Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer.

Background: Upregulated expression and aberrant activation of the epidermal growth-factor receptor (EGFR) are found in lung cancer, making EGFR a relevant target for non-small-cell lung cancer (NSCLC). Treatment with anti-EGFR monoclonal antibodies (mAbs) is associated with modest improvement in overall survival in patients with squamous cell lung cancer (SqCLC) who have a significant unmet need for effective treatment options. While there is evidence that using EGFR gene copy number, EGFR mutation, and EGFR protein expression as biomarkers can help select patients who respond to treatment, it is important to consider biomarkers for response in patients treated with combination therapies that include EGFR mAbs. Design: Randomized trials of EGFR-directed mAbs cetuximab and necitumumab in combination with chemotherapy, immunotherapy, or antiangiogenic therapy in patients with advanced NSCLC, including SqCLC, were searched in the literature. Results of associations of potential biomarkers and outcomes were summarized. Results: Data from phase III clinical trials indicate that patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein (H-score of ≥200) and/or gene copy numbers of EGFR (e.g. ≥40% cells with ≥4 EGFR copies as detected by fluorescence in situ hybridization; gene amplification in ≥10% of analyzed cells) derive greater therapeutic benefits from EGFR-directed mAbs. Biomarker data are limited for EGFR mAbs used in combination with immunotherapy and are absent when used in combination with antiangiogenic agents. Conclusions: Therapy with EGFR-directed mAbs in combination with chemotherapy is associated with greater clinical benefits in patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein and/or have increased EGFR gene copy number. These data support validating the role of these as biomarkers to identify those patients who derive the greatest clinical benefit from EGFR mAb therapy. However, data on biomarkers for EGFR-directed mAbs combined with immunotherapy or antiangiogenic agents remain limited.

Clusterin regulates ß-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway.

Although the mechanism of Aß action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aß neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aß/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aß toxicity and DKK1 upregulation and, conversely, Aß increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aß mediates neurotoxicity, we measured the effects of Aß and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aß neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aß-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aß-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aß induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aß in neurodegenerative diseases.

Conformational analysis of the eight-membered ring of the oxidized cysteinyl-cysteine unit implicated in nicotinic acetylcholine receptor ligand recognition.

Nicotinic acetylcholine receptors (nAChRs) are membrane-bound, pentameric ligand-gated ion channels associated with a variety of human disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, and pain. Most known nAChRs contain an unusual eight-membered disulfide-containing cysteinyl-cysteine ring, ox-[Cys-Cys], as does the soluble acetylcholine binding protein (AChBP) found in the snail Lymnaea stagnalis. The cysteinyl-cysteine ring is located in a region implicated in ligand binding, and conformational changes involving this ring may be important for modulation of nAChR function. We have studied the preferred conformations of Ac-ox-[Cys-Cys]-NH2 by NMR in water and computationally by Monte Carlo simulations using the OPLS-AA force field and GB/SA water model. ox-[Cys-Cys] adopts four distinct low-energy conformers at slightly above 0 degrees C in water. Two populations are dependent on the peptide omega2 dihedral angle, with the trans amide favored over the cis amide by a ratio of ca. 60:40. Two ox-[Cys-Cys] conformers with a cis amide bond (C+ and C-) differ from each other primarily by variation of the chi3 dihedral angle, which defines the orientation of the helicity about the S-S bond (+/- 90 degrees ). Two trans amide conformers have the same S-S helicity (chi3 approximately -90 degrees ), but are distinguished by a backbone rotation about phi2 and psi1 (T- and T'-). The ratio of T-/T'-/C+/C- is 47:15:29:9. The orientation of the pendant moieties from the eight-membered ring is more compact for the major trans conformer (T-) than for the extended conformations adopted by T'-, C+, and C-. These conformational preferences are also observed in tetrapeptide and undecapeptide fragments of the human alpha7 subtype of the nAChR that contains the ox-[Cys-Cys] unit. Conformer T- is nearly identical to the conformation seen in the X-ray structure of ox-[Cys(187)-Cys(188)] found in the unliganded AChBP, and is a Type VIII beta-turn.

Diversity and coverage of structural sublibraries selected using the SAGE and SCA algorithms.

