Grazia M. L. Consoli

Polycationic calix[8]arenes able to recognize and neutralize heparin

A mutual induced fit mechanism is responsible for the exceptional complexation performances exhibited by calix[8]arene polycations towards heparin. The recognition process was studied in comparison with two other heparin antagonists: protamine and polylysine. The arrangement of multiple functional groups on the flexible macrocyclic scaffold of calix[8]arene, with respect to the conformationally rigid protamine and low ordered polylysine, allowed a mutual adaptability between calixarene polycations and heparin, significantly enhancing the recognition performances. Fluorescence, NMR titration, and activated partial thromboplastin time (aPTT) experiments confirmed that these calixarene derivatives have a very high specificity and affinity towards heparin neutralization as in aqueous solution as in blood. Analogous results were obtained with low molecular weight heparin (LMWH) whose effect protamine is unable to completely reverse.

The KDEL receptor couples to Gαq/11 to activate Src kinases and regulate transport through the Golgi

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra‐Golgi trafficking and is initiated by the KDEL receptor (KDEL‐R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL‐R signalling mechanism. We show that the KDEL‐R is predicted to fold like a G‐protein‐coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G‐protein Gαq/11 which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR‐like mode of action of the KDEL‐R and shed light on a core molecular control mechanism of intra‐Golgi traffic.

Calix[4]arene Decorated with Four Tn Antigen Glycomimetic Units and P3CS Immunoadjuvant : Synthesis, Characterization, and Anticancer Immunological Evaluation

A novel anticancer vaccine candidate built on a nonpeptidic scaffold has been synthesized. Four S-Tn tumor-associated glycomimetic antigens have been clustered onto a calix[4]arene scaffold bearing an immunoadjuvant moiety (P3CS). The immunogenicity of the synthetic construct has been investigated by immunization of mice in vivo. ELISA assay has evidenced that the tetravalent construct stimulates a higher production of anti-Tn antigen IgG antibodies when compared to an analogous monovalent compound. This result is ascribable to an antigen cluster effect and makes the reported vaccine candidate a good mimic of the natural motifs present on the mucine surface.

First Self-Adjuvant Multicomponent Potential Vaccine Candidates by Tethering of Four or Eight MUC1 Antigenic Immunodominant PDTRP Units on a Calixarene Platform: Synthesis and Biological Evaluation

MUC1 protein overexpressed in human epithelial carcinoma is a target in development of novel anticancer vaccines. Multiple units of immunodominant B-cell epitope PDTRP MUC1 core sequence were conjugated to calix[4,8]arene platforms containing TLR2 ligand, to produce two novel anticancer self-adjuvant vaccine candidates. The immunogenicity of the synthetic constructs was investigated by immunization of mice in vivo. ELISA assay evidenced that the vaccine candidates stimulate anti MUC1 IgG antibody production (major for the octavalent construct) and no additive effect but a multivalency effect was observed when compared to an analogous monovalent. Octa- and tetravalent constructs lacking in PDTRP peptide moieties did not show anti MUC1 IgG antibody production in mice. The antibodies induced by the synthesized constructs are able to recognize the MUC1 structures present on MCF7 tumor cells. The results display that calixarenes are convenient platforms for building multicomponent self-adjuvant vaccine constructs promising as immunotherapeutic anticancer agents.

Alkali cation ‘conformational templation’ in 1,5-bridged calix[8]arenes: a single crystal X-ray proof

Abstract X-Ray crystallography proves that alkali cations can act as conformational templates for 1,5-bridged calix[8]arenes 1 H 6 – 3 H 6 , folding their skeleton in a ‘tub-shaped’ conformation composed of four 3/4-cone clefts. This templation occurs even for neutral 1 H 6 to give a ( 1 H 6 Cs) + ·Cl − complex, where Cs + was found perfectly fitting inside the eight calix[8]arene oxygens cavity. Conventional ‘electrostatic’ coordination with O atoms occurs whereas cation-π interactions can be excluded.

