Identification of Potential drugs for rare diseases by analyzing and characterizing waste fractions from industrial processing of human plasma

Rare diseases (RDs) such as alkaptonuria (AKU) face considerable challenges due to limited medical, economic, and research investment. AKU is an ultra-rare genetic disorder caused by a deficiency in homogentisate 1,2-dioxygenase (HGD). In 2020, based on the results of clinical trials, the EMA approved the use of 2-(2-nitro-4-trifluoromethylbenzoyl)- 1,3-cyclohexanedione (NTBC) for the treatment of AKU. However, NTBC has significant limitations, including severe side effects and the fact that there are still doubts about the right timing for starting treatment. Given these challenges, there is a growing interest in exploring alternative therapeutic strategies. In this context, human plasma, particularly plasma waste fractions could serve as a sustainable and economically viable source for developing protein-based therapeutics. In Chapter 1, we investigated this possibility by evaluating whether HGD could be extracted from plasma waste fractions as a basis for protein replacement therapy (PRT) in AKU. The project was carried out in collaboration with Kedrion S.p.A. with the main aim of identifying, if any, the most suitable plasma waste fraction for the isolation of active HGD. Despite extensive assay development and testing, however, HGD was not detected at measurable levels, indicating that plasma waste is not a feasible source for HGD as PRT. In order to keep in line with the principles inspiring this work, that is novel sustainable green approaches to AKU allowing circular economy, drug repurposing, as well as predictive and personalized medicine, we decided to shift our focus toward other plasma proteins that were already found to be enriched in plasma waste fractions from Kedrion. Among these proteins, Serum Amyloid A (SAA) could be a candidate of interest due to its involvement in AA amyloidosis, a condition driven by the pathological misfolding of SAA and found to be associated to AKU. However, before proceeding with experimental extraction and characterization of SAA, we first aimed to understand its misfolding mechanisms in detail through computational studies. In Chapter 2, we exploited a bioinformatics approach based on high-temperature molecular dynamics simulations combined with Harmonic Linear Discriminant Analysis (HLDA) and Parallel Tempering Metadynamics (PT-MetaD) to explore the early misfolding process of the disease-relevant SAA1−76 fragment. Our enhanced sampling approach revealed a stepwise misfolding pathway involving thirteen metastable intermediates, with α-helix III destabilizing first, followed by other two α-helices, while global compactness is preserved. Notably, transient exposure of the predicted aggregation prone region (APR) (residues 42–48) was observed in specific intermediates, suggesting that partial unfolding may initiate pathological aggregation. Building on these findings, Chapter 3 focused on structure-based drug discovery targeting the identified misfolding-prone states of SAA. Using a virtual docking screening approach, approximately 10,000 small compounds were docked against four metastable SAA conformers with exposed APR. A consensus scoring strategy was used to select the most promising candidate, which was then evaluated through further PT-MetaD simulations. The results showed that the ligand stabilized folded conformations of SAA and restricted transitions toward unfolded states, supporting its potential role as a misfolding inhibitor. As a future step, we plan to extract SAA from plasma waste fractions and experimentally validate the inhibitory effects of the identified ligand through in vitro assays. This final phase will complete the translational pipeline, from computational insight to therapeutic application, offering a promising strategy for transforming plasma waste into a valuable resource while advancing sustainable approaches to rare disease treatment.