The extracellular signal regulated kinases (ERK1 and ERK2) are important mediators of cell proliferation. Constitutive activation of the ERK proteins plays a critical role in the proliferation of many human cancers. Taking advantage of recently identified substrate docking domains on ERK2, we have used computer-aided drug design (CADD) to identify novel low molecular weight compounds that interact with ERK2 in an ATP-independent manner and disrupt substrate specific interactions. In the current study, a CADD screen of the 3D structure of active phosphorylated ERK2 protein was used to identify inhibitory compounds. We tested 13 compounds identified by the CADD screen in ERK-specific phosphorylation, cell proliferation, and binding assays. Of the 13 compounds tested, 4 compounds strongly inhibited ERK-mediated phosphorylation of ribosomal S6 kinase-1 (Rsk-1) and/or the transcription factor Elk-1 and inhibited the proliferation of HeLa cervical carcinoma cells with IC 50 values in the 2-10 μM range. These studies demonstrate that CADD can be used identify lead compounds for development of novel non-ATP dependent inhibitors selective for active ERK and its interactions with substrates involved in cancer cell proliferation.The mitogen activated protein (MAP) kinase family of enzymes regulates most biological processes including cell growth, proliferation, differentiation, inflammatory responses, and programmed cell death. Changes in MAP kinase activity have been implicated in the pathophysiology of cancer, inflammatory diseases, and neurodegenerative disorders. 1-4The three main members of MAP kinases include the extracellular signal regulated kinases (ERK), the c-Jun N-terminal kinases (JNK), and p38 MAP kinases. 5 Currently, there is much interest in understanding the role of MAP kinases in disease as these proteins may be promising targets of new chemotherapy and anti-inflammatory agents. 6The ERK proteins consist of 2 isoforms (ERK1 and ERK2; referred to as ERK1/2) that are linked to the proliferation and survival of cancer cells. 7 The ERK1/2 pathway is commonly activated by extracellular ligands, which stimulate plasma membrane receptors and the sequential activation of Ras G-protein isoforms (H, K, and N-Ras), Raf isoforms (A, B, and C-Raf), and the MAP or ERK kinases-1 and 2 (MEK1/2), which are currently the only known Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ERK1/2 regulation of dozens of different proteins underscores the importance of these proteins in regulating a variety of cellular functions associated with normal and diseased tissue. Thus, we and ...