It is often impractical to synthesize and test all compounds in a large exhaustive chemical library. Herein, we discuss rational approaches to selecting representative subsets of virtual libraries that help direct experimental synthetic efforts for diverse library design. We compare the performance of two stochastic sampling algorithms, Simulating Annealing Guided Evaluation (SAGE; Zheng, W.; Cho, S. J.; Waller, C. L.; Tropsha, A. J. Chem. Inf. Comput. Sci. 1999, 39, 738-746.) and Stochastic Cluster Analysis (SCA; Reynolds, C. H.; Druker, R.; Pfahler, L. B. Lead Discovery Using Stochastic Cluster Analysis (SCA): A New Method for Clustering Structurally Similar Compounds J. Chem. Inf. Comput. Sci. 1998, 38, 305-312.) for their ability to select both diverse and representative subsets of the entire chemical library space. The SAGE and SCA algorithms were compared using u- and s-optimal metrics as an independent assessment of diversity and coverage. This comparison showed that both algorithms were capable of generating sublibraries in descriptor space that are diverse and give reasonable coverage (i.e. are representative) of the original full library. Tests were carried out using simulated two-dimensional data sets and a 27 000 compound proprietary structural library as represented by computed Molconn-Z descriptors. One of the key observations from this work is that the algorithmically simple SCA method is capable of selecting subsets that are comparable to the more computationally intensive SAGE method.

Sites of phosphorylation in tau and factors affecting their regulation.

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

Clinical efficacy of rhIL-11.

Placebo-controlled clinical trials of recombinant human interleukin-11 (rhIL-11, also known as oprelvekin [Neumega]) in patients with nonmyeloid malignancies have demonstrated significant efficacy in preventing postchemotherapy platelet nadirs of < or = 20,000/microL, and reducing the need for platelet transfusions while continuing chemotherapy without dose reductions. The likelihood of requiring a platelet transfusion in rhIL-11-treated patients receiving chemotherapy is approximately 40% lower than the risk for untreated patients. Treatment with rhIL-11 appears to accelerate earlier recovery to platelet counts of 20,000/microL, 50,000/microL, and 100,000/microL, suggesting that patients treated with rhIL-11 are more likely to be able to receive their next chemotherapy cycle in a timely fashion. rhIL-11 shows sustained efficacy over multiple cycles of full-dose chemotherapy. Activity has also been demonstrated in pediatric patients with solid tumors. The use of rhIL-11 in combination with granulocyte colony-stimulating factor (G-CSF, filgrastim [Neupogen]) may also produce a synergistic hematopoietic effect, resulting in earlier neutrophil recovery. The recommended adult dose regimen for rhIL-11 is 50 micrograms/kg administered subcutaneously once daily beginning 6 to 24 hours after the administration of chemotherapy until a postnadir platelet count of > or = 50,000/microL is reached. The recommended pediatric dose of rhIL-11 is 75 micrograms/kg subcutaneously, once daily beginning 6 to 24 hours after the administration of chemotherapy until a postnadir platelet count of > or = 50,000/microL is reached. The administration of rhIL-11 for greater than 21 days has not been studied and therefore is not recommended.

GB/SA water model for the Merck molecular force field (MMFF).

A revised generalized Born/surface area (GB/SA) continuum solvation model has been developed for water that is compatible with the Merck molecular force field (MMFF). This model gives free energies of aqueous solvation that are comparable in accuracy to the original water model when the OPLS* force field is employed. The average unsigned error in aqueous deltaGsol using the new water model and MMFF is 0.62 kcal/mol for a training set of 82 solutes compared to 1.24 kcal/mol for the original GB/SA water model and MMFF. The average unsigned errors for 47 neutral solutes outside the training set and 10 ions are 0.96 and 2.32 kcal/mol, respectively. By comparison, the average errors for the test set and ions using the original GB/SA water model are 1.76 and 5.32 kcal/mol. This revised parameter set provides a more accurate representation of aqueous solvation for use with MMFF.

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta.

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.

Tau phosphorylation both in vitro and in cells.

Tau was originally isolated from brain microtubules and shown to be a microtubule-associated protein (MAP) that promoted tubulin polymerization (1). It is largely confined to axons, where it is the major MAP. It promotes microtubule nucleation, elongation, and bundling, and stabilizes microtubules by inhibiting depolymerization.

Improved AMBER torsional parameters for the N-N rotational barrier in diacylhydrazines.

The structure and rotational barrier for substituted diacylhydrazines are of significant interest given the role this functionality plays in peptidomimetics and ecdysone agonists, the latter of which have application as extremely selective insecticides. Ab initio calculations show that the lowest energy conformations are typically nonplanar with essentially perpendicular nitrogen lone pairs. Molecular mechanics calculations using the AMBER force field in MacroModel yield minima and rotational barriers that are both quantitatively and qualitatively inconsistent with the ab initio results. In this work the AMBER N-N rotational barriers for all configurations of the parent, methyl and di-methyl substituted diformylhydrazines have been fitted to MP2/6-31 + G relative energies. The resulting AMBER torsional parameters have been validated by calculating the rotational barriers for N-t-butyl substituted diformylhydrazine, dibenzoylhydrazine and an azadipeptide. In each case the new AMBER rotational barriers compare favorably with the corresponding MP2 calculated rotational barriers.

