Multivalent calixarenes for targeting of cell membrane receptors and intracellular cargo delivery

Abstract

The cell membrane plays important roles of protection and support, it regulates the transport of substances inside or outside the cell and, most importantly, it takes part through its components in many biological processes that occur on its surface. In particular, both physiological processes, like cell-cell communication, and pathological phenomena, such as the adhesion of viruses and bacteria and the migration of tumors, are based on molecular recognition events and are largely characterized by the presence of multivalent interactions. Multivalency is defined as the ability of an entity to bind another entity through the simultaneous formation of several host-guest complexes, resulting in an efficiency of binding which is higher than their simple sum. Taking this concept into account, it is then evident that the design and development of multivalent ligands to interfere in these kind of processes is an interesting approach, which has been widely exploited in last years. Different platforms have been and are currently used for the synthesis of multivalent ligands, with different sizes, valencies and geometries of exposition of the ligating units. Among the others, calixarenes represent a versatile and attractive scaffold. First of all they can be easily synthesized and functionalized using well-established procedures. Changing the conditions, calixarenes of different dimensions and valency can be obtained. Depending on the size and on the functionalization they can be conformationally mobile or, in the case of the calix[4]arenes, selectively blocked in four different conformations. Moreover, both the upper and the lower rim can be decorated with different groups, with the possibility to prepare a huge variety of molecules. The work reported in this thesis aims at the design and synthesis of multivalent ligands for two different purposes: the targeting of cell membrane receptors involved in important pathological conditions and the delivery of cargos into cells. In a first project, calixarenes functionalized with -mannosyl residues were prepared and studied as inhibitors of the receptor DC-SIGN (Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin), involved in the infection of the organism by the Human Immunodeficiency Virus (HIV). The receptor recognizes the high-mannose glycans of the glycoprotein gp120 present on the virus envelope and is exploited by the pathogen to transfer virions to the lymphocytes, following the so-called Trojan Horse model. Because of the tetrameric form of the receptor and its organization on cell surface into clusters a multivalent approach seems to be a valuable strategy to improve ligands efficiency and selectivity. We, therefore, decided to synthesize a small library of mannosylcalixarenes to find an efficient inhibitor of this process evaluating the influence of valency, geometry of exposition of the ligating units and distance of the saccharidic epitopes from the scaffold on the biological activity. The compounds were tested using SPR and demonstrated inhibitory activity towards the receptor. Small differences among the candidates IC50 were found, however the calix[4]arene blocked in the cone conformation resulted the best inhibitor, suggesting the orientation of all the ligating units in the same direction in space and a preorganized and rigid scaffold as important features. For this compound an enhanced relative potency with respect to the methyl-α-D-mannopyranoside used as monovalent reference was calculated, assessing the presence of a multivalent effect. Amphiphilic calixarenes were instead employed for the modulation of Toll-like Receptor 4 (TLR4) activity. Its involvement in the triggering of the innate immune response in the organism renders it an important biological and pharmacological target: molecules able to stimulate the receptor could be used as vaccines or immunotherapeutics, while inhibitors could be used as drugs against the septic shock or as anti-inflammatory agents. Based on the calix[4]arene blocked in the cone conformation, different ligands were synthesized bearing guanidinium or carboxylate groups at the upper rim and chains of different length and nature at the lower rim, or, with an opposite polarity, exposing guanidinium groups at the lower rim linked to the macrocyclic scaffold with spacers of different length. The self-assembly properties in water of the compounds were studied using DLS, fluorescence and NMR spectroscopy, to evaluate a possible correlation between their tendency to aggregate and their biological activity. Experiments on cells showed that all compounds act as inhibitors of TLR4 but compounds with guanidinium work much better than compounds with carboxylate. Quite surprisingly, the best candidates among the upper rim guanidinocalixarenes were the molecules with the shorter chains, moving to the background the importance of their aggregation properties in determining the potency of these derivatives. Finally, calixarenes functionalized with guanidinium groups were exploited in the development of cargo delivery systems. In one case three different calixarenes were used to decorate the membrane of liposomes and their uptake in cells was evaluated with respect to the uptake of regular non-modified liposomes. On the other side, a calixarene functionalized at the upper rim with guanidinylated valeric acid and bearing at the lower rim a biotin moiety was employed as molecular transporter, exploiting its interaction with a fluorescently labelled streptavidin as model cargo. Uptake experiments showed an enhanced internalization for the modified liposomes with respect to the regular ones. Moreover, the role of heparan sulfate proteoglycans in the mechanism of internalization was also assessed by comparing the results on cells lacking the proteoglycans. No significant toxicity was observed for the modified liposomes. The biotinylated calixarene, on the other hand, was not able to significantly promote the translocation of streptavidin used as model payload. However, at this stage of the work it is not clear if the problem could be in the complex formation between the biotin conjugate and the protein.