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41° Congresso Nazionale della SIF
TITLE: Drug off-target proteins identified on a proteome-wide scale by the innovative SPILLO-PBSS software and experimental validations.
BACKGROUND: The property of a drug to interact with multiple molecular targets is usually a major drawback both in the drug development process, where off-target interactions often lead to safety-related failures, and in medicine, where therapeutic agents often lead to side effects that can cause dose limitation or even treatment discontinuation. Representative examples are finasteride, widely used to treat benign prostatic hypertrophy and androgenetic alopecia, which can give rise to sexual dysfunction and psychological and physical disorders , and bortezomib, a first-line drug to treat multiple myeloma, which in about 50% of patients causes peripheral neuropathy often responsible for discontinuation of therapy . The use of innovative in silico methods, such as the SPILLO potential binding sites searcher (SPILLO-PBSS) software, may prove helpful for such challenging problems .
METHODS: Two unbiased 3D proteome-wide scale in silico screenings were performed using SPILLO-PBSS. This tool has unique capabilities in identifying targets and off-targets of any small molecule through a direct identification of their (often previously unknown) binding sites, which can be detected even when hidden or completely closed, and therefore not identifiable by traditional approaches (e.g., molecular docking simulations, QSAR, etc.). The identified off-targets and the proposed biomolecular mechanisms were validated by in vitro, in vivo and/or NMR experiments.
RESULTS: As for finasteride, the 'phenylethanolamine N-methyltransferase (PNMT)' enzyme was found 5th out of 17,900+ proteins analyzed. It catalyzes the conversion of noradrenaline to adrenaline and, therefore, it is potentially correlated to the considered adverse effects of finasteride. Importantly, the binding site of finasteride on PNMT was found to overlap with its catalytic site, thus suggesting a competitive inhibition of the enzyme activity by finasteride. Both the interaction with PNMT and the inhibitory nature of the binding were then confirmed in vitro and in vivo . As for bortezomib, tubulin was identified as one of its potential off-target proteins (4th out of 26,100+ proteins analyzed). In this case, it was possible to hypothesize an inhibition of the GTPase activity of tubulin by bortezomib and a reduction of 'microtubule catastrophe' as a possible cause of the neurotoxicity of this drug. Both the inhibition of GTPase activity and the reduction of 'microtubule catastrophe' have been experimentally confirmed in vitro, while the direct interaction with tubulin has been confirmed by NMR binding studies .
CONCLUSIONS: The results obtained, in addition to representing a step toward a deeper understanding of the molecular mechanisms potentially responsible for the adverse effects of the drugs considered, further confirm SPILLO-PBSS’s great potentialities in identifying targets and off-targets of any small molecule on a proteome-wide scale and its manifold applications in the drug R&D .
 Androgenetic alopecia; drug safety and therapeutic strategies. Expert Opin. Drug Saf. (2018); DOI: 10.1080/14740338.2018.1430765
 Safety of proteasome inhibitors for treatment of multiple myeloma. Expert Opin. Drug Saf. (2017); DOI: 10.1080/14740338.2017.1259310
 SPILLOproject: https://www.spilloproject.com/
 SPILLO-PBSS: Detecting Hidden Binding Sites within Protein 3D-Structures Through a Flexible Structure-Based Approach. J. Comput. Chem. (2014); DOI: 10.1002/jcc.23714
 Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target. J. Med. Chem. (2021); DOI: 10.1021/acs.jmedchem.0c02039
 Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect. Sci. Rep. (2021); DOI: 10.1038/s41598-021-89856-3
 3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line. Bioorg. Med. Chem. (2019); DOI: 10.1016/j.bmc.2019.115300
 MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines. Biology (2022); DOI: 10.3390/biology11010070