Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation.

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized ß-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of ß-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.

Oxy-imino saccharidic derivatives as a new structural class of aldose reductase inhibitors endowed with anti-oxidant activity.

Aldose reductase is a key enzyme in the development of long term diabetic complications and its inhibition represents a viable therapeutic solution for people affected by these pathologies. Therefore, the search for effective aldose reductase inhibitors is a timely and pressing challenge. Herein we describe the access to a novel class of oxyimino derivatives, obtained by reaction of a 1,5-dicarbonyl substrate with O-(arylmethyl)hydroxylamines. The synthesised compounds proved to be active against the target enzyme. The best performing inhibitor, compound (Z)-8, proved also to reduce both cell death and the apoptotic process when tested in an in vitro model of diabetic retinopathy made of photoreceptor-like 661w cell line exposed to high-glucose medium, counteracting oxidative stress triggered by hyperglycaemic conditions.

Synthesis and investigation of polyhydroxylated pyrrolidine derivatives as novel chemotypes showing dual activity as glucosidase and aldose reductase inhibitors.

Diabetes is a multi-factorial disorder that should be treated with multi-effective compounds. Here we describe the access to polyhydroxylated pyrrolidines, belonging to the d-gluco and d-galacto series, through aminocyclization reactions of two differentially protected d-xylo-hexos-4-ulose derivatives. The prepared compounds proved to inhibit both alpha-glucosidase, responsible for the emergence of hyperglycemic spikes, and aldose reductase, accountable for the development of abnormalities in diabetic tissues. Accordingly, they show the dual inhibitory profile deemed as ideal for diabetes treatment. Significantly, compound 17b reduced the process of cell death and restored the physiological levels of oxidative stress when tested in the photoreceptor-like 661w cell line, thus proving to be effective in an in vitro model of diabetic retinopathy.

N-(Aroyl)-N-(arylmethyloxy)-α-alanines: Selective inhibitors of aldose reductase.

Aldose reductase (ALR2), a NADPH-dependent reductase, is the first and rate-limiting enzyme of the polyol pathway of glucose metabolism and is implicated in the pathogenesis of secondary diabetic complications. In the last decades, this enzyme has been targeted for inhibition but despite the numerous efforts made to identify potent and safe ALR2 inhibitors, many clinical candidates have been a failure. For this reason the research of new ALR2 inhibitors highly effective, selective and with suitable pharmacokinetic properties is still of great interest. In this paper some new N-(aroyl)-N-(arylmethyloxy)alanines have been synthesized and tested for their ability to inhibit ALR2. Some of the synthesized compounds exhibit IC50 in the low micromolar range and all have proved to be highly selective towards ALR2. The N-(aroyl)-N-(arylmethyloxy)-α-alanines are a promising starting point for the development of new ALR2 selective drugs with the aim of delaying the onset of diabetic complications.

Synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl and sulfamoyl acetamides and ethyl acetates as potent COX-2 inhibitors.

We report herein the synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl, sulfamoyl acetamides and ethyl acetates that selectively inhibit cyclooxygenase-2 (COX-2) isoform. Among the newly synthesized compounds, some of them were endowed with a good activity against COX-2 and a good selectivity COX-2/COX-1 in vitro as well as a desirable analgesic activity in vivo, proving that replacement of the ester moiety with an amide group gave access to more stable derivatives, characterized by a good COX-inhibition.

Sampling protein motion and solvent effect during ligand binding.

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one.

PMMA/polysaccharides nanofilm loaded with adenosine deaminase inhibitor for targeted anti-inflammatory drug delivery.

A novel drug delivery vector, a free-standing polymeric ultrathin film (nanofilm) composed of PMMA and a polysaccharides multilayer, is presented. Chitosan and sodium alginate are alternatively deposited by spin-assisted LbL assembly onto a plasma-treated PMMA thin film. Hydrophobic anti-inflammatory drugs, an adenosine deaminase inhibitor (APP) and its fluorescent dansyl derivate (APP-Dns), are encapsulated inside the LbL multilayer using a simple casting deposition procedure. The resulting drug loaded nanofilm can be suspended in water upon dissolution of a PVA sacrificial layer. Morphological characterization of the nanofilm shows that PMMA/LbL nanofilms possess nanometric thickness (<200 nm) and very low surface roughness (1-2 nm for drug loaded nanofilms and <1 nm for blank nanofilm). Drug loaded films exhibit a diffusion controlled release mechanism following the Korsmayer-Peppas release model, confirmed by the fit of release data with a characteristic power law. Drug release is impaired through the PMMA layer, which acts effectively as a barrier for drug transport. This ultrathin polymer film can find application as a nanopatch for targeted inflammatory drug delivery to treat localized pathologies as inflammatory bowel disease.

