A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

Integrin α5 mediates intrinsic cisplatin resistance in three-dimensional nasopharyngeal carcinoma spheroids via the inhibition of phosphorylated ERK /caspase-3 induced apoptosis.

Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and ß subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G0/G1 and G2/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5ß1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

A novel protein encoded by circMAPK1 inhibits progression of gastric cancer by suppressing activation of MAPK signaling.

BACKGROUND: A novel type of noncoding RNA, circRNA has been reported to participate in the occurrence and development of diseases through many mechanisms. The MAPK pathway is a common signal transduction pathway involved in cell proliferation, inflammation and apoptosis and plays a particularly important role in cancers. However, the role of circRNAs related to the MAPK pathway in gastric cancer has not been explored. METHODS: A bioinformatics analysis was performed to profile and identify the circRNAs involved in the MAPK pathway in gastric cancer. The tumor-suppressive role of circMAPK1 was confirmed both in vitro and in vivo. Mass spectrometry, Western blot and immunofluorescence staining assays were used to validate the existence and expression of MAPK1-109aa. The molecular mechanism of circMAPK1 was investigated by mass spectrometry and immunoprecipitation analyses. RESULTS: In this study, we identified that circMAPK1 (hsa_circ_0004872) was downregulated in gastric cancer tissues compared with adjacent normal tissues. Importantly, lower circMAPK1 expression predicted poor survival in GC patients. CircMAPK1 inhibited the proliferation and invasion of gastric cancer cells in vitro and in vivo. Next, we found that circMAPK1 encoded a novel protein with 109 amino acids in length. Through a series of functional experiments, we confirmed that circMAPK1 exerted a tumor-suppressing effect via the encoded protein MAPK1-109aa. Mechanistically, the tumor suppressor MAPK1-109aa inhibited the phosphorylation of MAPK1 by competitively binding to MEK1, thereby suppressing the activation of MAPK1 and its downstream factors in MAPK pathway. CONCLUSIONS: Our study revealed that circMAPK1 inhibits the malignant biological behavior of gastric cancer cells through its encoded protein MAPK1-109aa. More importantly, circMAPK1 is a favorable predictor for gastric cancer patients and may provide a new therapeutic target in the treatment of gastric cancer.

Mechanistic Analysis of an Extracellular Signal-Regulated Kinase 2-Interacting Compound that Inhibits Mutant BRAF-Expressing Melanoma Cells by Inducing Oxidative Stress.

Constitutively active extracellular signal-regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1-mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound (SF-3-030) and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early gene levels, including DEF motif-containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the ROS inhibitor N-acetyl cysteine reversed SF-3-030-induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism. SIGNIFICANCE STATEMENT: Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the proliferation and survival of many cancer cell types. Given the diversity of cellular functions regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.

How CD40L reverse signaling regulates axon and dendrite growth.

CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.

Kinetic network model to explain gain-of-function mutations in ERK2 enzyme.

ERK2 is a kinase protein that belongs to a Ras/Raf/MEK/ERK signaling pathway, which is activated in response to a range of extracellular signals. Malfunctioning of this cascade leads to a variety of serious diseases, including cancers. This is often caused by mutations in proteins belonging to the cascade, frequently leading to abnormally high activity of the cascade even in the absence of an external signal. One such "gain-of-function" mutation in the ERK2 protein, called a "sevenmaker" mutation (D319N), was discovered in 1994 in Drosophila. The mutation leads to disruption of interactions of other proteins with the D-site of ERK2 and results, contrary to expectations, in an increase of its activity in vivo. However, no molecular mechanism to explain this effect has been presented so far. The difficulty is that this mutation should equally negatively affect interactions of ERK2 with all substrates, activators, and deactivators. In this paper, we present a semiquantitative kinetic network model that gives a possible explanation of the increased activity of mutant ERK2 species. A simplified biochemical network for ERK2, viewed as a system of coupled Michaelis-Menten processes, is presented. Its dynamic properties are calculated explicitly using the method of first-passage processes. The effect of mutation is associated with changes in the strength of interaction energy between the enzyme and the substrates. It is found that the dependence of kinetic properties of the protein on the interaction energy is nonmonotonic, suggesting that some mutations might lead to more efficient catalytic properties, despite weakening intermolecular interactions. Our theoretical predictions agree with experimental observations for the sevenmaker mutation in ERK2. It is also argued that the effect of mutations might depend on the concentrations of substrates.

