Novel Fluorescent Molecular Probes and Peptide-Drug Conjugates Targeting Integrin αvβ6 towards Precise Imaging and Treatment of Pulmonary Fibrosis

During my Ph.D. studies, I mainly focused on the design and synthesis of targeted covalent conjugates that incorporate cyclopeptides selectively targeting αvβ6 integrins. These conjugates include various types of linkers, both cleavable and non-cleavable, as well as payloads with diverse functions. These payloads comprised molecules intended for applications either in molecular imaging, or therapeutic fields. One domain of particular interest was the idiopathic pulmonary fibrosis, for which a clear correlation between αvβ6 integrin overexpression, transforming growth factor TGF-b overactivation, and fibrotic response enhancement has been widely recognized. We sought to develop small collections of αvβ6 integrin-targeted fluorescent molecular probes and peptide-drug conjugates (PDCs), with the final aim of evaluating their behavior in both in vitro and in vivo αvβ6 integrin-overexpressing pulmonary fibrosis models. Chapter 2 introduces the overall domain of integrins and, in particular, αvβ6 integrins; then, it is disclosed the rationale behind the development of covalent conjugates, with special emphasis on peptide-drug conjugates and peptide-fluorescent probe conjugates. In both cases, some examples on either cleavable or non-cleavable linkers are exposed. Chapter 2 finishes with the description of integrin-targeted conjugates which have been already developed over the years. During these years we focused firstly on the development of two small collections of αvβ6 integrin targeted fluorescent probes. Indeed, traditional methods for studying integrin function, such as antibody-based techniques, have limitations in terms of specificity, sensitivity, real-time monitoring capabilities, and additional issues also arise in terms of the ability to access to the stiff fibrotic tissues. For these reasons the need for the development of different approaches rose. By developing fluorescent probes specifically targeted towards αvβ6 integrin, we wanted firstly to evaluate the behavior and the selectivity of our cyclopeptides in vitro, trying to track integrin internalization, recycling, and trafficking in response to cellular signals. Moreover, we aimed to put the basis for developing further probes able to monitor the progress of the IPF disease in vivo. This last objective rose because IPF is directly correlated with αvβ6 integrin. In particular, the designed fluorescent conjugates feature a cyclopeptide moiety, intended to selectively target the αvβ6 integrin, a robust non-cleavable linker, and different kind of fluorescent moieties. The choice to incorporate different kinds of fluorophores was based on the possibility to differentiate the probes for in vitro and in vivo application. In Chapter 3, the selected fluorophore for our conjugates is the popular cyanine 5, featuring absorption and emission wavelengths of 651 nm and 670 nm, respectively. This fluorophore was chosen due to its favorable optical properties. In Chapter 4, given the need for simple, rapid, and effective diagnostic tools to study and assess pulmonary fibrosis, we proposed a near-infrared (NIR) fluorophore, the ZW800-1, as the active imaging unit of our conjugates. This fluorophore, whose wavelengths of absorption and emission fall into the NIR-I region, has superior properties compared to other NIR-I emitting fluorophores, largely thanks to its zwitterionic character. This unique feature enhances its stability, reduces non-specific binding, and improves overall imaging quality, making it more effective for in vivo applications. In general, with this approach, we aimed to monitor the progression of fibrosis in a pulmonary fibrosis mouse model and potentially, in the future, to monitor tumor progression as well. In Chapter 5, our work was aimed at the development of new peptide-drug conjugates connected by cleavable linkers. In particular, we proposed a series of novel PDCs, whereby a αvβ6 integrin-targeting cyclopeptide and a nintedanib-based moiety are connected by MMP2-cleavable linkers. The rationale underlying this approach is that, it would be possible for the targeted PDCs to selectively address the fibrotic lung tissue intact, they could be subsequently cleaved in the extracellular environment by overexpressed proteases such as MMP2, thus releasing the nintedanib-based drug and allowing its diffusion in both αvβ6-positive and αvβ6-negative cells. Finally, in Chapter 6, we describe a project to which I had the opportunity to participate during my six months-long secondment at the University of Edinburgh, at the Institute of the Regeneration and Repair in Scotland, under the supervision of Prof. Marc Vendrell Escobar and co-supervision of researchers Dr. Abigail Eleonor Rease and Dr. Fabio de Moliner. There, I worked on biosensor development, in particular in the ribosome-mediated incorporation of fluorescent amino acids into small proteins. The primary goal of this project was to screen a small library of newly synthesized amino acids, each containing the same fluorogenic fluorophore, but featuring linkers of varying lengths. In the next future, after this screening, we would like to apply the best-performing amino acid towards the construction of biosensors targeting the NorA efflux pump to detect the presence of methicillin-resistant Staphylococcus aureus, thereby advancing our understanding of antibiotic resistance mechanisms.