Synthesis and biological evaluation of 2'-oxo-2,3-dihydro-3'H- spiro[chromene-4,5'-[1,3]oxazolidin]-3'yl]acetic acid derivatives as aldose reductase inhibitors.

Aldose reductase (ARL2) is the first enzyme in the polyol pathway which catalyzes the NADPH-dependent reduction of glucose to sorbitol. Its involvement on diabetic complications makes this enzyme a challenge therapeutic target widely investigated to limit and/or prevent them. On this basis, a limited series of 4-spiro-oxazolidinone-benzopyran derivatives (1-7) were synthesized to evaluate them as potential ARL2 inhibitors. The activity was determined spectrophotometrically by monitoring the oxidation of NADPH catalyzed by ALR2. Within the series of compounds, the 4-methoxy derivative 1b showed to be the most active compound, exhibiting inhibitory levels in the submicromolar range. In addition, the activity against the aldehyde reductase isoform (ARL1) was also evaluated. Unlike sorbinil (reference drug) that lack of selectivity towards the two enzyme all the tested compounds resulted to be devoid of ARL1 inhibitory activity (IC(50) > 10 µM), thus proving to be selective.

Synthetic mycomelanin thin films as emergent bio-inspired interfaces controlling the fate of embryonic stem cells.

The fungal pathways of melanin synthesis have so far been little considered as a source of bio-inspiration in the field of functional materials, despite the interesting properties exhibited by Ascomycetes melanins from 1,8-dihydroxynaphthalene (1,8-DHN), including the ability to shield organisms from ionizing radiation. Herein, the processing techniques and characterizations of mycomelanin thin films obtained from the solid state polymerization of 1,8-DHN is reported for the first time. Overall, the results highlighted the role of synthetic mycomelanin thin films as a prototype of next generation bioinspired interfaces featuring high structural regularity and ultrasmooth morphology, high robustness against peroxidative bleaching and adhesion under water conditions, good biocompatibility and unprecedented effects in inducing the spontaneous differentiation of embryonic stem cells prevalently towards the endodermal lineages in the absence of added factors. These data open up new avenues towards the applications of this biomaterial in the fields of tissue engineering and regenerative medicine.

Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

Combined Use of Scanning Electron Microscopy-Energy-Dispersive X-ray Spectroscopy and Fourier Transform Infrared Imaging Coupled with Principal Component Analysis in the Study of Ancient Egyptian Papyri.

This paper reports the results of a spectroscopic study on the chemistry of some small papyrus fragments arising from three Egyptian excavation sites. The aim of this investigation was to verify the possibility to identify significant differences in ancient Egyptian papyri using noninvasive analytical methods, that is, ATR (attenuated total reflection)-FTIR (Fourier transform infrared) spectroscopic imaging and scanning electron microscopy-energy-dispersive X-ray spectroscopy. Differences in both lignin and cellulose compositions, which are difficult to detect with traditional FTIR and FTIR imaging spectral analysis, were revealed by the multivariate approach, and the second derivative spectroscopy was applied to enhance the spectrum resolution. Thus, it has been possible to recognize a fragment characterized by the presence of lead and of long chain aliphatic organic compound, which are not present in the other fragments, in the ink region. These data show not only that the combination of these techniques can provide important chemical information, such as to evidence the different compositions and manufacture of each papyrus, but also that metal inks were probably used also in ancient Egyptian papyri.

pH-Responsive Carboxymethylcellulose Nanoparticles for 68Ga-WBC Labeling in PET Imaging.

