Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC.

A new series of dithiocarbamates (DTCs) was prepared from primary/secondary amines incorporating amino/hydroxyl-alkyl, mono- and bicyclic aliphatic ring systems based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, and carbon disulfide. The compounds were investigated for the inhibition of four mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, that is, the human (h) hCA I, II, IX and XII, drug targets for antiglaucoma (hCA II and XII) or antitumor (hCA IX/XII) agents. The compounds were moderate or inefficient hCA I inhibitors (off-target isoform for both applications), efficiently inhibited hCA II, whereas some of them were low nanomolar/subnanomolar hCA IX/XII inhibitors. One DTC showed excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, with a two times better efficiency compared to the clinically used sulfonamide dorzolamide.

New N-acetyl/N-thiocarbamoylpyrazoline derivatives were designed and synthesized in high yields to assess their inhibitory activity and selectivity against human monoamine oxidase A and B. The most important chiral compounds were separated into their single enantiomers and tested. The impact of the substituents at N1, C3 and C5 positions as well the influence of the configuration of the C5 on the biological activity were analyzed. Bulky aromatic groups at C5 were not tolerated. p-Prenyloxyaryl moiety at C3 oriented the selectivity toward the B isoform. The results were also corroborated by molecular modelling studies providing new suggestions for the synthesis of privileged structures to serve as lead compounds for the treatment of mood disorders and neurodegenerative diseases.

Interfering with tumor metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Featuring a rapid proliferation rate and exacerbated glycolysis, hepatocellular carcinoma (HCC) creates a highly hypoxic microenvironment with excessive production of lactic and carbonic acids. These metabolic conditions promote disease aggressiveness and cancer-related immunosuppression. The pH regulatory molecules work as a bridge between tumor cells and their surrounding milieu. Herein, we show that the pH regulatory molecules CAIX, CAXII and V-ATPase are overexpressed in the HCC microenvironment and that interfering with their pathways exerts antitumor activity. Importantly, the V-ATPase complex was expressed by M2-like tumor-associated macrophages. Blocking ex vivo V-ATPase activity established a less immune-suppressive tumor microenvironment and reversed the mesenchymal features of HCC. Thus, targeting the unique cross-talk between tumor cells and the tumor microenvironment played by pH regulatory molecules holds promise as a strategy to control HCC progression and to reduce the immunosuppressive pressure mediated by the hypoxic/acidic metabolism, particularly considering the potential combination of this strategy with emerging immune checkpoint-based immunotherapies.

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK(i) ≥5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [(3)H]epibatidine on chimeric α7/5-HT(3) receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.

The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100 nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, ζ-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells.

Pomegranate is receiving renewed commercial and scientific interest, therefore a deeper knowledge of the chemical composition of the fruits of less studied varieties is required. In this work, juices from arils and decoctions from mesocarp plus exocarp were prepared from fifteen varieties. Samples were submitted to High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry, spectrophotometric and colorimetric CIEL*a*b* analyses. Antioxidant, antiradical and metal chelating properties, inhibitory activity against tyrosinase and α-amylase enzymes were also evaluated. All varieties presented the same main phenols; anthocyanins and ellagitannins were widely variable among varieties, with the richest anthocyanin content in the juices from the Wonderful and Soft Seed Maule varieties (approx. 660 mg/L) and the highest ellagitannin content in the peel of the Black variety (approx. 133 mg/g dry matter). A good correlation was shown between the colour hue and the delphinidin/cyanidin ratio in juices (R2 = 0.885). Total polysaccharide yield ranged from 3% to 12% of the peels' dry weight, with the highest content in the Black variety. Decoctions (24.44-118.50 mg KAE/g) showed better in vitro antioxidant properties and higher inhibitory capacity against tyrosinase than juices (not active-16.56 mg KAE/g); the inhibitory capacity against α-amylase was similar and quite potent for juices and decoctions. Knowledge about the chemical composition of different pomegranate varieties will allow for a more aware use of the different parts of the fruit.

