Abstract
PRIN-UNO focuses on the design and early-stage development of innovative Pin1 inhibitors, targeting a key enzyme involved in tumor growth, progression, and resistance to therapy. Starting from two chemotype series, the project combines structure-based drug design, biochemical screening, and preclinical evaluation in 2D cultures and 3D cancer models. A key component is the analytical profiling of candidate molecules, with in-depth assessment of their metabolic fate, bioavailability, and pharmacokinetic properties. This is achieved through original miniaturized sampling and mass spectrometry-based workflows, designed to ensure high sensitivity and reliability from limited sample volumes. These analytical strategies support the identification of drug-like compounds with favorable ADME-Tox profiles and guide their selection for in vivo testing. The ultimate goal is to deliver one or more validated lead compounds suitable for future development as targeted anticancer therapeutics.
Results achieved
Overall outcomes. The project addressed the design and preclinical characterization of innovative inhibitors of Pin1 (the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), an enzyme frequently overexpressed in aggressive tumors that concurrently activates multiple oncogenic pathways involved in cancer cell proliferation, metastasis and resistance to chemotherapy, and is therefore regarded as an attractive therapeutic target. The work was carried out by a consortium comprising the Universities of Torino, Trieste and Bologna together with an institute of the National Research Council (ICB-CNR), integrating medicinal chemistry, biochemical and cell-based assays, computational and structural studies, and analytical characterization. Starting from two chemotype series, a focused library of new inhibitors was rationally designed and synthesized on the basis of structure-activity relationship considerations, supported by molecular docking and molecular dynamics simulations that guided ligand optimization and characterized ligand-protein binding. Enzymatic assays on isolated Pin1 identified several compounds with pronounced inhibitory activity, comparable to that of all-trans retinoic acid (ATRA), a well-established reference Pin1 inhibitor. The most potent candidates were then evaluated on MDA-MB-231 triple-negative breast cancer cells, where they reduced cell viability with effectiveness comparable to published Pin1 inhibitors, and the expression of Pin1-regulated genes was quantified in the presence and absence of the most active compounds. A research article reporting these findings is in preparation. Triple-negative breast cancer, one of the most aggressive forms of the disease, still lacks effective targeted therapies, which makes the development of selective Pin1 inhibitors particularly relevant. UNIBO unit results. The Alma Mater Studiorum - University of Bologna Unit (Research group of Pharmaco-Toxicological Analysis, PTA Lab) was responsible for the metabolic and analytical characterization of the selected compounds, working on samples provided by the University of Torino. This contribution addressed a decisive step of the hit-to-lead process: information on chemical stability, metabolic fate and bioavailability is required to identify at an early stage the compounds with the most favourable ADME (absorption, distribution, metabolism and excretion) profile, since a molecule may display excellent target inhibition in vitro and yet prove unsuitable for further development because of insufficient stability or rapid biotransformation. An HPLC-DAD-MS/MS method (high-performance liquid chromatography with diode-array detection coupled to tandem mass spectrometry), originally established for the quali-quantitative analysis of ATRA and its derivatives, was optimized and validated according to international guidelines, demonstrating selectivity, accuracy, precision and robustness. The combination of the two detection modes allowed selective and reproducible quantitation together with spectral confirmation within a single analytical run, and the method was applied systematically to the newly synthesized compounds, with ATRA used throughout as a reference term of comparison ensuring inter-experiment comparability. On this basis, stability studies were carried out under a range of biologically relevant conditions, including working solutions, buffers, cell-culture media and surrogate biological fluids, in order to assess the chemical and metabolic behaviour of the compounds in environments representative of the downstream biological assays. Testing across multiple conditions is essential, since a compound may prove perfectly stable in a simple solution and yet undergo degradation once transferred to a more complex, biologically realistic medium; multi-condition screening therefore yields a more reliable prediction of the behaviour expected in vivo. Metabolic stability was investigated through incubation experiments in human liver microsomes, subcellular preparations that reproduce in vitro the hepatic phase-I biotransformations a compound would undergo in vivo. The incubates were analysed at UNIBO and stability was monitored as a function of incubation time, by following the progressive depletion of each parent compound and the concomitant formation of its transformation products. Selected samples were further characterized by ultra-high-performance liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-HRMS Q-TOF), whose accurate-mass capability enabled the tentative structural assignment of the main metabolites and provided a first description of the metabolic pathways involved, discriminating the more metabolically robust candidates from those prone to rapid transformation. The characterization of the metabolites is informative in itself, as a biotransformation product may be inactive or, occasionally, responsible for undesirable effects, and thus contributes to the early safety assessment of a candidate. A distinctive methodological advancement was the development, validation and implementation of a miniaturized, high-throughput sample-preparation and clean-up protocol based on StAGE-tip (stop-and-go extraction tip) microextraction, performed within a pipette tip. The procedure enabled the efficient processing of very small sample volumes while preserving analytical sensitivity and reproducibility, and proved fully compatible with the downstream UHPLC-MS and UHPLC-HRMS platforms. It was assessed in terms of extraction recovery, repeatability and freedom from interfering signals, and proved robust across the different media investigated, making it particularly suited to an early-discovery setting in which compound amounts are limited and a large number of samples must be processed rapidly and reproducibly. The analytical platform was successfully validated and applied to the characterization of the selected compounds. The metabolic profile obtained for ATRA was fully consistent with literature data, thereby validating the overall workflow, while the newly developed Pin1 inhibitors exhibited an overall acceptable metabolic stability in human liver microsomes, supporting their potential for further pharmacokinetic development. The robustness of the data was ensured through replicate experiments and continuous method refinement, providing a solid analytical foundation for the pharmacokinetic and in vivo investigations envisaged beyond the project. The activities of the Bologna Unit were embedded in a continuous, iterative exchange with the synthesis group: newly synthesized compounds were progressively delivered for analytical and metabolic evaluation, and the resulting data were fed back into the optimization cycle. In this way the analytical results did not merely describe the compounds in isolation, but actively supported their prioritization within the consortium, complementing the enzymatic and cellular data with quantitative information on chemical and metabolic robustness. Consistently with green analytical chemistry principles, the workflows were designed to minimise sample volumes, solvent and reagent consumption, and to be transferable: the validated StAGE-tip and UHPLC-MS platform is directly applicable to other early drug-discovery campaigns requiring sustainable, low-volume stability and metabolic screening. Overall, the Bologna Unit delivered a reliable, sustainable and transferable analytical framework for the chemical and metabolic characterization of Pin1 inhibitors, underpinning the medicinal-chemistry effort of the consortium and providing reliable data and methodologies for the future preclinical development of the most promising candidates.Dettagli del progetto
Responsabile scientifico: Michele Protti
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università degli Studi di TORINO(Italy)
Contributo totale Unibo: Euro (EUR) 58.514,00
Durata del progetto in mesi: 24
Data di inizio
30/11/2023
Data di fine:
28/02/2026
