MAPK-Activated Protein Kinases: Servant or Partner?

Mitogen-activated protein kinase (MAPK)-activated protein kinases (MAPKAPKs) are defined by their exclusive activation by MAPKs. They can be activated by classical and atypical MAPKs that have been stimulated by mitogens and various stresses. Genetic deletions of MAPKAPKs and availability of highly specific small-molecule inhibitors have continuously increased our functional understanding of these kinases. MAPKAPKs cooperate in the regulation of gene expression at the level of transcription; RNA processing, export, and stability; and protein synthesis. The diversity of stimuli for MAPK activation, the crosstalk between the different MAPKs and MAPKAPKs, and the specific substrate pattern of MAPKAPKs orchestrate immediate-early and inflammatory responses in space and time and ensure proper control of cell growth, differentiation, and cell behavior. Hence, MAPKAPKs are promising targets for cancer therapy and treatments for conditions of acute and chronic inflammation, such as cytokine storms and rheumatoid arthritis.

Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling.

BACKGROUND: Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. METHODS: We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. RESULTS: The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. CONCLUSIONS: The model can be used to systematically motivate which dabrafenib-trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

Structural basis for the action of the drug trametinib at KSR-bound MEK.

The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry.

Mitogen-activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three-tiered kinase module-MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase-that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine-glutamic acid-tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal-regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)-selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID-SRM-MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono-phosphorylatable mutants ERKT202A and ERKY204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.

Cell growth inhibition by 3-deoxysappanchalcone is mediated by directly targeting the TOPK signaling pathway in colon cancer.

BACKGROUND: Colorectal cancer is one of the most common causes of cancer death worldwide. Unfortunately, chemotherapies are limited due to many complications and development of resistance and recurrence. The T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays an important role in inflammation, proliferation, and survival of cancer cells. Therefore, suppressing TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy against colon cancers. PURPOSE: 3-Deoxysappanchalcone (3-DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In this study, we investigated the effects of 3-DSC on colon cancer cell growth and elucidated its underlying molecular mechanism of targeting TOPK. STUDY DESIGN AND METHODS: To evaluate the effects of 3-DSC against colon cancer, we performed cell proliferation assays, propidium iodide- and annexin V-staining analyses and Western blotting. Targeting TOPK by 3-DSC was identified by a kinase-binding assay and computational docking models. RESULTS: 3-DSC inhibited the kinase activity of TOPK, but not mitogen-activated protein kinase (MEK). The direct binding of 3-DSC with TOPK was explored using a computational docking model and binding assay in vitro and ex vivo. 3-DSC inhibited colon cancer cell proliferation and anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with 3-DSC induced expression of protein that are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3, and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK), and c-Jun, which are regulated by the upstream kinase, TOPK. CONCLUSION: 3-DSC suppresses colon cancer cell growth by directly targeting the TOPK- mediated signaling pathway.

In silico docking studies of Lupeol with MAPK pathway proteins- Raf-1, MEK & ERK.

OBJECTIVE: Lupeol, A triterpenoid found in variety of plants is reported to have beneficial medicinal effects on several ailments. Lupeol is also found to show inhibitory effect on proliferation of breast cancer cells. Metastasis is considered to be a major cause for worldwide deaths related to cancer. Ras related MAPK Signaling Pathway is one of the crucial pathways leading to metastasis. Lupeols binding possibility with Ras is already reported. In present study, Interaction between with downstream proteins of Ras- MAPK pathway, Raf ,MEK ,ERK1/2 and their corresponding domains are studied using STRING Database and their structures are retrieved in PDB Format. Lupeols binding affinity with downstream proteins of these signaling proteins at their interacting domains are analyzed. Here in silico docking approach to identify binding sites of each of these proteins with Lupeol is used. FDA approved standard drug molecule CH5126766 was used as reference ligand. Lupeol shows potent binding at significant sites with extremely high affinity. Since it binds with all the proteins involved in the pathway with high efficiency it is an important compound which can be developed as a therapeutic molecule.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

ADA-07 Suppresses Solar Ultraviolet-Induced Skin Carcinogenesis by Directly Inhibiting TOPK.

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the MAPK cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell-originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK. Mol Cancer Ther; 16(9); 1843-54. ©2017 AACR.

