Abstract B132: Functionalization of the primary amino group of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione enhances selectivity and specificity of the inhibition of ERK substrate docking domains.
Authors: Fletcher Steven , Nevels Kerrick , Jeong Kwan-Young , Samadani Ramin , Worlikar Shilpa , Smith Sarice , Yap Jeremy L. , Shukla Sagar , MacKerell Alexander D. , Shapiro Paul 
Published: June 2012

Abstract
Extracellular signal-related kinase-1 and 2 (ERK1/2) are hyperactivated in several human cancers, predominantly due to gain-of function mutations in the upstream Ras and Raf proteins. However, its complete inhibition through targeting its active site is expected to be detrimental, since ERK is crucial in various “housekeeping” roles in normal cells. Therefore, our aim is to block specific substrate docking domains on the surface of ERK and, hence, inhibit functions involved in cancer cell proliferation, such as the phosphorylation (activation) of the transcription factor Elk-1 and the enzyme ribosomal S6 kinase-1 (RSK-1). We have previously used computer-aided drug design (CADD) to identify ERK docking domain inhibitors, which led to the discovery of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione (referred to in the literature as 76) that is believed to target the groove in between the common docking (CD) and ED domains. A fluorescence quenching assay showed that 76 binds ERK2 with a KD of 5 μM, exhibits minimal selectivity over the structurally related p38α MAP kinase, and inhibits the proliferation of A375 melanoma cells, which harbor constitutive ERK pathway activation owing to a B-Raf mutation, with an IC50 value of about 20 μM.
Through functionalization of the primary amino group of our lead to afford a focused library of amides, carbamates, sulfonamides and anilines, we have been able to improve binding affinities for ERK2 to as low as 70 nM. Importantly, selectivity for ERK2 over p38α was increased to more than 70-fold. Mutation studies suggested that several of our best compounds bind in the vicinity of Thr157 (located at the ED domain) and, to a lesser extent, Asp319 of the CD domain (a residue that was predicted by CADD to bind the primary amino group of 76). Immunoblot analysis demonstrated that new lead SF-2-054 (ERK2: KD = 170 nM) is a more potent inhibitor of Elk-1 phosphorylation than the parent compound 76. Furthermore, negligible effects on the levels of activated ERK2 suggested that SF-2-054 does not operate upstream of ERK2. Consistent with the mutation binding data, SF-2-054 exhibits minimal effects of the levels of phosphorylated Rsk-1, which is known to bind the CD site, opening the door for the development of substrate-specific ERK inhibitors. At the same time, it is noteworthy that this data suggests that SF-2-054 does not bind the catalytic domain of ERK2. In addition, SF-2-054 was found to inhibit proliferation of A375 cells with an IC50 value of &lt; 5 μM, around 5 times more potent than in the HeLa cell line. In contrast to B-Raf (and MEK) inhibitors that afford complete blockade of the B-Raf/MEK/ERK signaling pathway, our approach demonstrates that only partial inhibition of ERK signaling function is necessary to inhibit the proliferation of melanoma cells. Moreover, it is envisaged that targeting ERK docking sites will lead to inhibitors with improved selectivities to which the development of resistance is less likely.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B132.