Prolyl isomerase PIN1 inhibitors: a drug design strategy against aggressive tumors - PRIN-UNO (PRIN 2022 PNRR)

Post-translational modifications (PTMs) of proteins are critical for appropriate cellular responses to intra- and extra-cellular stimuli. A crucial PTM in signal transduction is provided by phosphorylation-dependent cis-trans prolyl isomerization of phosphorylated Ser/Thr-Pro motifs, which results in conformational changes affecting the structure of specific domains with impact on protein function. In humans, the only known enzyme that specifically isomerizes phospho-Ser/Thr-Pro motifs is the peptidyl-prolyl isomerase Pin1. High Pin1 levels are prevalent in tumors,1 and we and others found that it modulates multiple signaling pathways relevant for cancer growth, metastasis, and treatment resistance.2 Abnormal Pin1 activation simultaneously boosts multiple oncogenes, while depletion or pharmacological inhibition of Pin1 in experimental mouse models curbs tumor growth and metastasis, making Pin1 an ideal drug target.3 Thus, considerable effort for developing Pin1 target therapies has been made, yet the provided inhibitors mainly display low potency, specificity, stability, and permeability.2, 4 A research program dedicated to designing Pin1 inhibitors is active in our consortium since 2015. Such program already identified KPT-6566, a potent Pin1 inhibitor able to force its degradation. Acting as covalent inhibitor, it exhibits significant antitumor activity in nude mice (5 mg/kg, ip injection).5 In recent years, our drug development program identified other new hits able to inhibit Pin1 with the same potency of all-trans retinoic acid (ATRA), one of the most studied Pin1 specific inhibitor.6 The hits could be clustered into two groups: ATRA analogs and pyrazole chemotypes derived from UniTO library screening. This proposal is dedicated to identifiy new Pin1 inhibitors, characterized up to preclinical level. We will pursue a lead optimization campaign, by using a structure-based computationally driven approach, starting from the above-described hits (see preliminary results). We will apply an iterative optimization cycle, supporting the drug optimization process by structural studies and state of the art in silico analysis. The best candidates will be then evaluated for in vitro metabolic profile and stability in biological fluids. Antitumor activity of the best compounds will be investigated in 2D and 3D models of breast cancer (2D cultures, tumor-derived spheroids and mouse- and patient-derived organoids). Once the lead molecule(s) will be identified, toxicity and pharmacokinetic profile in mice will be endorsed. Analysis will be performed exploiting advanced miniaturized biosampling and sample treatment technologies coupled to original mass spectrometry-based methods7 in order to reduce sample volumes, solvents and reagents, in the general framework of sustainability.