Glycycoumarin exerts anti-liver cancer activity by directly targeting T-LAK cell-originated protein kinase.

Glycycoumarin (GCM) is a major bioactive coumarin compound isolated from licorice and the anti-cancer activity of GCM has not been scientifically addressed. In the present study, we have tested the anti-liver cancer activity of GCM using both in vitro and in vivo models and found for the first time that GCM possesses a potent activity against liver cancer evidenced by cell growth inhibition and apoptosis induction in vitro and tumor reduction in vivo. Mechanistically, GCM was able to bind to and inactivate oncogenic kinase T-LAK cell-originated protein kinase (TOPK), which in turn led to activation of p53 pathway. Our findings supported GCM as a novel active compound that contributed to the anti-cancer activity of licorice and TOPK could be an effective target for hepatocellular carcinoma (HCC) treatment.

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases.

Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.

The crystal structure of an inactive dimer of PDZ-binding kinase.

The overexpression of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) has been associated with hematologic tumors, breast cancer and various other cancers. However, the three-dimensional structure of PBK has not been solved. In this study, we determined the crystal structure of human PBK, which has two phospho-mimicking mutations T9E and T198E. The structural data indicated that PBK may assemble into an inactive dimer in alkaline conditions. Analytical size-exclusion chromatography and analytical ultracentrifugation confirmed that PBK exists in a conformational transition between dimers and monomers at different pH conditions. Co-IP and kinase assays suggested that the active state of PBK is a monomer and does not form a dimer even under alkaline conditions. These results showed that the conformational transition of PBK is important for its kinase activity regulation. Collectively, our observations may provide a novel starting point for structure-based functional studies.

Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades, which consist of three functionally associated protein kinases, namely MEKKs, MKKs and MPKs, are universal signaling modules in all eukaryotes and have been shown to play critical roles in many physiological and biochemical processes in plants. However, little or nothing is known about the MPK and MKK families in watermelon. RESULTS: In the present study, we performed a systematic characterization of the ClMPK and ClMKK families including the identification and nomenclature, chromosomal localization, phylogenetic relationships, ClMPK-ClMKK interactions, expression patterns in different tissues and in response to abiotic and biotic stress and transient expression-based functional analysis for their roles in disease resistance. Genome-wide survey identified fifteen ClMPK and six ClMKK genes in watermelon genome and phylogenetic analysis revealed that both of the ClMPK and ClMKK families can be classified into four distinct groups. Yeast two-hybrid assays demonstrated significant interactions between members of the ClMPK and ClMKK families, defining putative ClMKK2-1/ClMKK6-ClMPK4-1/ClMPK4-2/ClMPK13 and ClMKK5-ClMPK6 cascades. Most of the members in the ClMPK and ClMKK families showed differential expression patterns in different tissues and in response to abiotic (e.g. drought, salt, cold and heat treatments) and biotic (e.g. infection of Fusarium oxysporum f. sp. niveum) stresses. Transient expression of ClMPK1, ClMPK4-2 and ClMPK7 in Nicotiana benthamiana resulted in enhanced resistance to Botrytis cinerea and upregulated expression of defense genes while transient expression of ClMPK6 and ClMKK2-2 led to increased susceptibility to B. cinerea. Furthermore, transient expression of ClMPK7 also led to hypersensitive response (HR)-like cell death and significant accumulation of H2O2 in N. benthamiana. CONCLUSION: We identified fifteen ClMPK and six ClMKK genes from watermelon and analyzed their phylogenetic relationships, expression patterns and protein-protein interactions and functions in disease resistance. Our results demonstrate that ClMPK1, ClMPK4-2 and ClMPK7 positively but ClMPK6 and ClMKK2-2 negatively regulate the resistance to B. cinerea when transiently expressed in N. benthamiana and that ClMPK7 functions as a regulator of HR-like cell death through modulating the generation of H2O2.

The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins.

BACKGROUND: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. RESULTS: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. CONCLUSIONS: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.

In silico identification of novel kinase inhibitors by targeting B-Raf(v660e) from natural products database.

