George McAllister

Behavioral and Neurochemical Alterations in Mice Deficient in Anaplastic Lymphoma Kinase Suggest Therapeutic Potential for Psychiatric Indications

The receptor tyrosine kinase product of the anaplastic lymphoma kinase (ALK) gene has been implicated in oncogenesis as a product of several chromosomal translocations, although its endogeneous role in the hematopoietic and neural systems has remained poorly understood. We describe that the generation of animals homozygous for a deletion of the ALK tyrosine kinase domain leads to alterations in adult brain function. Evaluation of adult ALK homozygotes (HOs) revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain. ALK HO animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object-recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. Altogether, these results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia and depression, with an underlying deregulated monoaminergic signalling.

Design, Synthesis, and Biological Evaluation of Potent and Selective Class IIa Histone Deacetylase (HDAC) Inhibitors as a Potential Therapy for Huntington’s Disease

Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntingtons disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a closed-loop form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.

A Monoclonal Antibody TrkB Receptor Agonist as a Potential Therapeutic for Huntington’s Disease

Huntington’s disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

Identification of signal transduction pathways used by orphan g protein-coupled receptors.

The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.

Edg2 receptor distribution in adult rat brain.

The Edg (endothelial differentiation gene) receptors are recently discovered G-protein coupled receptors which are activated by endogenous lysophospholipids. The cellular activities mediated by Edg receptors are reminiscent of those normally associated with Trk receptor activation and include modulation of cell growth, differentiation, proliferation and migration as well as apoptotic and cytoskeletal effects. In this study we have investigated immunohistochemically the distribution of one family member, the Edg2 receptor, within the adult rat brain and shown the protein expression to be most prominent in white matter tract regions. This suggests a possible role for the Edg2 receptor in nerve cell myelination.

Native and Recombinant Human Edg4 Receptor-Mediated Ca2+ Signalling

We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptors downstream effects.

Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors.

Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.

L19 Optimisation of potent, selective and brain penetrant atm inhibitors as potential therapeutic agents for HD

Emerging evidence shows that the ATM signalling pathway is dysregulated in neurodegenerative disorders including Huntington’s disease (HD). ATM signalling has been shown to be elevated in cells derived from HD patients and mouse models of HD. Genetic and pharmacological evidence from cellular and animal models of HD suggests that reduction of ATM signalling can ameliorate mHTT toxicity. Thus inhibitor of ATM kinase presents a promising therapeutic intervention strategy for the treatment of HD. We present data from our medicinal chemistry program which aims to develop potent, selective and brain-penetrant ATM inhibitors. Chemical optimisation of CNS-compliant physical chemical properties led to the identification of compounds suitable for a proof-of-concept study in HD models. Our lead compound, CHDI-00485194, displayed excellent oral bioavailability and pharmacokinetics. PO administration to mice showed distribution into brain (brain:plasma ratio of 1.3) and linear pharmacokinetics in a dose escalation study. An acute PK/PD biomarker study to evaluate the effect of CHDI-00485194 on irradiation induced DNA damage biomarkers showed a significant dose-dependent inhibition of KAP1 phosphorylation by CHDI-00485194 at 30 and 60 minutes post-irradiation.