The mitogen-activated protein kinase (MAPK) pathway regulates crucial biological responses, including cell proliferation and cell survival, in response to extracellular stimuli. Its dysregulation due to alterations in key components leading to the upregulation of extracellular signal-regulated kinases 1/2 (ERK1/2) is a driver of many human cancers. ERK1/2 have emerged as a potential therapeutic target for overcoming acquired resistance to FDA-approved targeted therapies. We have previously identified a highly potent and selective ERK1/2 inhibitor, BI78D3, with a novel mechanism of action. Bi78D3 acts as a substrate mimetic, binding covalently to an exposed Cys159 within the D-recruitment site (DRS) of ERK, a conserved region distal to the active site which mediates protein-protein interactions. In this study, we prepared a library of compounds in which we independently modified the benzodioxane, thiazole, or triazalone rings of BI78D3 to better understand the features of chemical structure which influence both the initial reversible binding step to the DRS of ERK and the subsequent rate of covalent bond formation with C159. We identified 13 compounds from our library with micromolar potency against ERK2 (IC₅₀’s 0.7 - 22 µM). Using linear-free energy relationships, we then estimated the rate-limiting transition state involves a build-up of positive charge (ρ = -0.15), which is consistent with our proposed mechanism of C159 thiolate addition occurs through a low pK_a formamidinium ion. Of these, novel compound ERK-In-3 displays an in vitro potency against ERK2 of k_inact/K_i = 2.4 × 10² M^-1s^-1 and suppresses ERK signaling at concentrations as low as 100 nM in BRAF-mutant A375 melanoma cells. ERK-In-3 possesses an electron-donating methyl group at N2 of the reactive triazalone core. ERK-In-3 additionally promotes apoptosis more potently than BI78D3 in cells. These studies provide a path forward for designing covalent substrate mimetics for the DRS of ERK to effect ERK signaling and induce apoptosis in MAPK-dependent cancers.

Ramakrishna Edupuganti

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