Selection, characterization, and engineering of nanomedicines inhibiting proteases

Cystic fibrosis (CF) is an autosomal recessive disease that reflects the consequences of pathogenic mutations in the CFTR gene. The consequences of CFTR dysfunction are airways obstruction, inflammation and infection in the lungs and upper airways. The resulting inflammatory process observed in CF is dominated by the presence of neutrophils, driving a vicious cycle of bacterial colonization, airways inflammation and structural damage of the lung tissue. Neutrophils of CF patients release their intracytoplasmic granules content, which comprises a set of neutrophil serine proteases (NSPs). These NSPs are externalized in an active form during the neutrophil activation at the inflammatory sites and their excessive secretion leads to a massive airways tissue damage and prolonged inflammation. In lungs there are protease inhibitors involved in the prevention of deleterious effects of NSPs, but the protease/antiprotease balance is disrupted during the progress of the disease. Therefore, NSPs are one of the main therapeutic targets. The specific inhibition of these proteases with nanomedicine to control unwanted proteolysis activity could be a therapy for inflammatory and infectious lung disease. In this regard, this project aimed at selecting, producing and characterizing a nanomedicine able to inhibit one of the NSPs (protease-1). Starting from a previously identified nanomedicine, its affinity to the mouse counterpart of protease-1 was studied with the Bio-layer interferometry (BLI) technique. In order to find a variant of nanomedicine-1 with improved properties for the mouse proteinase-1, six mutants were designed according to the 3D X-rays crystal structure of nanomedicine-1 in complex with protease-1. These nanomedicine-1 mutants were selected, produced in E. coli and purified with IMAC and SEC chromatography. To evaluate their inhibitory profile to mouse proteinase-1 several inhibition assays were performed, while their binding was evaluated with BLI experiments. The analysis revealed that none of these new nanomedicines was able to inhibit the protease of interest, neither of binding it. The mutations carried out were not advantageous for the inhibition of mouse protease-1 and were also counterproductive for the binding itself. A second goal of the project was to select a new nanomedicine that functions as an inhibitor for another NSP, protease-2. Starting from the results of the phage display and panning carried out prior to this project, two nanomedicines were selected, produced in E. coli and purified. Their inhibitory activity to protease-2 was evaluated, but no positive results were obtained. Their affinity for protease-2 was studied, as well as their stability in presence of protease -2. During the course of the experiments, the non-specific binding of protease-2 to the biosensors used for BLI experiments has been noted. All the results previously obtained have been questioned and the non-specific binding of protease -2 to the biosensors was studied in order to find a solution to reduce it.