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Novel sulfonamidoindole-based hydrazones with a 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide scaffold were synthesized and tested in enzyme inhibition assays against the tumor-associated carbonic anhydrase isoforms, hCA IX and XII, and the off-targets, hCA I and II. The compounds showed selectivity against hCA IX and XII over hCA I and II. Six compounds showed KI values lower than 10 nM against hCA IX or XII. Molecular modeling studies were performed to suggest binding interactions between the ligand and the hCA active sites.

Even though Salvia genus is one of the most known and studied taxa of Lamiaceae family, the knowledge regarding the chemical composition and health-related benefits of some locally used Salvia species (mostly endemic) is still scarce. In this regard, the present work aims to evaluate the chemical profile and potential bioactivities of 70% (v/v) ethanolic extracts obtained from the less-studied S. transsylvanica and S. glutinosa in comparison with S. officinalis. HPLC-PDA analysis revealed the presence of rutin and catechin as the main compounds in the extracts of the three studied species (using the employed HPLC method), whereas the presence of naringenin was highlighted only in S. glutinosa extract. Chlorogenic acid, rutin and quercetin were identified and quantified for the first time in S. transsylvanica extracts. The in vitro antioxidant capacity of each extract was tested through complementary methods (phosphomolybdenum assay, DPPH, ABTS, CUPRAC and FRAP assays), and correlated with the presence of phenolics (especially flavonoids) in high amounts. The neuroprotective and antidiabetic abilities of S. officinalis (the most active as AChE, BChE and α-glucosidase inhibitor), S. glutinosa (the most active as α-amylase inhibitor) and S. transsylvanica were also studied. For each extract it was determined the antimicrobial, antifungal and cytotoxic effects using in vitro assays. The obtained results confirm the potential of S. transsylvanica and S. glutinosa as promising sources of bioactive compounds and as a starting point for further analyses.

Industrial hemp (Cannabis sativa) is traditionally cultivated as a valuable source of fibers and nutrients. Multiple studies also demonstrated antimicrobial, anti-proliferative, phytotoxic and insecticide effects of the essential oil from hemp female inflorescences. On the other side, only a few studies explored the potential pharmacological application of polar extracts from inflorescences. In the present study, we investigated the water extract from inflorescences of industrial hemp Futura 75 variety, from phytochemical and pharmacological point of view. The water extract was assayed for phenolic compound content, radical scavenger/reducing, chelating and anti-tyrosinase effects. Through an ex vivo model of toxicity induced by lipopolysaccharide (LPS) on isolated rat colon and liver, we explored the extract effects on serotonin, dopamine and kynurenine pathways and the production of prostaglandin (PG)E2. Anti-proliferative effects were also evaluated against human colon cancer HCT116 cell line. Additionally, antimycotic effects were investigated against Trichophyton rubrum, Trichophyton interdigitale, Microsporum gypseum. Finally, in silico studies, including bioinformatics, network pharmacology and docking approaches were conducted in order to predict the putative targets underlying the observed pharmacological and microbiological effects. Futura 75 water extract was able to blunt LPS-induced reduction of serotonin and increase of dopamine and kynurenine turnover, in rat colon. Additionally, the reduction of PGE2 levels was observed in both colon and liver specimens, as well. The extract inhibited the HCT116 cell viability, the growth of T. rubrum and T. interdigitale and the activity of tyrosinase, in vitro, whereas in silico studies highlighting the inhibitions of cyclooxygenase-1 (induced by carvacrol), carbonic anhydrase IX (induced by chlorogenic acid and gallic acid) and lanosterol 14-α-demethylase (induced by rutin) further support the observed pharmacological and antimycotic effects. The present findings suggest female inflorescences from industrial hemp as high quality by-products, thus representing promising sources of nutraceuticals and cosmeceuticals against inflammatory and infectious diseases.

