• Novel Extracellular Signal-Regulated Kinase (ERK) Targeted Inhibitors Effects on Apoptotic Signaling: A Potential Cancer Therapy

      Boston, Sarice Renee; Shapiro, Paul, Ph.D. (2011)
      The extracellular signal-regulated kinase 1 and 2 (ERK 1/2) proteins potently mediate cell proliferation and survival signals via substrate phosphorylation events. Aberrant activation of ERK due to mutations in upstream activators such as B-Raf is a hallmark of a variety of cancers. Thus, inhibition of substrates involved in these mechanisms provides a promising target for anti-cancer therapies. Small molecules designed to interfere with five putative binding sites on ERK2, which are potentially involved in substrate interactions, were developed using computer-aided drug design (CADD) and assessed for their ability to inhibit ERK-mediated signaling events. The inhibitors identified successfully inhibited cell proliferation and survival signals as demonstrated by inhibition of cell cycle progression and/or the induction of apoptosis in cell based assays. Evaluation of the mechanism of action of these ERK-targeted inhibitors revealed inhibitory effects on ERK mediated phosphorylation of p90Rsk-1, which plays a role in cell growth and survival as well as caspase-9, which is involved in the activation of the intrinsic apoptotic pathway resulting in the induction of apoptosis. Additionally, the ERK-targeted inhibitors appeared to enhance chemotherapeutic drugs by inducing a cytostatic effect through selective inhibition of p90Rsk-1 phosphorylation and cyclin D1 expression indicative of a G1-phase arrest in a melanoma model harboring a B-Raf mutation. These studies suggest that selective inhibition of ERK functions is an alternative approach to complete ERK pathway inhibition and can be used to enhance the sensitivity of melanoma cells to chemotherapeutics drugs potentially reducing toxicity to normal cells and the development of drug resistance.
    • Studies on Extracellular Signal-Regulated Kinase-1/2 (ERK1/2) Function and Identification of Small Molecule Inhibitors that Selectively Target ERK1/2 Regulation of Activator Protein-1 (AP-1) Function in Cancer Cell Proliferation

      Zhang, Jun; Shapiro, Paul, Ph.D. (2014)
      Excessive activation of the Raf/MEK/ERK pathway participates in the pathogenesis of numerous human tumors. In this pathway, extracellular signal regulated kinase 1/2 (ERK1/2) occupy a unique position on which signals from various membrane receptors converge. Therefore, ERK1/2 have been appealing targets for the development of anticancer drugs. In the recent years, careful examination of ATP binding site of protein kinases coupled with structure-based drug design methods has largely facilitated development of specific protein kinase inhibitors. The crystal structures of ERK2 with ATP complex refined to 2.3 Å have been reported previously. However, the details of interactions between ERK2 and ATP binding pocket were not certain because of low resolution. We have obtained high resolution crystals and refined the structures of the apoenzyme, ATP-bound ERK2, ADP-bound ERK2 to 1.8, 1.7 and 1.8 Å respectively. We then described the identification of a novel lead compound with a thienyl benzenesulfonate scaffold that targets the F-domain Recruitment site (FRS) of ERK1/2. Our crystal structure data suggested that this compound interacted with ERK2 in the vicinity of the FRS. Biological analyses further showed that this compound, along with a few structurally similar analogs derived from computational methods based on information provided by the crystal structure, preferentially inhibited F-site containing substrates that form the activator protein-1 (AP-1) transcription factor complex including c-Fos, Fra1, and FosB. Cell viability analysis revealed that melanoma cell lines that harbor constitutively activated ERK1/2 driven by activating B-Raf mutants were more sensitive to growth inhibition by these compounds. Lastly, we discovered a novel mechanism by which ERK1/2 activation regulates the phosphorylation of protein kinase C (PKC) isoforms PKCβII/δ. Our studies demonstrated that growth factor mediated ERK1/2 activation regulated PKCβII/δ activity as inhibition of ERK1/2 affected PKC substrate phosphorylation. Moreover, we provide evidence that inhibition of ERK1/2 reduced cell migration through a mechanism involving regulation of PKCβII. Thus, these data indicate that ERK1/2 proteins act as upstream regulators of PKC proteins and that inhibition of this regulation may have utility in preventing cancer cell migration and metastasis.
    • Study of the Interactions Between Mitogen-Activated Protein Kinases and Small Molecule Inhibitors of Substrate Proteins Using Fluorescence Spectroscopy

      Nevels, Kerrick; Shapiro, Paul, Ph.D. (2011)
      The extracellular-signal-regulated kinase (ERK) proteins belong to the mitogen-activated protein kinase (MAPK) family. In addition to participating in important cell signaling pathways involved in normal cellular functions, unregulated activation of the ERK pathway promotes the proliferation and survival of cancer cells. As such, targeted inhibition of the ERK pathway is a goal in the development of new anti-cancer drugs. Given the importance of the ERK pathway in regulating normal cellular processes, we sought to identify small molecules that can selectively inhibit ERK interactions with protein substrates that promote cancer cell proliferation, but preserve ERK functions in normal cells. To accomplish this, computer-aided drug design (CADD) was used to identify low molecular weight compounds that are predicted to interact with specific substrate docking sites on ERK2. In addition, chemical modifications were made to explore structure-activity relationships. The results of the studies demonstrate how steady-state fluorescence spectroscopy methods can be used to rapidly identify ERK-targeted compounds, determine binding affinities, and evaluate the specificity for the ERK proteins as compared to the structurally related MAPK proteins.
    • Towards Targeted Anti-Neoplastics: The Disruption of Aberrant Protein-Protein Interactions with Low Molecular-Weight Proteomimetics

      Lanning, Maryanna Elizabeth; Fletcher, Steven; 0000-0002-9511-4435 (2017)
      Protein-protein interactions (PPIs) play pivotal roles in a range of cellular processes including proliferation, differentiation, metabolism and apoptosis. Dysregulations of certain PPIs can lead to the development and progression of human cancers. In particular, the overexpression of the anti-apoptotic BCL-2 family members, specifically Mcl-1, have been linked to pancreatic, colorectal and lung cancers as well as leukemia and lymphoma. When over-expressed, MCL-1 prevents cell death by binding and sequestering the BH3 "death" domain of its pro-apoptotic counterpart, such as Bim. Mcl-1 has become an important target for the development of novel antineoplastics. As with many, helix-mediated PPIs, several key residues are hydrophobic and located on one face of the BH3 α-helix, specifically at the i, i + 3/4, i + 7 residues. In addition to exhibiting a hydrophobic face, Asp67 on the "other" face of the Bim-BH3 helix forms a salt bridge with the protein (Arg263). In an improved effort to develop more potent and more selective agents to disrupt the MCL-1-BH3 PPI, we used structure-based design, and developed two complementary strategies: synthetic α-helix (purine based) and BH3 mimetics (naphthoate based). Both scaffolds yielded molecules that disrupted the MCL-1-BIM PPI consistently, and subsequent studies were undertaken towards second-generation molecules. The designed molecules were subjected to biological assays and further structure-activity relationship (SAR) studies to increase affinity and potency in an effort to translate in vitro activity to on target cell activity. It is expected anticipated that the SAR developed in the present study will facilitate the development of novel therapeutics capable of inhibiting Mcl-1 will be identified, which can be advanced to preclinical evaluation.