Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.

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Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is currently under investigation as a new approach mainly for the treatment of pain. In this study, a series of N-substituted tryptamines was prepared to explore the structural requirements determining TRPM8 modulation. As a result of a fluorescence-based screening assay, we identified two compounds acting as an activator (2-(1H-indol-3-yl)-N-(4-phenoxybenzyl) ethanamine, compound 21) or an inhibitor (N,N-dibenzyl-2-(1H-indol-3-yl) ethanamine, compound 12) of calcium influx in HEK293 cells. In patch-clamp recordings, compound 21 displayed a significantly higher potency (EC 50 = 40±4 µM) and a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentrationdependent inhibition of menthol-induced TRPM8 currents (IC 50 = 367±24 nM). Molecular modelling studies using a homology model of a single TRPM8 subunit identified a putative binding site located between the VSD and the TRP box, disclosing two different binding modes for the agonist and the antagonist.

Here, we report the design of new analogues of spirooxoindolepyrrolidine nucleus as modulators of p53 activity. Compounds (3R,7aR)-6-(4-chlorobenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (9c) and (3R,7aR)-5'-methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (10d) are the most potent compounds of this series, inhibiting cell growth of different human tumor cells at submicromolar and micromolar concentrations, respectively. Compound 9c induces apoptotic cell death in human melanoma cell line M14 at 24 h, while in the same condition, treatment with 10d showes a clear arrest at G2/M phase inducing delay of cell cycle progression. Possibly, these activities may be due to inhibition of p53-MDM2 interaction and subsequent p53 release and activation.

Analogues of the previously described spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione p53 modulators were prepared to explore new structural requirements at the thiazolidine domain for the antiproliferative activity and p53 modulation. In cell, antiproliferative activity was evaluated against two human tumor cell lines. Derivative 5-bromo-3'-(cyclohexane carbonyl)-1-methyl-2-oxospiro[indoline-3,2'-thiazolidine] (4n) emerged as the most potent compound of this series, inhibiting in vitro 30% of p53-MDM2 interaction at 5 μM and the cell growth of different human tumor cells at nanomolar concentrations. Docking studies confirmed the interactions of 4n with the well-known Trp23 and Phe19 clefts, explaining the reasons for its binding affinity for MDM2. 4n at 50 nM is capable of inducing the accumulation of p53 protein, inducing significant apoptotic cell death without affecting the cell cycle progression. Comparative studies using nutlin in the same cellular system confirm the potential of 4n as a tool for increasing understanding of the process involved in the nontranscriptional proapoptotic activities of p53.

Kv7 K+ channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the EC50s for Kv7.2 current enhancements by compound 23a (0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for 24a (0.63 ± 0.07 μM). In addition, compared to retigabine, 23a and 24a showed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of 23a and 24a in comparison with retigabine. Overall, the present results highlight that the N5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.

A series of 3-substituted spiro[(dihydropyrazine-2,5-dione)-6,3'-(2',3'-dihydrothieno[2,3-b]naphtho-4',9'-dione)] derivatives were prepared using an easy synthetic route via condensation of the 3-amino-3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]naphtho-4,9-dione system and amino acids followed by intramolecular lactamization. Amino acids containing alkyl and aryl, linear and cyclic, polar and apolar, and basic and acid residues were incorporated. Evaluation of these analogues against the MCF-7 human breast carcinoma and SW 620 human colon carcinoma cell lines revealed, for the 3S,3'R isomers derived from Pro (7a), Cys (11a), and Met (12a) and the 3R,3'S isomer derived from D-Pro (7c), a cytotoxic potency comparable to or greater than that of doxorubicin. Some of these selected analogues were potent cytotoxic agents in several other sensible and resistant human solid tumor cell lines and may be able to circumvent the multiple-drug-resistance mechanism. In particular, only a partial cross-resistance to the compounds 7, 11, and 12 was observed in selected tumor cell sublines known to be resistant to doxorubicin (MCF-7/Dx and A2780/Dx), whereas a very low level of cross-resistance to compounds 7 and 11 was found in a tumor cell subline selected for resistance to cisplatin (A2780/DDP). In addition, the topoisomerase II inhibition activity and DNA-binding properties were investigated.

Arthrospira platensis, better known as Spirulina, is one of the most important microalgae species. This cyanobacterium possesses a rich metabolite pattern, including high amounts of natural pigments. In this study, we applied a combined strategy based on Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and Ultra High-Performance Liquid Chromatography (UHPLC) for the qualitative/quantitative characterization of Spirulina pigments in three different commercial dietary supplements. FT-ICR was employed to elucidate the qualitative profile of Spirulina pigments, in both direct infusion mode (DIMS) and coupled to UHPLC. DIMS showed to be a very fast (4 min) and accurate (mass accuracy ≤ 0.01 ppm) tool. 51 pigments were tentatively identified. The profile revealed different classes, such as carotenes, xanthophylls and chlorophylls. Moreover, the antioxidant evaluation of the major compounds was assessed by pre-column reaction with the DPPH radical followed by fast UHPLC-PDA separation, highlighting the contribution of single analytes to the antioxidant potential of the entire pigment fraction. β-carotene, diadinoxanthin and diatoxanthin showed the highest scavenging activity. The method took 40 min per sample, comprising reaction. This strategy could represent a valid tool for the fast and comprehensive characterization of Spirulina pigments in dietary supplements, as well as in other microalgae-based products.