetrics and Gynecology, University Hospital of Messina. The data were compared with those from a control group of pregnant women who did not have ICP. One hundred twenty-nine and eighty-five patients were included, respectively, in the study and in the control group. There was a significant difference between the two groups in the incidence of hypothyroidism, thrombophilia, gestational diabetes, gestational hypertension, postpartum hemorrhage, and preterm delivery, which were more frequent in the ICP patients. No neonatal adverse events were recorded, although a significant difference in the meconium-stained amniotic fluid condition was noted. After a 24-month follow-up, 48/129 patients with ICP accepted to be reassessed by liver ultrasound, elastographic examination, and liver function blood tests. No patient showed signs of chronic liver disease. This study confirmed a higher probability of adverse short-term maternal outcomes in ICP pregnant patients, but a lower probability of adverse short-term fetal outcomes and the absence of a long-term maternal risk of chronic liver disease.

Overview of current detection methods and microRNA potential in Clostridioides difficile infection screening.

Clostridioides difficile (formerly called Clostridium difficile, C. difficile) infection (CDI) is listed as an urgent threat on the 2019 antibiotic resistance threats report in the United States by the Centers for Disease Control and Prevention. Early detection and appropriate disease management appear to be essential. Meanwhile, although the majority of cases are hospital-acquired CDI, community-acquired CDI cases are also on the rise, and this vulnerability is not limited to immunocompromised patients. Gastrointestinal treatments and/or gastrointestinal tract surgeries may be required for patients diagnosed with digestive diseases. Such treatments could suppress or interfere with the patient's immune system and disrupt gut flora homeostasis, creating a suitable microecosystem for C. difficile overgrowth. Currently, stool-based non-invasive screening is the first-line approach to CDI diagnosis, but the accuracy is varied due to different clinical microbiology detection methods; therefore, improving reliability is clearly required. In this review, we briefly summarised the life cycle and toxicity of C. difficile, and we examined existing diagnostic approaches with an emphasis on novel biomarkers such as microRNAs. These biomarkers can be easily detected through non-invasive liquid biopsy and can yield crucial information about ongoing pathological phenomena, particularly in CDI.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9.

Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported "pharm-druglike2" 3D structure-based pharmacophore model, introducing it as "pharm-druglike2.1". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

Tetrasubstituted Pyrrole Derivative Mimetics of Protein-Protein Interaction Hot-Spot Residues: A Promising Class of Anticancer Agents Targeting Melanoma Cells.

A new series of tetrasubstituted pyrrole derivatives (TSPs) was synthesized based on a previously developed hypothesis on their ability to mimic hydrophobic protein motifs. The resulting new TSPs were endowed with a significant toxicity against human epithelial melanoma A375 cells, showing IC50 values ranging from 10 to 27 µM, consistent with the IC50 value of the reference compound nutlin-3a (IC50 = 15 µM). In particular, compound 10a (IC50 = 10 µM) resulted as both the most soluble and active among the previous and present TSPs. The biological investigation evidenced that the anticancer activity is related to the activation of apoptotic cell-death pathways, supporting our rational design based on the ability of TSPs to interfere with PPI involved in the cell cycle regulation of cancer cells and, in particular, the p53 pathway. A reinvestigation of the TSP pharmacophore by using DFT calculations showed that the three aromatic substituents on the pyrrole core are able to mimic the hydrophobic side chains of the hot-spot residues of parallel and antiparallel coiled coil structures suggesting a possible molecular mechanism of action. A structure-activity relationship (SAR) analysis which includes solubility studies allows us to rationalize the role of the different substituents on the pyrrole core.

The role of High-mobility group box-1 and Psoriasin in multiple myeloma: Analysis of a population affected by monoclonal gammopathies and review of the literature.

Multiple myeloma (MM) is a plasma cells neoplasm which is often preceded by a preneoplastic condition called monoclonal gammopathy of unknown significance (MGUS). A protein called High-mobility group box-1 (HMGB-1) controls transcription and genomic stability. Both pro- and anti-tumor properties of HMGB1 have been described during tumor growth. The S100 protein family includes a protein known as psoriasin. Poorer prognosis and survival were linked to higher psoriasin expression in cancer patients. The goal of the current investigation was to compare the plasma levels of HMGB-1 and psoriasin in patients with MM and MGUS significance, as well as in a group of healthy controls. According to our research, patients with MGUS have higher HMGHB-1 concentrations than healthy controls (846.7 ± 287.6 pg/ml vs. 176.9 ± 204.8 pg/ml for controls, p < 0.001). Similarly, we found a huge difference in HMGB-1 levels for MM patients with respect to controls (928.0 ± 551.4 pg/ml vs. 176.9 ± 204.8 pg/ml; p = 0.001). No difference was found as for the Psoriasin levels in the three groups considered. Additionally, we tried to evaluate the knowledge already present in the literature about putative mechanisms of action for these molecules in the onset and development of these disorders.