The Ras/Raf/MEK/ERK (MAPK) signaling pathway has gained much attention from scientific community for therapeutic intervention in the past decades, specifically in oncology. Notably, a most prevalent B-Raf(v600e) mutant in Raf kinase family exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, virtual screening is applied to identify its potential inhibitors. Following the 25 ns molecular dynamic (MD) simulations, ZINC38541768, ZINC38541767, and ZINC12496469 are identified as B-Raf(v600e) potential inhibitors in a DFG-in conformation. Furthermore, according to the molecular mechanics/generalized born surface area (MM/GBSA) method, these three small molecules exhibit similar and good binding affinity toward B-Raf(v600e) (-38.76 kcal mol(-1), -42.60 kcal mol(-1), and -39.04 kcal mol(-1)). At the same time, several critical residues, such as I463, V471 in the P-loop, and DFG motif residue D594 within the A-loop, are also well clarified. All these results may not only indicate some future applications of inhibitors targeting B-Raf(v600e), but also benefit B-Raf(v600e) harboring cancer patients.

Bilberry-derived anthocyanins prevent IFN-γ-induced pro-inflammatory signalling and cytokine secretion in human THP-1 monocytic cells.

BACKGROUND/AIMS: Anthocyanins are plant-derived dietary components that are highly abundant, for example, in bilberries. We have previously demonstrated that anthocyanins exert anti-inflammatory properties in mouse colitis models and ameliorate disease activity in ulcerative colitis patients. Here, we studied the molecular mechanisms through which anthocyanin-containing bilberry extract (BE) exerts anti-inflammatory effects in human monocytic THP-1 cells. METHODS: THP-1 cells were pre-incubated with BE 20 min prior to TNF-α or IFN-γ (100 ng/ml each) stimulation. Signalling protein activation was studied by Western blotting, mRNA expression by quantitative PCR and cytokine secretion by ELISA. RESULTS: IFN-γ-induced phosphorylation of STAT1 and STAT3 was significantly reduced by BE co-treatment. Consequently, levels of mRNA expression and/or cytokine secretion of MCP-1, IL-6, TNF-α, ICAM-1, and T-bet were lower with BE co-treatment. In contrast, BE enhanced TNF-α-mediated p65-NF-κB phosphorylation but reduced ERK1/2 phosphorylation. BE co-treatment further increased TNF-α-induced mRNA expression and secretion of NF-κB target genes, such as IL-6, IL-8, and MCP-1, while mRNA levels of ICAM-1 were reduced. CONCLUSIONS: BE co-treatment reduced IFN-γ-induced signal protein activation, pro-inflammatory gene expression, and cytokine secretion, whereas it enhanced TNF-α-induced responses. These findings suggest a distinct role for anthocyanins in modulating inflammatory responses that need to be further studied to fully understand anthocyanin-mediated effects.

Comparative molecular dynamics simulations of mitogen-activated protein kinase-activated protein kinase 5.

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

In silico structural and functional analysis of the human TOPK protein by structure modeling and molecular dynamics studies.

Over expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) has been associated with leukemia, myeloma tumors and various other cancers. The function and regulatory mechanism of TOPK in tumor cells remains unclear. Structural studies that could reveal the regulatory mechanism have been a challenge because of the unavailabity of TOPK's crystal structure. Hence, in this study, the 3D structure of TOPK protein has been constructed by using multiple templates. The quality and reliability of the generated model was checked and the molecular dynamics method was utilized to refine the model. APBS method was employed to know the electrostatic potential surface of the modeled protein and it was found that the optimum pH for protein stability is 3.4 which will further help in mechanistic hypothesis of TOPK protein. Active site of TOPK was identified from available literature and HTVS was employed to identify the lead molecules. The expected binding modes of protein-ligand complexes were reproduced in the MD simulation which indicates that the complex is relatively stable. The pharmacokinetic properties of the lead molecules are also under acceptable range. TOPK act as a substrate for CDK1 and the protein-protein docking and dynamics studies were carried out to analyze the effect of Thr9Ala mutation of TOPK in the two protein complex formation. It shows that the wild type complex is more stable when compared with the mutant type. Such structural information at atomic level not only exhibits the action modes of TOPK inhibitors but also furnishes a novel starting point for structure based drug design of TOPK inhibitors.

193-nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization.

193-nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge-reduced radical products generated by UV photoexcitation were also subjected to collision-induced dissociation (termed, activated-electron photodetachment dissociation (a-EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine-modified mobile phase, LC-MS/UVPD was implemented and resulted in the successful analysis of mitogen-activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).