Vincenzi, Marian (2016) TARGETING PROTEIN INTERACTIONS WITH BIOTECHNOLOGICAL ORIGINAL MOLECULES: A NMR AND MOLECULAR MODELLING INTEGRATED APPROACH. [Tesi di dottorato]

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This PhD thesis work has been covered in the application of an integrated approach of different experimental and computational techniques, to get a better understanding about the solution structure details and the intermolecular interactions of two systems: Akt-heterocyclic ligand (Workpackage 1) and the chemokine, CXCR4 (Workpackage 2). It is well known that the dysregulation of Akt and CXCR4 proteins, normally involved in regulation processes in cell, such as proliferation, is overexpressed in different human cancers. For this reason, they are considered as valuable targets in the development of potential cancer therapeutic agents. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt, a serine/threonine protein kinase, have led to the identification of novel inhibitors with various heterocycle scaffolds. Among heterocyclic compounds that attracted a lot of attention because of its wide spread biological activities, the pyrrolo[1,2-a]quinoxaline heterocyclic framework has been identified as interesting scaffolds for antiproliferative activity against various human cancer cell lines. Thus, based on previous results obtained on the antiproliferative activities of new pyrrolo[1,2-a]quinoxalines, a novel series of pyrrolo[1,2-a]quinoxaline derivatives 1a-l have been designed and synthesized via multistep heterocyclization process. Their cytotoxicities were evaluated against five different leukemia cell lines, including Jurkat and U266 (lymphoid cell lines), and K562, U937, HL60 (myeloid cell lines), as well as normal human peripheral blood mononuclear cells. The new series showed promising cytotoxic potential against all leukemia cell lines tested, and some compounds showed better results than the reference compound A6730. Some compounds, such as 1a, 1e, 1g and 1h are promising because of their high activity against leukemia and their low activity against normal hematopoietic cells. Structure-activity relationships of these new synthetic compounds 1a-l are here also discussed. In addition, docking results, conducted on the isolated PH domain, showed that these new compounds could be considered as allosteric inhibitors. The second workpackage reports on the design and the synthesis of two new peptidic sequences containing a few amino acids with disorder propensity. In addition, they present a CPC unit: binding motif of the specific ligand of CXCR4 namely the CXCL12. The peptide structural preferences were analysed by CD and NMR techniques that highlighted their flexibility. These results were also confirmed by MD simulations that demonstrated the ability of these peptides to assume conformational ensembles stabilized by a network of transient and dynamic H-bonds and characterized by interactions with water molecules. Afterwards we have explored the possibility that these peptides could be used as polar heads connected to alkyl chains to generate new molecular buildings (named peptide amphiphiles, PAs) for drug delivery vehicles in cells over-expressing the CXCR4 receptor. For these reasons, we firstly studied the alkyl chain effect on the conformational rearrangement of intrinsically disordered peptides. Solution fluorescence and DLS studies have been performed to evaluate critical micellar concentrations and the dimension of supramolecular aggregates. Biological tests pointed out that a hydrophobic moiety connected to our peptides could improve their activity on CXR4. In fact, these molecular buildings show promising activities, even higher than the reference molecule (AMD3100). According to these preliminary results, these PA systems could be further modified to realize supramolecular peptide-based compounds, able to be encapsulated by living cells for biomedical application.

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