Carboxymethylcellulose (CMC) is a well-known pharmaceutical polymer, recently gaining attention in the field of nanomedicine, especially as a polyelectrolyte agent for the formation of complexes with oppositely charged macromolecules. Here, we report on the application of pH-sensitive pharmaceutical grade CMC-based nanoparticles (NP) for white blood cells (WBC) PET imaging. In this context and as an alternative to 99mTc-HMPAO SPECT labeling, the use of 68Ga3+ as PET radionuclide was investigated since, at early time points, it could provide the greater spatial resolution and patient convenience of PET tomography over SPECT clinical practices. Two operator-friendly kit-type formulations were compared, with the intention of radiolabeling within a short time (10 min), under mild conditions (physiological pH, room temperature) and in agreement with the actual clinically applied guidelines. NP were labeled by directly using 68Ga3+ eluted in HCL 0.05 N, from hospital suited 68Ge/68Ga generator and in absence of chelator. The first kit type approach involved the application of 68Ga3+ as an ionotropic gelation agent for in-situ forming NP. The second kit type approach concerned the re-hydration of a proper freeze-dried injectable NP powder. pH-sensitive NP with 250 nm average diameter and 80% labeling efficacy were obtained. The NP dispersant medium, including a cryoprotective agent, was modulated in order to optimize the Zeta potential value (-18 mV), minimize the NP interaction with serum proteins and guarantee a physiological environment for WBC during NP incubation. Time-dependent WBC radiolabeling was correlated to NP uptake by using both confocal and FT-IR microscopies. The ready to use lyophilized NP formulation approach appears promising as a straightforward 68Ga-WBC labeling tool for PET imaging applications.

Dual Kit/Aur Inhibitors as Chemosensitizing Agents for the Treatment of Melanoma: Design, Synthesis, Docking Studies and Functional Investigation.

Melanoma is the most serious form of skin cancer but its medication is still far from being safe and thoroughly effective. The search of novel therapeutic approaches represents therefore a health emergency to push through eagerly. In this study, we describe a novel class of dual c-Kit/Aur inhibitors, characterized by a 1,2,4-triazole core and developed by a structure-based optimization of a previously developed hit, and report the evidence of their significance as drug candidates for the treatment of melanoma. Compound 6a, merging the best inhibitory profile against the target kinases, showed anti-proliferative efficacy against the human melanoma cell lines A2058, expressing the BRAF V600D mutation, and WM266-4, expressing BRAF V600E. Significantly, it displayed also a highly synergistic profile when tested in combination with vemurafenib, thus proving its efficacy not only per se but even in a combination therapy, which is nowadays acknowledged as the cornerstone approach of the forthcoming tumour management.

Evidence for a novel binding site conformer of aldose reductase in ligand-bound state.

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of how the slight change of a chemically extended side-chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships.

Acetic acid aldose reductase inhibitors bearing a five-membered heterocyclic core with potent topical activity in a visual impairment rat model.

A number of 1,2,4-oxadiazol-5-yl-acetic acids and oxazol-4-yl-acetic acids were synthesized and tested for their ability to inhibit aldose reductase (ALR2). The oxadiazole derivatives, 7c, 7f, 7i, and 8h, 8i, proved to be the most active compounds, exhibiting inhibitory levels in the submicromolar range. In this series, the phenyl group turned out to be the preferred substitution pattern, as its lengthening to a benzyl moiety determined a general reduction of the inhibitory potency. The lead compound, 2-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]acetic acid, 7c, showed an excellent in vivo activity, proving to prevent cataract development in severely galactosemic rats when administered as an eye-drop solution in the precorneal region of the animals. Computational studies on the ALR2 inhibitors were performed to rationalize the structure-activity relationships observed and to provide the basis for further structure-guided design of novel ALR2 inhibitors.

Chiral ionic liquids supported on natural sporopollenin microcapsules.

