Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3

• DOI: 10.1016/j.ejmech.2022.114683
• Publication Date: 2022-09-08T02:38:43Z
• Authors: Marco Daniele Par...; Marina Naldi; Elisabetta Manoni; Edoardo Fabini; Daniela Cederfelt; Vladimir O. Talibov; Valeria Gressani; Ummu Guven; Valentina Grossi; Candida Fasano; Paola Sanese; Katia De Marco; Alexander A. Shtil; Alexander V. Kurkin; Andrea Altieri; U. Helena Danielson; Giuseppina Caretti; Cristiano Simone; Greta Varchi; Manuela Bartolini; Alberto Del Rio
• Keywords: SMYD3; 0303 health sciences; 610; Lysine methyltransferase; Breast Neoplasms; Histone-Lysine N-Methyltransferase; Cancer target therapy; Epigenetic inhibitor; 3. Good health; Histones; Covalent inhibitor; SMYD3; Lysine methyltransferase; Epigenetic inhibitors; Cancer target therapy; 03 medical and health sciences; Cell Line, Tumor; Epigenetic inhibitors; Humans; Cancer target therapy; Covalent inhibitor; Epigenetic inhibitors; Lysine methyltransferase; SMYD3;; Female; Covalent inhibitor

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 μM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.