Exploiting the bile acid binding protein as transporter of a Cholic Acid/Mirin bioconjugate for potential applications in liver cancer therapy.

Bioconjugation is one of the most promising strategies to improve drug delivery, especially in cancer therapy. Biomolecules such as bile acids (BAs) have been intensively explored as carriers, due to their peculiar physicochemical properties and biocompatibility. BAs trafficking is regulated by intracellular lipid-binding proteins and their transport in the liver can be studied using chicken liver Bile Acid-Binding Proteins (cL-BABPs) as a reference model. Therefore, we conceived the idea of developing a BA-conjugate with Mirin, an exonuclease inhibitor of Mre11 endowed with different anticancer activities, to direct its transport to the liver. Following computational analysis of various BAs in complex with cL-BABP, we identified cholic acid (CA) as the most promising candidate as carrier, leading to the synthesis of a novel bioconjugate named CA-M11. As predicted by computational data and confirmed by X-ray crystallographic studies, CA-M11 was able to accommodate into the binding pocket of BABP. Hence, it can enter BAs trafficking in the hepatic compartment and here release Mirin. The effect of CA-M11, evaluated in combination with varying concentrations of Doxorubicin on HepG2 cell line, demonstrated a significant increase in cell mortality compared to the use of the cytotoxic drug or Mirin alone, thus highlighting chemo-sensitizing properties. The promising results regarding plasma stability for CA-M11 validate its potential as a valuable agent or adjuvant for hepatic cancer therapy.

Infesting Seaweeds as a Novel Functional Food: Analysis of Nutrients, Antioxidants and ACE Inhibitory Effects.

Globalization and climate change are both contributing to an increase in the number of potentially invasive algae in coastal areas. In terms of biodiversity and financial losses, the invasiveness of algae has become a significant issue in Orbetello Lagoon. Indeed, studies from the Tuscany Regional Agency for Environmental Protection show that the reduction in dissolved oxygen caused by algal diffusion is detrimental to fisheries and biodiversity. Considering that wakame and numerous other potentially invasive seaweeds are consumed as food in Asia, we assess the nutritional and nutraceutical qualities of two potentially invasive seaweeds: Valonia aegagrophila and Chaetomorpha linum. We found that both algae are a valuable source of proteins and essential amino acids. Even if the fat content accounts for less than 2% of the dried weight, its quality is high, due to the presence of unsaturated fatty acids. Both algae are rich in antioxidants pigments and polyphenols, which can be exploited as nutraceuticals. Most importantly, human gastrointestinal digestion increased the quantity of polyphenols and originated secondary metabolites with ACE inhibitory activity. Taken together, our data strongly promote the use of Valonia aegagrophila and Chaetomorpha linum as functional foods, with possible application in the treatment of hypertension and cardiovascular diseases.

Serendipitous Identification of Azine Anticancer Agents Using a Privileged Scaffold Morphing Strategy.

The use of privileged scaffolds as a starting point for the construction of libraries of bioactive compounds is a widely used strategy in drug discovery and development. Scaffold decoration, morphing and hopping are additional techniques that enable the modification of the chosen privileged framework and better explore the chemical space around it. In this study, two series of highly functionalized pyrimidine and pyridine derivatives were synthesized using a scaffold morphing approach consisting of triazine compounds obtained previously as antiviral agents. Newly synthesized azines were evaluated against lymphoma, hepatocarcinoma, and colon epithelial carcinoma cells, showing in five cases acceptable to good anticancer activity associated with low cytotoxicity on healthy fibroblasts. Finally, ADME in vitro studies were conducted on the best derivatives of the two series showing good passive permeability and resistance to metabolic degradation.

A novel bioresponsive self-immolative spacer based on aza-quinone methide reactivity for the controlled release of thiols, phenols, amines, sulfonamides or amides.

A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.

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.

Sweet Cherry Extract as Permeation Enhancer of Tyrosine Kinase Inhibitors: A Promising Prospective for Future Oral Anticancer Therapies.

Although patients would rather oral therapies to injections, the gastrointestinal tract's low permeability makes this method limiting for most compounds, including anticancer drugs. Due to their low bioavailability, oral antitumor therapies suffer from significant variability in pharmacokinetics and efficacy. The improvement of their pharmacokinetic profiles can be achieved by a new approach: the use of natural extracts enriched with polyphenolic compounds that act as intestinal permeability enhancers. Here, we propose a safe sweet cherry extract capable of enhancing oral absorption. The extract was characterized by the HPLC-UV/MS method, evaluated for in vitro antioxidant activity, safety on the Caco-2 cell line, and as a potential permeation enhancer. The sweet cherry extract showed a high antioxidant capacity (ABTS and DPPH assays were 211.74 and 48.65 µmol of Trolox equivalent/g dried extract, respectively), high content of polyphenols (8.44 mg of gallic acid per gram of dry extract), and anthocyanins (1.80 mg of cyanidin-3-glucoside equivalent per g of dry extract), reassuring safety profile (cell viability never lower than 98%), and a significant and fully reversible ability to alter the integrity of the Caco-2 monolayer (+81.5% of Lucifer yellow permeability after 2 h). Furthermore, the ability of the sweet cherry extract to improve the permeability (Papp) and modify the efflux ratio (ER) of reference compounds (atenolol, propranolol, and dasatinib) and selected pyrazolo[3,4-d]pyrimidine derivatives was investigated. The obtained results show a significant increase in apparent permeability across the Caco-2 monolayer (tripled and quadrupled in most cases), and an interesting decrease in efflux ratio when compounds were co-incubated with sweet cherry extract.

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.

Progress towards Adjuvant Development: Focus on Antiviral Therapy.

In recent decades, vaccines have been extraordinary resources to prevent pathogen diffusion and cancer. Even if they can be formed by a single antigen, the addition of one or more adjuvants represents the key to enhance the response of the immune signal to the antigen, thus accelerating and increasing the duration and the potency of the protective effect. Their use is of particular importance for vulnerable populations, such as the elderly or immunocompromised people. Despite their importance, only in the last forty years has the search for novel adjuvants increased, with the discovery of novel classes of immune potentiators and immunomodulators. Due to the complexity of the cascades involved in immune signal activation, their mechanism of action remains poorly understood, even if significant discovery has been recently made thanks to recombinant technology and metabolomics. This review focuses on the classes of adjuvants under research, recent mechanism of action studies, as well as nanodelivery systems and novel classes of adjuvants that can be chemically manipulated to create novel small molecule adjuvants.

Biological Evaluation and In Vitro Characterization of ADME Profile of In-House Pyrazolo[3,4-d]pyrimidines as Dual Tyrosine Kinase Inhibitors Active against Glioblastoma Multiforme.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

The Pyrazolo[3,4-d]Pyrimidine Derivative Si306 Encapsulated into Anti-GD2-Immunoliposomes as Therapeutic Treatment of Neuroblastoma.

Si306, a pyrazolo[3,4-d]pyrimidine derivative recently identified as promising anticancer agent, has shown favorable in vitro and in vivo activity profile against neuroblastoma (NB) models by acting as a competitive inhibitor of c-Src tyrosine kinase. Nevertheless, Si306 antitumor activity is associated with sub-optimal aqueous solubility, which might hinder its further development. Drug delivery systems were here developed with the aim to overcome this limitation, obtaining suitable formulations for more efficacious in vivo use. Si306 was encapsulated in pegylated stealth liposomes, undecorated or decorated with a monoclonal antibody able to specifically recognize and bind to the disialoganglioside GD2 expressed by NB cells (LP[Si306] and GD2-LP[Si306], respectively). Both liposomes possessed excellent morphological and physio-chemical properties, maintained over a period of two weeks. Compared to LP[Si306], GD2-LP[Si306] showed in vitro specific cellular targeting and increased cytotoxic activity against NB cell lines. After intravenous injection in healthy mice, pharmacokinetic profiles showed increased plasma exposure of Si306 when delivered by both liposomal formulations, compared to that obtained when Si306 was administered as free form. In vivo tumor homing and cytotoxic effectiveness of both liposomal formulations were finally tested in an orthotopic animal model of NB. Si306 tumor uptake resulted significantly higher when encapsulated in GD2-LP, compared to Si306, either free or encapsulated into untargeted LP. This, in turn, led to a significant increase in survival of mice treated with GD2-LP[Si306]. These results demonstrate a promising antitumor efficacy of Si306 encapsulated into GD2-targeted liposomes, supporting further therapeutic developments in pre-clinical trials and in the clinic for NB.

Synthesis and Antiproliferative Activity of Nitric Oxide-Donor Largazole Prodrugs.

The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery, many research groups have been attracted by the structural complexity and the peculiar anticancer activity, due to its capability to discriminate between tumor cells and normal cells. Herein, we discuss the synthesis and the in vitro biological profile of hybrid analogues of Largazole, as dual HDAC inhibitor and nitric oxide (NO) donors, potentially useful as anticancer agents. In particular, the metabolic stability of the modified thioester moiety of Largazole, bearing the NO-donor function/s, the in vitro release of NO, and the antiproliferative activity in tumor cell lines are presented.

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.