Effects of peripheral flicker masking on M- and P-pathway response.

PURPOSE: The effects of metahue and metacontrast masking on the responses of the M- and P-pathways of the human visual system were investigated using temporal analysis of the multifocal flash visually evoked potential (VEP) (VERIS system). METHODS: The experiments were carried out at three achromatic luminance contrasts, 24, 70 and 96%, with three peripheral masks: a steady 65% grey mask, a grey flicker mask and an isoluminant red/green flicker mask. The stimuli were viewed monocularly and the mask and VERIS stimuli superposed with a mirror-aperture system. RESULTS/CONCLUSIONS: The masks had no significant effect on the behaviour of the M-pathway as stimulus contrast increased. The amplitude of the P-pathway was found to be approximately level at all contrasts of the stimulus and with all three masks rather than showing the expected steady increase in activity with increasing stimulus contrast.

Reactivating kinase/p38 phosphorylates tau protein in vitro.

Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer-diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated tau protein. Tau from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate tau in vitro; those that require or accept prolines include GSK3 and members of the mitogen-activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase-beta/JNK. In this report, we show that another member of the MAP kinase family, the stress-activated kinase p38/RK, can phosphorylate tau in vitro. Western blots with phosphorylation-sensitive antibodies showed that p38, like ERK2 and SAP kinase-beta/JNK, phosphorylated tau at sites found phosphorylated physiologically (Thr181, Ser202, Thr205, and Ser396) and also at Ser422, which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to tau hyperphosphorylation during the formation of PHFs, and hence, to the development of tau pathology.

Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly.

To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Oxidative stress induces dephosphorylation of tau in rat brain primary neuronal cultures.

Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of beta-amyloid is mediated via free radicals, and as the deposition of beta-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein tau that resemble those found in PHF-tau. Contrary to causing an increase in tau phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of tau and so we conclude that oxidative stress is not the direct cause of tau hyperphosphorylation and hence of PHF formation.

Stress-activated protein kinase/c-jun N-terminal kinase phosphorylates tau protein.

A proportion of the neuronal microtubule-associated protein (MAP) tau is highly phosphorylated in foetal and adult brain, whereas the majority of tau in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate tau at such sites in vitro. We have now shown that purified recombinant stress-activated protein kinase/c-Jun N-terminal kinase, a proline-directed kinase of the MAP kinase extended family, phosphorylates recombinant tau in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation-dependent tau epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of tau protein. Unlike glycogen synthase kinase-3, the c-Jun N-terminal kinase readily phosphorylates Thr205 and Ser422, which are more highly phosphorylated in Alzheimer tau than in foetal or adult tau. Glycogen synthase kinase-3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult tau, whereas stress-activated/c-Jun N-terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline-directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated tau found in neurofibrillary tangles.

Equilibria for the adsorption of antibiotics onto neutral polymeric sorbents: Experimental and modeling studies.

The objective of this work was to study the equilibria for adsorption of three antibiotics (penicillin V, tetracycline, and cephalosporin C) from water onto commercially available neutral polymeric sorbents. The pH was observed to be an important factor in adsorption as our results suggest that the neutral forms of penicillin V and cephalosporin C are preferentially adsorbed onto the neutral sorbents. Also, sorbent surface chemistry was observed to be important for adsorption, as the antibiotics adsorbed more favorably (both in terms of affinities and enthalpies) onto the aromatic sorbent as compared to the aliphatic ester sorbent. In addition to these thermodynamic measurements, molecular modeling studies and Monte Carlo simulations suggest that adsorption onto aromatic sorbents may involve specific interactions between the planar regions of the antibiotic molecules and the phenyl rings of the aromatic sorbent. The interaction energies predicted from Monte Carlo simulations were observed to provide qualitative agreement with experimentally determined adsorption affinities. (c) 1995 John Wiley & Sons, Inc.

Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau.

A proportion of the microtubule-associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimer's disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase-3 beta (GSK-3 beta) when co-transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK-3 beta may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells.

BACKGROUND: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3 alpha, GSK-3 beta and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3 alpha and GSK-3 beta can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3. RESULTS: Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3 alpha or GSK-3 beta decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies. CONCLUSIONS: Our data indicate that GSK-3 alpha and/or GSK-3 beta, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.

Comparison of baculovirus-expressed c-Abl and BCR/ABL protein tyrosine kinases.

Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 microM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.