Surgical site infections (SSIs) represent the most common nosocomial infections, and surgical sutures are optimal surfaces for bacterial adhesion and biofilm formation. Staphylococcus spp., Enterococcus spp., and Escherichia coli are the most commonly isolated microorganisms. The aim of this research was to evaluate the antibiofilm activity of a medical device (MD) containing TIAB, which is a silver-nanotech patented product. The antibacterial effect was evaluated against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and E. coli ATCC 25922 by assessing the minimum inhibitory concentration (MIC) by the Alamar Blue® (AB) assay. The antibiofilm effect was determined by evaluation of the minimum biofilm inhibitory concentration (MBIC) and colony-forming unit (CFU) count. Subsequently, the MD was applied on sutures exposed to the bacterial species. The antimicrobial and antibiofilm effects were evaluated by the agar diffusion test method, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). The MIC was determined for S. aureus and E. faecalis at 2 mg/mL, while the MBIC was 1.5 mg/mL for S. aureus and 1 mg/mL for E. faecalis. The formation of an inhibition zone around three different treated sutures confirmed the antimicrobial activity, while the SEM and CLSM analysis performed on the MD-treated sutures underlined the presence of a few adhesive cells, which were for the most part dead. The MD showed antimicrobial and antibiofilm activities versus S. aureus and E. faecalis, but a lower efficacy against E. coli. Surgical sutures coated with the MD have the potential to reduce SSIs as well as the risk of biofilm formation post-surgery.

The enormous burden of the COVID-19 pandemic in economic and healthcare terms has cast a shadow on the serious threat of antimicrobial resistance, increasing the inappropriate use of antibiotics and shifting the focus of drug discovery programmes from antibacterial and antifungal fields. Thus, there is a pressing need for new antimicrobials involving innovative modes of action (MoAs) to avoid cross-resistance rise. Thiosemicarbazones (TSCs) stand out due to their easy preparation and polypharmacological application, also in infectious diseases. Recently, we reported a small library of TSCs (1-9) that emerged for their non-cytotoxic behaviour. Inspired by their multifaceted activity, we investigated the antibacterial, antifungal, and antidermatophytal profiles of derivatives 1-9, highlighting a new promising research line. Furthermore, the ability of these compounds to inhibit selected microbial and human carbonic anhydrases (CAs) was assessed, revealing their possible involvement in the MoA and a good selectivity index for some derivatives.

Industrial hemp is a multiuse crop that has been widely cultivated to produce fibers and nutrients. The capability of the essential oil (EO) from inflorescences as antimicrobial agent has been reported. However, literature data are still lacking about the hemp EO antiprotozoal efficacy in vivo. The present study aims to unravel this concern through the evaluation of the efficacy of hemp EOs (2.5 mL/kg, intraperitoneally) of three different cultivars, namely Futura 75, Carmagnola selezionata and Eletta campana, in mice intraperitoneally infected with Leishmania tropica. A detailed description of EO composition and targets-components analysis is reported. Myrcene, α-pinene and E-caryophyllene were the main components of the EOs, as indicated by the gas-chromatographic analysis. However, a prominent position in the scenario of the theoretical interactions underlying the bio-pharmacological activity was also occupied by selina-3,7(11)-diene, which displayed affinities in the micromolar range (5.4-28.9) towards proliferator-activated receptor α, cannabinoid CB2 receptor and acetylcholinesterase. The content of this compound was higher in Futura 75 and Eletta campana, in accordance with their higher scavenging/reducing properties and efficacy against the tissue wound, induced by L. tropica. Overall, the present study recommends hemp female inflorescences, as sources of biomolecules with potential pharmacological applications, especially towards infective diseases.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC50) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