Triple Negative Breast Cancer Preclinical Therapeutic Management by a Cationic Ruthenium-Based Nucleolipid Nanosystem.

Based on compelling preclinical evidence concerning the progress of our novel ruthenium-based metallotherapeutics, we are focusing research efforts on challenging indications for the treatment of invasive neoplasms such as the triple-negative breast cancer (TNBC). This malignancy mainly afflicts younger women, who are black, or who have a BRCA1 mutation. Because of faster growing and spreading, TNBC differs from other invasive breast cancers having fewer treatment options and worse prognosis, where existing therapies are mostly ineffective, resulting in a large unmet biomedical need. In this context, we benefited from an experimental model of TNBC both in vitro and in vivo to explore the effects of a biocompatible cationic liposomal nanoformulation, named HoThyRu/DOTAP, able to effectively deliver the antiproliferative ruthenium(III) complex AziRu, thus resulting in a prospective candidate drug. As part of the multitargeting mechanisms featuring metal-based therapeutics other than platinum-containing agents, we herein validate the potential of HoThyRu/DOTAP liposomes to act as a multimodal anticancer agent through inhibition of TNBC cell growth and proliferation, as well as migration and invasion. The here-obtained preclinical findings suggest a potential targeting of the complex pathways network controlling invasive and migratory cancer phenotypes. Overall, in the field of alternative chemotherapy to platinum-based drugs, these outcomes suggest prospective brand-new settings for the nanostructured AziRu complex to get promising goals for the treatment of metastatic TNBC.

Survival Outcomes in Squamous Cell Carcinoma of the External Auditory Canal: A Systematic Review and Meta-Analysis.

Squamous cell carcinomas (SCC) of the external auditory canal (EAC) are rare tumors representing a surgical challenge. Current knowledge is based largely on case series; thus, the level of evidence is weak. This study sought to systematically review the available SCC of the EAC literature and to identify risk factors for overall survival (OS) and disease-specific survival (DSS). A systematic review and meta-analysis of papers searched up to December 2022 through PubMed, Scopus, Web of Science, and Cochrane Library databases was conducted. Quality assessment of the eligible studies was done according to the Newcastle-Ottawa Scale. Pooled univariate and multivariable analyses and meta-analysis using a random-effects or fixed-effects Mantel-Haenszel model were performed. Fifteen articles (282 patients) met the inclusion criteria and were included in the quantitative analysis. The pooled multivariable analysis revealed cT3 and cT4 as independent prognostic factors for OS (p = 0.005, and p < 0.001, respectively) and DSS (p = 0.002, and p < 0.001, respectively). Local recurrence rate was 32.3%. The meta-analysis estimated significantly higher odds ratios for advanced T categories, than cT1-T2 tumors for OS and DSS (OR = 3.55; 95% CI, 1.93-6.52, and OR = 3.73; 95% CI, 2.00-6.97, respectively). In conclusion, locally advanced tumors were associated with poor prognosis. Poor outcomes mostly occurred due to local recurrence.

Sustained-Release Powders Based on Polymer Particles for Pulmonary Delivery of Beclomethasone Dipropionate in the Treatment of Lung Inflammation.

Inhaled corticosteroids are the mainstay in the management of lung inflammation associated to chronic lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Nonetheless, available inhalation products are mostly short-acting formulations that require frequent administrations and do not always produce the desired anti-inflammatory effects. In this work, the production of inhalable beclomethasone dipropionate (BDP) dry powders based on polymeric particles was attempted. As starting material, the PHEA-g-RhB-g-PLA-g-PEG copolymer was chosen, obtained by grafting 0.6, 2.4 and 3.0 mol%, respectively, of rhodamine (RhB), polylactic acid (PLA) and polyethylene glycol 5000 (PEG) on alpha,beta-poly(N-2-hydroxyethyl)DL-aspartamide (PHEA). The drug was loaded into the polymeric particles (MP) as an inclusion complex (CI) with hydroxypropyl-cyclodextrin (HP-ß-Cyd) (at a stoichiometric ratio of 1:1) or as free form. The spray-drying (SD) process to produce MPs was optimized by keeping the polymer concentration (0.6 wt/vol%) constant in the liquid feed and by varying other parameters such as the drug concentration. The theoretical aerodynamic diameter (daer) values among the MPs are comparable and potentially suitable for inhalation, as confirmed also through evaluation of the experimental mass median aerodynamic diameter (MMADexp). BDP shows a controlled release profile from MPs that is significantly higher (more than tripled) than from Clenil®. In vitro tests on bronchial epithelial cells (16HBE) and adenocarcinomic human alveolar basal epithelial cells (A549) showed that all the MP samples (empty or drug-loaded) were highly biocompatible. None of the systems used induced apoptosis or necrosis. Moreover, the BDP loaded into the particles (BDP-Micro and CI-Micro) was more efficient than free BDP to counteract the effects of cigarette smoke and LPS on release of IL-6 and IL-8.