Isobavachalcone exerts anti­proliferative and pro­apoptotic effects on human liver cancer cells by targeting the ERKs/RSK2 signaling pathway.

Aberrant activation of the extracellular signal­regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer­aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP­competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit­8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase­dependent mitochondria pathway. The computer prediction was confirmed with pull­down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose­dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor­induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element­binding protein, activating transcription factor 1, histone H3 and activating protein­1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC­induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.

Computational Models for Activated Human MEK1: Identification of Key Active Site Residues and Interactions.

MEK1 is a protein kinase in the MAPK cellular signaling pathway that is notable for its dual specificity and its potential as a drug target for a variety of cancer therapies. While much is known about the key role of MEK1 in signaling events, understanding of the structural features that sustain MEK1 function remains limited because of the absence of crystal or NMR structural insights into the phosphorylated and activated form of MEK1. In this work, homology modeling was used to overcome this limitation and generate computational models of the doubly phosphorylated active MEK1 conformation. A variety of models were generated using crystal structures of active protein kinases as homology model templates. These models were equilibrated using molecular dynamics simulations, and each model was validated against several known structural characteristics of activated kinases. The best model structures were used in docking studies with ATP and a small peptide sequence that represents the activation loop of ERK2 to identify the most important residues in stabilizing protein docking and phosphorylation. These results provide insights for the pursuit of structure-guided mutagenesis and drug design.

Ligand-induced activation of ERK1/2 signaling by constitutively active Gs-coupled 5-HT receptors.

5-HT4R, 5-HT6R, and 5-HT7AR are three constitutively active Gs-coupled 5-HT receptors that have key roles in brain development, learning, memory, cognition, and other physiological processes in the central nervous system. In addition to Gs signaling cascade mediated by these three 5-HT receptors, the ERK1/2 signaling which is dependent on cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation downstream of Gs signaling has also been widely studied. In this study, we investigated these two signaling pathways originating from the three Gs-coupled 5-HT receptors in AD293 cells. We found that the phosphorylation and activation of ERK1/2 are ligand-induced, in contrast to the constitutively active Gs signaling. This indicates that Gs signaling alone is not sufficient for ERK1/2 activation in these three 5-HT receptors. In addition to Gs, we found that ß-arrestin and Fyn are essential for the activation of ERK1/2. Together, these results put forth a novel mechanism for ERK1/2 activation involving the cooperative action of Gs, ß-arrestin, and Fyn.

Quantitative proteomic analyses of dynamic signalling events in cortical neurons undergoing excitotoxic cell death.

Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death. Our data, available in ProteomeXchange with identifier PXD008353, identified over 100 changed proteins exhibiting significant alterations in abundance and/or phosphorylation levels at different time points (5-240 min) in neurons after glutamate overstimulation. Bioinformatic analyses predicted that many of them are components of signalling networks directing defective neuronal morphology and functions. Among them, the well-known neuronal survival regulators including mitogen-activated protein kinases Erk1/2, glycogen synthase kinase 3 (GSK3) and microtubule-associated protein (Tau), were selected for validation by biochemical approaches, which confirmed the findings of the proteomic analysis. Bioinformatic analysis predicted Protein Kinase B (Akt), c-Jun kinase (JNK), cyclin-dependent protein kinase 5 (Cdk5), MAP kinase kinase (MEK), Casein kinase 2 (CK2), Rho-activated protein kinase (Rock) and Serum/glucocorticoid-regulated kinase 1 (SGK1) as the potential upstream kinases phosphorylating some of the changed proteins. Further biochemical investigation confirmed the predictions of sustained changes of the activation states of neuronal Akt and CK2 in excitotoxicity. Thus, future investigation to define the signalling pathways directing the dynamic alterations in abundance and phosphorylation of the identified changed neuronal proteins will help elucidate the molecular mechanism of neuronal death in excitotoxicity.

Design, synthesis and biological evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-mutant melanoma.

