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Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor.

ABSTRACT: The MAP kinase (Ras/MEK/ERK) and PI3K/Akt/mTOR oncogenic signaling pathways are central regulators of KRAS-mediated transformation. Molecular reciprocity between the Ras/MEK/ERK and PI3K/Akt/mTOR pathways provides cancer cells with the ability to evade treatment when targeting only one pathway with monotherapy. Multi-kinase targeting was explored through the development of a single bivalent chemical entity by covalent linking of high-affinity MEK and PI3K inhibitors. A prototype dual-acting agent (compound 8) designed using the PI3K inhibitor ZSTK474 and the Raf/MEK inhibitor RO5126766 as scaffolds displayed high in vitro inhibition of both PI3K (IC50=172nM) and MEK1 (IC50=473nM). Additionally, compound 8 demonstrated significant modulation of MEK and PI3K signaling pathway activity in human A549 human lung adenocarcinoma cells and pancreatic cancer cells (PANC-1) and also decreased cellular viability in these two cell lines.

SUBMITTER: Van Dort ME

PROVIDER: S-EPMC4370322 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor.

Van Dort Marcian E ME Galbán Stefanie S Wang Hanxiao H Sebolt-Leopold Judith J Whitehead Christopher C Hong Hao H Rehemtulla Alnawaz A Ross Brian D BD

Bioorganic & medicinal chemistry 20150306 7

The MAP kinase (Ras/MEK/ERK) and PI3K/Akt/mTOR oncogenic signaling pathways are central regulators of KRAS-mediated transformation. Molecular reciprocity between the Ras/MEK/ERK and PI3K/Akt/mTOR pathways provides cancer cells with the ability to evade treatment when targeting only one pathway with monotherapy. Multi-kinase targeting was explored through the development of a single bivalent chemical entity by covalent linking of high-affinity MEK and PI3K inhibitors. A prototype dual-acting agen ...[more]

PMID: 25766633

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Project description:Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) β-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and β-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While β-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the β-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and β-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.

Project description:Insulin resistance plays a key role in the pathogenesis of type 2 diabetes and is also related to other health problems like obesity, hypertension, and metabolic syndrome. Imbalance between insulin vascular actions via the phosphatidylinositol 3-Kinase (PI3K) and the mitogen activated protein kinase (MAPK) signaling pathways during insulin resistant states results in impaired endothelial PI3K/eNOS- and augmented MAPK/endothelin 1 pathways leading to endothelial dysfunction and abnormal vasoconstriction. The role of PI3K, MAPK, and protein kinase C (PKC) in Ca2+ handling of resistance arteries involved in blood pressure regulation is poorly understood. Therefore, we assessed here whether PI3K, MAPK, and PKC play a role in the Ca2+ signaling pathways linked to adrenergic vasoconstriction in resistance arteries. Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle (VSM) and tension were performed in endothelium-denuded branches of mesenteric arteries from Wistar rats mounted in a microvascular myographs. Responses to CaCl2 were assessed in arteries activated with phenylephrine (PE) and kept in Ca2+-free solution, in the absence and presence of the selective antagonist of L-type Ca2+ channels nifedipine, cyclopiazonic acid (CPA) to block sarcoplasmic reticulum (SR) intracellular Ca2+ release or specific inhibitors of PI3K, ERK-MAPK, or PKC. Activation of α1-adrenoceptors with PE stimulated both intracellular Ca2+ mobilization and Ca2+ entry along with contraction in resistance arteries. Both [Ca2+]i and contractile responses were inhibited by nifedipine while CPA abolished intracellular Ca2+ mobilization and modestly reduced Ca2+ entry suggesting that α1-adrenergic vasoconstriction is largely dependent Ca2+ influx through L-type Ca2+ channel and to a lesser extent through store-operated Ca2+ channels. Inhibition of ERK-MAPK did not alter intracellular Ca2+ mobilization but largely reduced L-type Ca2+ entry elicited by PE without altering vasoconstriction. The PI3K blocker LY-294002 moderately reduced intracellular Ca2+ release, Ca2+ entry and contraction induced by the α1-adrenoceptor agonist, while PKC inhibition decreased PE-elicited Ca2+ entry and to a lesser extent contraction without affecting intracellular Ca2+ mobilization. Under conditions of ryanodine receptor (RyR) blockade to inhibit Ca2+-induced Ca2+-release (CICR), inhibitors of PI3K, ERK-MAPK, or PKC significantly reduced [Ca2+]i increases but not contraction elicited by high K+ depolarization suggesting an activation of L-type Ca2+ entry in VSM independent of RyR. In summary, our results demonstrate that PI3K, ERK-MAPK, and PKC regulate Ca2+ handling coupled to the α1-adrenoceptor in VSM of resistance arteries and related to both contractile and non-contractile functions. These kinases represent potential pharmacological targets in pathologies associated to vascular dysfunction and abnormal Ca2+ handling such as obesity, hypertension and diabetes mellitus, in which these signaling pathways are profoundly impaired.

Dataset Information

Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor.

Publications

Dual inhibition of allosteric mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) oncogenic targets with a bifunctional inhibitor.

Similar Datasets

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