Tyndallized Bacteria Preferentially Induce Human Macrophage M1 Polarization: An Effect Useful to Balance Allergic Immune Responses and to Control Infections.

Macrophage polarization is a dynamic process through which macrophages acquire specific features whose extremes are represented by M1 and M2 polarization. Interleukin (IL)-6, IL-1ß, IL-12 and IL-8 belong to M1 macrophages while transforming growth factor-beta (TGF-ß belongs to M2 cytokines. M2 polarization prevalence is observed in allergic diseases. Tyndallization is a thermal process able to inactivate microorganisms and to allow their use for chronic respiratory disease treatment via immune response modulation. The present study explores the effects of a blend of tyndallized bacteria (TB) on macrophage polarization. THP-1-derived macrophages were exposed to different concentrations of TB (106, 5 × 106, 107, 5 × 107, 108 CFU/mL) and then cell viability and TB phagocytosis, and IL-8, IL-1ß, IL-6, IL-12 and TGF-ß1 gene expression and release were assessed. TB were tolerated, phagocyted and able to increase IL-8, IL-1ß and IL-6 gene expression and release IL-12 gene expression, as well as decrease TGF-ß1 gene expression and release. The effects on IL-8, IL-6 and TGF-ß1 release were confirmed in human monocyte-derived macrophages (hMDMs) exposed to TB. In conclusion, TB promote M1 polarization, and this mechanism might have valuable potential in controlling allergic diseases and infections, possibly preventing disease exacerbations.

Oxidative Stress, Environmental Pollution, and Lifestyle as Determinants of Asthma in Children.

Exposure to cigarette smoke, allergens, viruses, and other environmental contaminants, as well as a detrimental lifestyle, are the main factors supporting elevated levels of airway oxidative stress. Elevated oxidative stress results from an imbalance in reactive oxygen species (ROS) production and efficiency in antioxidant defense systems. Uncontrolled increased oxidative stress amplifies inflammatory processes and tissue damage and alters innate and adaptive immunity, thus compromising airway homeostasis. Oxidative stress events reduce responsiveness to corticosteroids. These events can increase risk of asthma into adolescence and prompt evolution of asthma toward its most severe forms. Development of new therapies aimed to restore oxidant/antioxidant balance and active interventions aimed to improve physical activity and quality/quantity of food are all necessary strategies to prevent asthma onset and avoid in asthmatics evolution toward severe forms of the disease.

Lipophilicity profiling and cell viability assessment of a selected panel of endocrine disruptors.

Endocrine disruptors are chemicals widely used worldwide by industries in a variety of applications. Routinely exposure to these chemicals, even if at low doses, can cause damage effects on human health. In the present study, we evaluated toxic effects of nine chemicals, among which phthalates, using various cell lines to inspect their capability to interfere with cell proliferation and viability. Alongside, we investigated their affinity for phospholipids to assess the possible passage through biomembranes. Experimentally determined logkwIAM.MG values ranged from 1.37 to 3.49 whilst calculated log kwIAM.DD2 spanned from 1.80 to 5.21, supporting the target contaminants to exhibit lipophilicity moderate or very high. The achieved results were related to pharmacokinetic and toxicological properties by ADMET predictor™ and EPI Suite™ software. Triclosan and 4-Nonylphenol were found to be the most toxic against all cell lines screened, showing an IC50 of 30 µM for triclosan on human keratinocytes and of 50 µM for 4-Nonylphenol on human colorectal adenocarcinoma cells. Overall, even if the phthalates showed higher IC50 values (ranging from 170 µM to 280 µM), we can assert that all contaminants herein tested were able to interfere with cell growth and viability.

Identification of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based small molecules as selective BRD9 binders.

Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for the development of new agents endowed with anticancer properties. With this aim, a set of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following a combined approach that relied on in silico studies, chemical synthesis, biophysical and biological evaluation of the most promising items. The protocol was initially based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested in silico against the bromodomain of BRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bind BRD9 in the submicromolar range (IC50 = 0.35 ± 0.18 µM and IC50 = 0.14 ± 0.03 µM, respectively) showing a promising selectivity profile when tested against further nine bromodomains. Taking advantage of 3D structure-based pharmacophore models, additional 10 derivatives were selected in silico for the synthetic step and binding assessment, highlighting seven compounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9 among different bromodomains. The ability of the identified BRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very good passive permeability profile. Preliminary studies were carried out on a panel of healthy and cancer human cell lines to explore the biological behavior of the selected compounds, disclosing a moderate activity and significant selectivity profile towards leukaemia cells. These results highlighted the applicability of the reported multidisciplinary approach for accelerating the selection of promising items and for driving the chemical synthesis of novel selective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for further exploring the chemical space in order to obtain new analogues with improved potency.