Scott Hobson

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.

Functional interaction of metabotropic glutamate receptor 5 and NMDA-receptor by a metabotropic glutamate receptor 5 positive allosteric modulator.

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

T40. GPR52 AGONISTS REPRESENT A NOVEL APPROACH TO TREAT UNMET MEDICAL NEED IN SCHIZOPHRENIA

Abstract Background There are currently no treatment options for key symptom domains in certain psychiatric and neurological diseases. For example, antipsychotics effectively treat the positive symptoms of schizophrenia, however both the cognitive impairments associated with schizophrenia (CIAS) and negative symptoms, both key predictors of functional outcome, are not treated by current therapies. Additionally, psychotic symptoms associated with neurological diseases such as Alzheimer’s Disease (AD) are not adequately treated with current antipsychotics. Therefore, novel mechanisms to address these unmet medical needs are urgently required and are under investigation. GPR52 is a Gs-coupled orphan g-protein coupled receptor which has an intriguing pattern of brain expression. In cortex, GPR52 is expressed primarily on glutamatergic neurons and co-localized with the Gs-coupled D1 receptor (D1R). Deficiencies in D1R activation are associated with both cognitive deficit and negative symptoms of schizophrenia. In contrast, in the striatum, GPR52 is almost exclusively co-expressed with the Gi-coupled D2 receptor (D2R), which mediates the reduction in positive symptoms by antipsychotics. Based on GPR52’s functional coupling and co-localization, agonists may be predicted to resemble D1R agonists in cortical regions, thus treating cognitive or negative symptoms, while resembling D2R antagonists in striatal regions. Thus, GPR52 agonists have the potential to provide a novel therapeutic strategy for the currently untreated CIAS and negative symptom domains in addition to the psychotic symptoms of AD. Methods To assess the antipsychotic potential of GPR52 agonists, they were tested for their ability to decrease psychostimulant-induced hyperlocomotion. The efficacy of GPR52 agonists for CIAS and sociability, an aspect of negative symptoms, was assessed in the sub-chronic phencyclidine (scPCP) model for schizophrenia, known to induce long-lasting cognitive and social behaviour deficits, in addition to a reduction in parvalbumin-positive GABAergic interneurons in hippocampus and pre-frontal cortex. Rats were treated with PCP twice daily for 7 days followed by 7 days washout and then tested in the attentional set shifting task (ASST) for executive function and the social interaction test for sociability respectively following treatment with a GPR52 agonist. Results GPR52 agonist 1 dose-dependently reversed psychostimulant-induced hyperlocomotion in rats at doses which were behaviorally quiescent when administered alone. Additionally, GPR52 agonist 2 showed a robust, dose-dependent rescue of scPCP induced deficits in the extra dimensional shift phase of the ASST, achieving significance after a 4 mg/kg p.o. application. Likewise, GPR52 agonist 2 significantly rescued scPCP induced deficits in social interaction at identical doses as in ASST without effects on object exploration or locomotor activity. Discussion GPR52 agonists were efficacious in animal models assessing the three main symptoms domains associated with schizophrenia. Efficacy in ASST and SI demonstrate both pro-cognitive efficacy and restoration of an aspect of negative symptoms, respectively, in a well-established model inducing behavioral and neuropathological deficits associated with schizophrenia. Furthermore, GPR52 agonists reduced psychostimulant-induced hyperlocomotion, an effect associated with antipsychotic efficacy. Taken together, these data demonstrate the potential of this innovative mechanism to simultaneously treat the three core symptoms domains of schizophrenia as well as potentially treat the psychotic symptoms associated with other neurological disorders.

Conditioned contextual fear memory to assess natural forgetting and cognitive enhancement in rats

Aversively established contextual fear memory manifests itself in robust freezing behavior, often lasting several weeks or months. Therefore, this approach is amenable to investigate the underlying neural circuitries by lesion or inactivation of specific brain regions or to test efficacy of substances that disrupt either the ability to acquire the association or to retrieve memories. In contrast, investigation of memory enhancement using this technique is time intensive since the non-treated control group naturally forgets the learned association only weeks after acquisition. Pharmacological interventions have been used to overcome this time span by disrupting memory at any time point, however, limiting it a mechanistic model of reversal of impairments instead of studying memory enhancement. Here, we investigated several parameters of the cued and contextual fear conditioning (CFC) protocol such that, while memory acquisition is established, loss of fear association occurs within a shorter time frame, allowing studies of memory enhancement in the context of natural forgetting. We found that three predictive tone-cues, each separated from a 0.3 mA foot shock by an interstimulus interval of 2 s and a pre-exposure to the context enables the investigation of enhanced contextual memory 7 d post training without the necessity of inducing pharmacological lesions.

Effects of the bace1 inhibitor bi 1181181 and the anti-abeta antibody m266 on abeta in rat brain homogenate and CSF

mice compared with wild-type mice, were significantly attenuated in FMeC1-treated APPswe/PS1dE9 mice. In the in vitro study, both curcumin and FMeC1 modulated the formation of Ab aggregates, however, only FMeC1 significantly attenuated cell toxicity of Ab. Conclusions: These results indicated that FMeC1 treatment inhibited the cognitive deficits and reduced Ab deposition and glial cell activation in the brain in APPswe/PS1dE9 mice. Thus we propose that the C-4 curcumin derivative, FMeC1 may be a useful candidate for preventing AD.

RESTORING LONG-TERM POTENTIATION IMPAIRED BY AMYLOID-BETA OLIGOMERS: COMPARISON OF A BROADLY-SPECIFIC ANTI AMYLOID-BETA NANOBODY AND A MONOMER-SPECIFIC ANTIBODY

P2-399 RESTORING LONG-TERM POTENTIATION IMPAIRED BYAMYLOID-BETA OLIGOMERS: COMPARISON OFA BROADLY-SPECIFIC ANTI AMYLOID-BETA NANOBODYAND A MONOMERSPECIFIC ANTIBODY Katja Kroker, Scott Hobson, Holger Rosenbrock, Thorsten Lamla, Jo Vercammen, Gerald Beste, Pascal Merchiers, Cornelia DornerCiossek, Martin Lenter, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany; Ablynx NV, Zwijnaarde, Gent, Belgium. Contact e-mail: scott.hobson@boehringer-ingelheim.com

Affinity Purification of Proteins Binding to Kinase Inhibitors Immobilized on Self-Assembling Monolayers

Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the ∼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.