Pomegranate peel is a natural source of phenolics, claimed to possess healing properties, among which are antioxidant and antidiabetic. In the present study, an ethyl acetate extract, obtained by Soxhlet from the peel of Dente di Cavallo DC2 pomegranate (PGE) and characterized to contain 4% w/w of ellagic acid, has been evaluated for its hypoglycemic, antiglycation, and antioxidative cytoprotective properties, in order to provide possible evidence for future nutraceutical applications. The α-amylase and α-glucosidase enzyme inhibition, interference with advanced glycation end-products (AGE) formation, and metal chelating abilities were studied. Moreover, the possible antioxidant cytoprotective properties of PGE under hyperglycemic conditions were assayed. Phenolic profile of the extract was characterized by integrated chromatographic and spectrophotometric methods. PGE resulted able to strongly inhibit the tested enzymes, especially α-glucosidase, and exerted chelating and antiglycation properties. Also, it counteracted the intracellular oxidative stress under hyperglycemic conditions, by reducing the levels of reactive oxygen species and total glutathione. Among the identified phenolics, rutin was the most abundant flavonoid (about 4 % w/w). Present results suggest PGE to be a possible remedy for hyperglycemia management and encourage further studies to exploit its promising properties.

A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives.

A library of sulfonate and sulfonamide derivatives of Resveratrol was synthesized and tested for its aromatase inhibitory potential. Interestingly, sulfonate derivatives were found to be more active than sulfonamide bioisosteres with IC50 values in the low micromolar range. The sulfonate analogues 1b-c and 1j exhibited good in vitro antiproliferative activity on the MCF7 cell line, evidenced by MTT and LDH release assays. Structure-activity relationships suggested that electronic and lipophilic properties could have a different role in promoting the biological response for sulfonates and sulfonamides, respectively. Docking studies disclosed the main interactions at a molecular level of detail behind the observed inhibition of the more active compounds whose chemical stability has been evaluated with nano-liquid chromatography. Finally, 1b-c and 1j were highlighted as sulfonates to be further developed as novel and original aromatase inhibitors.

In this study, sixty novel coumarin-monoterpene compounds were synthesized in two series [thirty-two compounds (12-43) bearing a triazole ring in the first series, and twenty-eight compounds (44-71) bearing an alkyl chain in the second one]. Their inhibitory effects on the human carbonic anhydrase (hCA) isoforms I, II, IX, and XII and anticancer potentials were determined. All synthesized molecules selectively inhibited CA IX and XII. 23 and 42 were found to be the strongest inhibitors, with K i values of 1.9 nM against hCA IX. Also, 70 showed the highest inhibitory activity with a K i value of 4.9 nM against hCA XII. Moreover, their cytotoxic effects on colon adenocarcinoma (HT-29), prostate adenocarcinoma (PC-3), and breast adenocarcinoma (MCF-7) cell lines were evaluated. According to the cytotoxicity results, 14 (IC50 = 2.48 μM) and 63 (IC50 = 3.91 μM) exhibited the highest cytotoxicity on the MCF-7 cells, while 23 showed the strongest cytotoxic effect on both PC-3 (IC50 = 9.40 μM) and HT-29 (IC50 = 12.10 μM) cell lines. 14, 23, and 66 decreased CA IX and CA XII protein expression in HT-29 cells, while 23 and 66 showed the strongest reduction of both CA IX and CA XII in MCF-7 cells. All of the selected compounds increased total apoptosis in a concentration-dependent manner in HT-29 and MCF-7 cells. 14 has the strongest apoptotic effect in MCF-7 cells. 23 increased early apoptosis primarily, while 14 and 66 increased total apoptosis in HT-29. In addition, PI/Hoechst staining proves that apoptotic cells are increased in HT-29 with an effect of 14, 23, and 66. As a result of the modeling studies, it has been shown that only the open coumarin form of the compounds can interact directly with the active-site Zn2+ ion. It has been shown that coumarin-monoterpene structures with different alkyl and monoterpene groups both specifically inhibit CA IX and XII and exhibit specific cytotoxicity in different cell lines.