New integrin-targeted small molecule peptidomimetics and their covalent conjugates as potential biomedical tools in oncology- and fibrosis-related diseases

Abstract

The work described in this PhD dissertation concerns the design and the synthesis of new small molecule cyclic peptidomimetics to be used as potent and selective integrin ligands, as both refined αVβ3 binders and novel αVβ6-targeting ligands. In addition, their covalent integration with active components, be they chemotherapeutics, antiangiogenic or antinflammatory agents, would result in the fabrication of novel conjugates to be evaluated for their potential as biomedical tools in oncology-, fibrosis- or inflammation-related diseases.

After an opening general introduction, Chapter 2 is focused on the design and the synthesis of a panel of novel cyclic peptidomimetics targeting the αVβ6 integrin, which is overexpressed in many epithelial tumors, as well as in liver and pulmonary fibrotic tissues, and has emerged as a biomarker of the Epithelial-to-Mesenchymal Transition (EMT). Inspired by previously established aminoproline (Amp)-RGD based cyclopeptidomimetics with attracting αVβ3 integrin affinity and selectivity, the design and synthesis of eighteen new AmpRGD chemotypes bearing additional structural variants were implemented, to shift toward peptide-like αVβ6 integrin targeted binders. All the newly synthesized peptidomimetics were tested for their aVb6 binding affinity by competitive solid phase binding receptor assays and the most potent candidates were also tested towards αVβ3 receptors. Four of the synthesized compounds gave promising results, with IC50 αVβ6) values in the low nanomolar range (4.6-58.5 nM) and selectivity indexes (reported as the ratio between IC50(αVβ3) and IC50 αVβ6)) in the range 243-628.

In Chapter 3 the work done during my period abroad is described. The project carried out at the University of Erlangen-Nürnberg, in the research group of Prof. Dr. Gmeiner was focused on the development of novel peptidomimetics as ligands of Proteinases-Activated Receptor 2 (PAR2), which it is implicated in the progression of different pathological events including cancers, arthritis and inflammation, but also exerts protective effects in certain other diseases (e.g. ischemia, wound healing and colitis). Starting from preliminary results by the Prof. Gmeiner’s research group, a panel of twenty novel compounds was designed, synthesized and characterized. All the final compounds were tested by IP-One-HEK assay and the most promising ones were also subjected to the arrestin-HEK-PS1K assay. The best performing PAR2 agonist among the twenty new synthesized compounds showed IC50 (tested by IP-One-HEK assay) in the nanomolar range, confirming that the proposed structural modifications can drive the research towards a full understanding of the receptor binding/activation properties of new ligands targeting PAR2.

Chapter 4 is focused on the synthesis of nine different covalent conjugates as potential antitumor and antifibrotic agents, based on the substantial evidence of the cross-talk between integrins and Growth Factor Receptors, which seems to determine drug resistance and sustain the EMT. The projected conjugates are constituted by an analogue of the kinase inhibitor nintedanib, which is linked to an RGD-based cyclopeptidomimetic as the targeting unit by means of a robust linker moiety. The panel of nine compounds was realized by conjugating the same kinase inhibitor to one out of three different integrin ligands, two of them targeting αVβ6 integrin and bearing the sequence c(AmpLRGDL), and one targeting αVβ3 integrin and possessing the c(AmpRGD) cyclopeptide. Another point of variability was the linker, which was selected among three possible structures differing in length, polarity and valency. Preliminary results towards TGFB-treated melanoma tumor cells seemed to confirm that antagonizing both kinase receptor and αVβ6 integrin could be a good strategy to address cancer related diseases.

Finally, Chapter 5 concerns the design of MMP2/MMP9-cleavable conjugates addressing both PAR1 and αVβ6 receptors, since it is well known that these receptors are spatially and functionally related and their cross-talk has emerged as a possible target for the treatment of Idiopathic Pulmonary Fibrosis (IPF). The designed conjugates comprise the aVb6-targeting ligand c(AmpLRGDL), the properly modified PAR1 antagonist RWJ-58259 and the MMP2/9-sensitive peptide sequence PLG-LAG, which is supposed to be cleaved at the disease site, thus releasing the two independent active units. The opening move of this project was the parallel synthesis of the two separated active modules, intended to be evaluated first in in-vitro assays towards the corresponding receptors, with the aim to prove that the independent moieties maintain their biological activity upon the proteolytic release.

For clarity’s sake, this thesis is organized into four main chapters following the opening introduction with self-consistent numbering of compound formulas, figures, schemes and references.