Approximately 60% of human cancers exhibit enhanced activity of ERK1 and ERK2, reflecting their multiple roles in tumor initiation and progression. Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma and several other cancers. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Herein, we report the design and synthesis of novel series of fused naphthofuro[3,2-c]quinoline-6,7,12-triones 3a-f and pyrano[3,2-c]quinoline-6,7,8,13-tetraones 5a,b and 6, as potential ERK inhibitors. New inhibitors were synthesized and identified by different spectroscopic techniques and X-ray crystallography. They were evaluated for their ability to inhibit ERK1/2 in an in vitro radioactive kinase assay. 3b and 6 inhibited ERK1 with IC50s of 0.5 and 0.19 µM, and inhibited ERK2 with IC50s of 0.6 and 0.16 µM respectively. Kinetic mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where 6 inhibited ERK2 with a Ki of 0.09 µM. Six of the new inhibitors were tested for their in vitro anticancer activity against the NCI-60 panel of tumor cell lines. Compound 3b and 6 were the most potent against most of the human tumor cell lines tested. Moreover, 3b and 6 inhibited the proliferation of the BRAF mutant A375 melanoma cells with IC50s of 3.7 and 0.13 µM, respectively. In addition, they suppressed anchorage-dependent colony formation. Treatment of the A375 cell line with 3b and 6 inhibited the phosphorylation of ERK substrates p-90RSK and ELK-1 and induced apoptosis in a dose dependent manner. Finally, a molecular docking study showed the potential binding mode of 3b and 6 within the ATP catalytic binding site of ERK2.

Antitumor effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study.

BACKGROUND: Berberine is an alkaloid plant-based DNA intercalator that affects gene regulation, particularly expression of oncogenic and tumor suppressor proteins. The effects of berberine on different signaling proteins remains to be elucidated. The present study aimed to identify the effects of berberine against key oncogenic proteins in breast cancer cells. METHODS: Molecular docking and molecular dynamics simulations were used for EGFR, p38, ERK1/2, and AKT. The effects of berberine and lapatinib on MAPK and PI3K pathways in MDA-MB231 and MCF-7 cells were evaluated using immunoflorescence assays, and the amounts of phosphorylated kinases were compared to total kinases after treating with different concentrations of berberine. RESULTS: Simulations showed berberine accurately interacted with EGFR, AKT, P38, and ERK1/2 active sites in silico (scores = -7.57 to -7.92 Kcal/mol) and decreased the levels of active forms of corresponding enzymes in both cell lines; however, berberine binding to p38 showed less stability. Cytotoxicity analysis indicated that MDA-MB231 cells were resistant to berberine compared to MCF-7 cells [72 h IC50 = 50 versus 15 µM, respectively). Also, lapatinib strongly activated AKT but suppressed EGFR in MDA-MB231 cells. The activity of EGFR, AKT, P38, and ERK1/2 were affected by berberine; however, berberine dramatically reduced EGFR and AKT phosphorylation. CONCLUSION: By way of its multikinase inhibitory effects, berberine might be a useful replacement for lapatinib, an EGFR inhibitor which can cause acquired drug resistance in patients.

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1-Disrupting Peptides.

Protein phosphatase-1 (PP1)-disrupting peptides (PDPs) are selective chemical modulators of PP1 that liberate the active PP1 catalytic subunit from regulatory proteins; thus allowing the dephosphorylation of nearby substrates. We have optimized the original cell-active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23-mer peptide for cellular applications. The optimized PDP-Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we have demonstrated that, in human osteosarcoma (U2OS) cells, phosphoMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of phosphoERK1/2 is indirect and likely mediated through enhanced tyrosine phosphatase activity after PDP-mediated PP1 activation. Thus, as liberators of PP1 activity, PDPs represent a valuable tool for identifying the substrates of PP1 and understanding its role in diverse signaling cascades.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Reversible versus irreversible inhibition modes of ERK2: a comparative analysis for ERK2 protein kinase in cancer therapy.

AIM: Irreversible covalent drug inhibition is an emerging paradigm; however, critical gaps in unraveling the efficacy of molecular determinants still persist. METHODOLOGY: We compare two ERK2 inhibitors with different binding modes. A 5-7-Oxozeaenol is selective inhibitor which irreversibly binds ERK2 by the formation of covalent bond with Cys166 while 5-iodotubercidin binds noncovalently. Result & discussion: Covalent inhibition showed greater protein stability, favorable binding energetics (irreversible inhibition binding free energy [ΔGbind] = -40.4354 kcal/mol and reversible inhibition ΔGbind = -26.2515 kcal/mol); higher correlation in residual movement and multiple van der Waals interactions as evident from residue interaction analysis. CONCLUSION: This investigation of the different inhibition modes of ERK2 would assist toward the design of more potent and highly site-specific covalent inhibitors in cancer therapy.

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase.

19F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments.