New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from ß-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying ß-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from ß-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for ß-thalassemia.

Xylitol as a Hydrophilization Moiety for a Biocatalytically Synthesized Ibuprofen Prodrug.

Biocatalyzed synthesis can be exploited to produce high-value products, such as prodrugs. The replacement of chemical approaches with biocatalytic processes is advantageous in terms of environmental prevention, embracing the principles of green chemistry. In this work, we propose the covalent attachment of xylitol to ibuprofen to produce an IBU-xylitol ester prodrug. Xylitol was chosen as a hydrophilizer for the final prodrug, enhancing the water solubility of ibuprofen. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) extensively used as an analgesic, anti-inflammatory, and antipyretic. Despite being the third-most-prescribed medicine in the world, the aqueous solubility of ibuprofen is just 21 mg/L. This poor water solubility greatly limits the bioavailability of ibuprofen. We aimed to functionalize ibuprofen with xylitol using the reusable immobilized N435 biocatalyst. Instead of a biphasic media, we proposed a monophasic reaction environment. The characterization of the IBU-xylitol ester was performed by 1H, 13C-NMR, DEPT, COSY, HMQC, HMBC, FTIR, and MS spectroscopy. Preliminary in vitro tests showed that this enzymatically synthesized prodrug of ibuprofen reduced the expression of the interleukin 8 genes in human bronchial epithelial cells (IB3-1) from cystic fibrosis (CF) patients.

Improved Trimethylangelicin Analogs for Cystic Fibrosis: Design, Synthesis and Preliminary Screening.

A small library of new angelicin derivatives was designed and synthesized with the aim of bypassing the side effects of trimethylangelicin (TMA), a promising agent for the treatment of cystic fibrosis. To prevent photoreactions with DNA, hindered substituents were inserted at the 4 and/or 6 positions. Unlike the parent TMA, none of the new derivatives exhibited significant cytotoxicity or mutagenic effects. Among the synthesized compounds, the 4-phenylderivative 12 and the 6-phenylderivative 25 exerted a promising F508del CFTR rescue ability. On these compounds, preliminary in vivo pharmacokinetic (PK) studies were carried out, evidencing a favorable PK profile per se or after incorporation into lipid formulations. Therefore, the selected compounds are good candidates for future extensive investigation to evaluate and develop novel CFTR correctors based on the angelicin structure.

New TMA (4,6,4'-Trimethyl angelicin) Analogues as Anti-Inflammatory Agents in the Treatment of Cystic Fibrosis Lung Disease.

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.

Design, Synthesis and Evaluation of New Multifunctional Benzothiazoles as Photoprotective, Antioxidant and Antiproliferative Agents.

A current trend of research in the health field is toward the discovery of multifunctional compounds, capable of interacting with multiple biological targets, thus simplifying multidrug therapies and improving patient compliance. The aim of this work was to synthesize new multifunctional chemical entities bearing a benzothiazole nucleus, a structure that has attracted increasing interest for the great variety of biological actions that it can perform, and already used as a scaffold in several multifunctional drugs. Compounds are reported, divided into two distinct series, synthetized and tested in vitro for the antioxidant, and include UV-filtering and antitumor activities. DPPH and FRAP tests were chosen to outline an antioxidant activity profile against different radical species. The UV-filtering activity was investigated, pre- and post-irradiation, through evaluation of a O/W sunscreen standard formulation containing 3% of the synthetic compounds. The antitumor activity was investigated both on human melanoma cells (Colo-38) and on immortalized human keratinocytes as a control (HaCat). A good antiproliferative profile in terms of IC50 was chosen as a mandatory condition to further investigate apoptosis induction as a possible cytotoxicity mechanism through the Annexin V test. Compound BZTcin4 was endowed with excellent activity and a selectivity profile towards Colo-38, supported by a good antioxidant capacity and an excellent broad-spectrum photoprotective profile.

Bi-allelic expression of the RyR1 p.A4329D mutation decreases muscle strength in slow-twitch muscles in mice.

Mutations in the ryanodine receptor 1 (RYR1) gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive RYR1 myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic versus mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.

Synthesis and Biological Activity Assessment of 2-Styrylbenzothiazoles as Potential Multifunctional Therapeutic Agents.

A current trend in healthcare research is to discover multifunctional compounds, able to interact with multiple biological targets, in order to simplify multi-drug therapies and improve patient compliance. The aim of this work was to outline the growing demand for innovative multifunctional compounds, achieved through the synthesis, characterisation and SAR evaluation of a series of 2-styrylbenzothiazole derivatives. The six synthesised compounds were studied for their potential as photoprotective, antioxidant, antiproliferative, and anti-inflammatory agents. In order to profile antioxidant activity against various radical species, in vitro DPPH, FRAP and ORAC assays were performed. UV-filtering activity was studied, first in solution and then in formulation (standard O/W sunscreen containing 3% synthesised molecules) before and after irradiation. Compound BZTst6 proved to be photostable, suitable for broad-spectrum criteria, and is an excellent UVA filter. In terms of antioxidant activity, only compound BZTst4 can be considered a promising candidate, due to the potential of the catechol moiety. Both also showed exceptional inhibitory action against the pro-inflammatory enzyme 5-lipoxygenase (LO), with IC50 values in the sub-micromolar range in both activated neutrophils and under cell-free conditions. The results showed that the compounds under investigation are suitable for multifunctional application purposes, underlining the importance of their chemical scaffolding in terms of different biological behaviours.

Multifunctional Profiling of Moringa oleifera Leaf Extracts for Topical Application: A Comparative Study of Different Collection Time.

This research investigated plant extracts as a source of potential new actives in the nutritional, cosmetic, and pharmaceutical fields. Moringa oleifera, which is extensively known for its nutritional properties, has been investigated in this work by preparation, characterization, and evaluation of the antioxidant (FRAP, DPPH, ORAC, and PCL test), antifungal, photoprotective, and cytotoxicity profile against human melanoma Colo38 cell line of two different extracts (hydroalcoholic and methanolic) and one infusion of dry leaves collected from Paraguay in four distinct harvest times (February, March, April, and May 2017). The outcomes of this study highlight Moringa oleifera as a potential ally to counteract skin aging and oxidative stress, as indicated by the favorable antioxidant profile of the extracts and infusions of Paraguay, which was, in all cases, superior to that provided by the same plant species when collected from Senegal. Moreover, some samples were more efficient in preventing the photodegradation of UVA filter butyl methoxydibenzoylmethane (Avobenzone) compared to commercial filters, thus suggesting an interesting future role as natural additives in sunscreens.

Combined Treatment of Cancer Cells Using Allyl Palladium Complexes Bearing Purine-Based NHC Ligands and Molecules Targeting MicroRNAs miR-221-3p and miR-222-3p: Synergistic Effects on Apoptosis.

Combined treatments employing lower concentrations of different drugs are used and studied to develop new and more effective anticancer therapeutic approaches. The combination therapy could be of great interest in the controlling of cancer. Regarding this, our research group has recently shown that peptide nucleic acids (PNAs) that target miR-221 are very effective and functional in inducing apoptosis of many tumor cells, including glioblastoma and colon cancer cells. Moreover, in a recent paper, we described a series of new palladium allyl complexes showing a strong antiproliferative activity on different tumor cell lines. The present study was aimed to analyze and validate the biological effects of the most active compounds tested, in combination with antagomiRNA molecules targeting two miRNAs, miR-221-3p and miR-222-3p. The obtained results show that a "combination therapy", produced by combining the antagomiRNAs targeting miR-221-3p, miR-222-3p and the palladium allyl complex 4d, is very effective in inducing apoptosis, supporting the concept that the combination treatment of cancer cells with antagomiRNAs targeting a specific upregulated oncomiRNAs (in this study miR-221-3p and miR-222-3p) and metal-based compounds represents a promising therapeutic strategy to increase the efficacy of the antitumor protocol, reducing side effects at the same time.

Pro-apoptotic activity of novel synthetic isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in vitro.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.