Inhibition of rat glioma cell migration and proliferation by a calix[8]arene scaffold exposing multiple GlcNAc and ureido functionalities

β1,4‐Galactosyltransferases (β1,4‐GalTase) exposed on the cell surface are involved in cell migration. Specifically, β1,4‐GalTase V is highly expressed in glioma and promotes invasion, growth, and survival of glioma cells. A glycocalix[8]arene exposing N‐acetylglucosamine (GlcNAc) residues (compound 1) inhibited rat C6 glioma cell migration as assessed in a scratch wound model. This effect was related to inhibition of focal adhesion kinase phosphorylation, measured by western blot analysis, and specifically observed in the area bordering the scratch wound. Compound 1 inhibited also C6 cell proliferation, an effect unrelated to its ability to interact with GalTase as it was mimicked by different calix[8]arene derivatives, all characterized by multivalency and ureido groups. Compound 1 did not induce apoptotic death, but caused a different distribution of C6 cells within the cell cycle. The results here reported identify compound 1 as a molecule able to exert inhibitory effects on C6 cell migration and proliferation, independently, because of distinct components in its structure.

On the Occurrence of the 1,3,5-trisubstitution Pattern In the O-alkylation of P-tert-butylcalix[6]arene

Abstract 1,3,5-Trisubstitution at the phenolic hydroxyls of p-tert-butylcalix[6]arene (1), previously obtained in sizeable yield only with MeI (Casnati et al. J. Chem. Soc., Chem. Commun. 1991, 1413), has been now achieved by direct O-alkylation with alkyl iodides (Et, n-Pr, n-Bu) and p-X-benzyl bromides, (X = tert-Bu, Me, Br, NO2) in the presence of a weak base (K2CO3 or CsF). These results suggest that 1,3,5-trisubstitution can also be extended to other alkylating agents provided that an appropriate weak base is used. The 1,3,5-tris(p-X-benzyl) ethers 6–9, whose structure have been established by 1H-NMR spectral analysis and chemical correlation, in CDCl3 solution adopt a conformation with only a 2-fold symmetry element bisecting two aromatic rings.

Diester intrabridging of p-tert-butylcalix[8]arene and unexpected formation of the monospirodienone derivative

Abstract Reaction of p - tert -butylcalix[8]arene 1 with adipoyl chloride in the presence of NaH as the base yielded singly and doubly intrabridged esters 2 – 4 and 6 . Surprisingly, calix[8]arene monospirodienone derivative 7 was also isolated, which was originated by O 2 oxidation. The conditions of this oxidation were optimized leading to a novel synthetic approach to calixarene monospirodienones based on the O 2 /NaH/acyl-chloride oxidizing system. Xantheno calix[8]arenes 8 – 8a were obtained by rearrangement of 7 .

Double-two-point and four-point intramolecular bridging of p-tert-butylcalix[8]arene

Abstract Calix[8]arenes bis-bridged at the lower rim with ortho - or meta -xylene unit ( 3a-b and 4a-b ) have been obtained in high yields by reaction of 1,3,5,7-tetra- O -substituted calix[8]arenes with 1,2- or 1,3-bis(bromomethyl)benzene. The use of 1,2,4,5-tetrakis)bromomethyl)benzene afforded the four-point intrabridged calix[8]arenes 5a-b in yields up to 41%. VT NMR studies indicated that the flipping motion of aromatic rings in these compounds is inhibited by the intramolecular bridging. Molecular modeling suggested an unprecedented pseudo pleated loop conformation for compounds 5a-b .

Potential Eye Drop Based on a Calix[4]arene Nanoassembly for Curcumin Delivery: Enhanced Drug Solubility, Stability, and Anti-Inflammatory Effect

Curcumin is an Indian spice with a wide spectrum of biological and pharmacological activities but poor aqueous solubility, rapid degradation, and low bioavailability that affect medical benefits. To overcome these limits in ophthalmic application, curcumin was entrapped in a polycationic calix[4]arene-based nanoaggregate by a simple and reproducible method. The calix[4]arene-curcumin supramolecular assembly (Calix-Cur) appeared as a clear colloidal solution consisting in micellar nanoaggregates with size, polydispersity index, surface potential, and drug loading percentage meeting the requirements for an ocular drug delivery system. The encapsulation in the calix[4]arene nanoassembly markedly enhanced the solubility, reduced the degradation, and improved the anti-inflammatory effects of curcumin compared to free curcumin in both in vitro and in vivo experiments. Calix-Cur did not compromise the viability of J774A.1 macrophages and suppressed pro-inflammatory marker expression in J774A.1 macrophages subjected to LPS-induced oxidative stress. Histological and immunohistochemical analyses showed that Calix-Cur reduced signs of inflammation in a rat model of LPS-induced uveitis when topically administrated in the eyes. Overall, the results supported the calix[4]arene nanoassembly as a promising nanocarrier for delivering curcumin to anterior ocular tissues.