Supported chiral ionic liquids (SILs) were prepared choosing the starting material for the ionic liquid part from the enantiopure stock of the chiral pool (monoterpenoids and an amino acid) and the sporopollenin as an environmentally friendly support. Sporopollenins are microcapsules with naturally well-defined sizes and shapes that can be obtained from pollen grains after removal of the internal cytoplasm and the second shell layer. As thermally stable organic biocompatible structures, sporopollenins have attracted increasing interest in recent years for several applications. Herein, bio-based ILs were anchored onto the surface of sporopollenins obtained from the pollen of Populus deltoides, selected as a model pollen grain. These new structures, which present an external positively charged shell, were characterized by physico-chemical techniques (ATR-FTIR, TGA, SEM, EDX, and solid-state 13C NMR). A metathesis reaction was also performed on selected bio-based IL modified sporopollenins, demonstrating the possibility to switch the surface properties by exploiting well-known IL chemistry.

Acid Derivatives of Pyrazolo[1,5-a]pyrimidine as Aldose Reductase Differential Inhibitors.

Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually AKR1B1 is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part in the antioxidant defense mechanism of the body. In this work we report the evidence that a class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and an ionizable fragment, modulates differently the catalytic activity of the enzyme, depending on the presence of specific substrates such as sugar, toxic aldehydes, and glutathione conjugates of toxic aldehydes. The study stands out as a systematic attempt to generate aldose reductase differential inhibitors (ARDIs) intended to target long-term diabetic complications while leaving unaltered the detoxifying role of the enzyme.

Challenging clinically unresponsive medullary thyroid cancer: Discovery and pharmacological activity of novel RET inhibitors.

It is now known that "gain of function" mutations of RET (REarranged during Transfection) kinase are specific and key oncogenic events in the onset of thyroid gland cancers such as the Medullary Thyroid Carcinoma (MTC). Although a number of RET inhibitors exist and are capable of inhibiting RET variants, in which mutations are outside the enzyme active site, the majority becomes dramatically ineffective when mutations are within the protein active site (V804L and V804M). Pursuing a receptor-based virtual screening against the kinase domain of RET, we found that compound 5 is able to inhibit efficiently both wild type and V804L mutant RET. Compound 5 was able to significantly reduce proliferation of both commercially available TT cell lines and surgical thyroid tissues obtained from patients with MTC and displayed a suitable drug-like profile, thus standing out as a promising candidate for further development towards the treatment of clinically unresponsive MTC.

Pyrido[1,2-a]pyrimidin-4-one derivatives as a novel class of selective aldose reductase inhibitors exhibiting antioxidant activity.

2-Phenyl-pyrido[1,2-a]pyrimidin-4-one derivatives bearing a phenol or a catechol moiety in position 2 were tested as aldose reductase (ALR2) inhibitors and exhibited activity levels in the micromolar/submicromolar range. Introduction of a hydroxy group in position 6 or 9 gave an enhancement of the inhibitory potency (compare 18, 19, 28, and 29 vs 13 and 14). Lengthening of the 2-side chain to benzyl determined a general reduction in activity. The lack or the methylation of the phenol or catechol hydroxyls gave inactive (10-12, 21, 22, 25-27) or scarcely active (15, 17, 20) compounds, thus demonstrating that the phenol or catechol hydroxyls are involved in the enzyme pharmacophoric recognition. Moreover, all the pyridopyrimidinones displayed significant antioxidant properties, with the best activity shown by the catechol derivatives. The theoretical binding mode of the most active compounds obtained by docking simulations into the ALR2 crystal structure was fully consistent with the structure-activity relationships in the pyrido[1,2-a]pyrimidin-4-one series.

Cyclodextrin-based nanosponges for the targeted delivery of the anti-restenotic agent DB103: A novel opportunity for the local therapy of vessels wall subjected to percutaneous intervention.

Nano-sized colloidal carriers represent innovative drug delivery systems, as they allow a targeted and prolonged release of poorly water-soluble drugs, improving their bioavailability and modifying their pharmacokinetic parameters. In this work we describe cyclodextrin-based nanosponges, obtained through polimerization of ß-cyclodextrin with diphenyl carbonate as the cross-linking agent, loaded with a novel multi-effective heterocyclic compound, DB103, able to regulate key cellular events involved in the remodelling of vessels wall. Fabrication and drug-loading procedures, as well as physical-chemical characterization and drug-release profile of the novel colloidal system are reported. Results achieved demonstrate the ability of nanosponges to enclose efficiently the target drug and release it slowly and continuously, thus suggesting the exploitability of the novel system for the local therapy of vessels wall subjected to percutaneous intervention.