Discovery And In Vitro And In Vivo Characterization Of Aminopyrazoline-Based Smyd2 Inhibitors

SMYD2 (SET and MYND domain-containing protein 2) is a protein lysine methyltransferase (PKMT) which was initially described as a histone H3K36 and H3K4 methyltransferase involved in transcriptional regulation. SMYD2 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as p53, retinoblastoma protein (Rb) and the estrogen receptor alpha. Given the reports that overexpression of SMYD2 is linked to poor prognosis in certain cancers, SMYD2 is proposed to be an oncogene and an attractive cancer drug target. Here we report the discovery of a novel potent and selective SMYD2 inhibitor, SMYD2-BAY-01, by high throughput screening and extensive biophysical validation. The co-crystal structure revealed that SMYD2-BAY-01 binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays is consistent with the binding mode observed in the crystal structure. Further optimization generated SMYD2-BAY-02, which shows improved low nanomolar potency and is selective against kinases and other PKMTs. Furthermore, SMYD2-BAY-02 specifically inhibits SMYD2 methylation activity in a cellular assay with similar potency and reduces methylation of the tumor suppressor protein p53. Based on promising in vitro and in vivo DMPK data, SMYD2-BAY-02 was further characterized in vivo for SMYD2-specific methylation inhibition. In vivo activity could be shown upon in vivo administration at doses as low as 30 mg/kg p.o. in a SMYD2 overexpressing esophageal squamous cell carcinoma model. In summary, SMYD2-BAY-02 is a promising selective and potent SMYD2 inhibitor in vitro and in vivo and may represent a new treatment option for cancers overexpressing SMYD2. Citation Format: Carlo Stresemann, Ingo Hartung, Timo Stellfeld, Naomi Barak, Jeffrey Mowat, Clara Christ, Antonius ter Laak, Silke Koehr, Jorg Weiske, Roman Hillig, Volker Badock, Detlef Stoeckigt, Karl Ziegelbauer, Hilmar Weinmann, Volker Gekeler. Discovery and in vitro and in vivo characterization of aminopyrazoline-based SMYD2 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2829. doi:10.1158/1538-7445.AM2015-2829