Garrick Paul Smith

Discovery of 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): a novel multimodal compound for the treatment of major depressive disorder.

The synthesis and structure-activity relationship of a novel series of compounds with combined effects on 5-HT(3A) and 5-HT(1A) receptors and on the serotonin (5-HT) transporter (SERT) are described. Compound 5m (Lu AA21004) was the lead compound, displaying high affinity for recombinant human 5-HT(1A) (K(i) = 15 nM), 5-HT(1B) (K(i) = 33 nM), 5-HT(3A) (K(i) = 3.7 nM), 5-HT(7) (K(i) = 19 nM), and noradrenergic β(1) (K(i) = 46 nM) receptors, and SERT (K(i) = 1.6 nM). Compound 5m displayed antagonistic properties at 5-HT(3A) and 5-HT(7) receptors, partial agonist properties at 5-HT(1B) receptors, agonistic properties at 5-HT(1A) receptors, and potent inhibition of SERT. In conscious rats, 5m significantly increased extracellular 5-HT levels in the brain after acute and 3 days of treatment. Following the 3-day treatment (5 or 10 (mg/kg)/day) SERT occupancies were only 43% and 57%, respectively. These characteristics indicate that 5m is a novel multimodal serotonergic compound, and 5m is currently in clinical development for major depressive disorder.

Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats

RationaleA growing body of evidence suggests that negative modulation of γ-aminobutyric acid (GABA) GABAA α5 receptors may be a promising strategy for the treatment of certain facets of cognitive impairment; however, selective modulators of GABAA α5 receptors have not yet been tested in “schizophrenia-relevant” cognitive assay/model systems in animals.ObjectivesThe objectives of this study were to investigate the potential of RO4938581, a negative modulator of GABAA α5 receptors, and to attenuate cognitive impairments induced following sub-chronic (sub-PCP) and early postnatal PCP (neo-PCP) administration in the novel object recognition (NOR) and intra-extradimensional shift (ID/ED) paradigms in rats. Complementary in vitro, ex vivo and in vivo studies were performed to confirm negative modulatory activity of RO4938581 and to investigate animal model validity, concept validity and potential side effect issues, respectively.ResultsIn vitro studies confirmed the reported negative modulatory activity of RO4938581, whilst immunohistochemical analyses revealed significantly reduced parvalbumin-positive cells in the prefrontal cortex of sub-PCP- and neo-PCP-treated rats. RO4938581 (1xa0mg/kg) ameliorated both sub-PCP- and neo-PCP-induced cognitive deficits in NOR and ID/ED performance, respectively. In contrast, QH-II-066 (1 and 3xa0mg/kg), a GABAA α5 receptor positive modulator, impaired cognitive performance in the NOR task when administered to vehicle-treated animals. Additional studies revealed that both RO4938581 (1xa0mg/kg) and QH-II-066 (1 and 3xa0mg/kg) attenuated amphetamine-induced hyperactivity in rats.ConclusionsTaken together, these novel findings suggest that negative modulation of GABAA α5 receptors may represent an attractive treatment option for the cognitive impairments, and potentially positive symptoms, associated with schizophrenia.

Discovery and SAR of a Series of Agonists at Orphan G Protein-Coupled Receptor 139

GPR139 is an orphan G-protein coupled receptor (GPCR) which is primarily expressed in the central nervous system (CNS). In order to explore the biological function of this receptor, selective tool compounds are required. A screening campaign identified compound 1a as a high potency GPR139 agonist with an EC50 = 39 nM in a calcium mobilization assay in CHO-K1 cells stably expressing the GPR139 receptor. In the absence of a known endogenous ligand, the maximum effect was set as 100% for 1a. Screening against 90 diverse targets revealed no cross-reactivity issues. Assessment of the pharmacokinetic properties showed limited utility as in vivo tool compound in rat with a poor whole brain exposure of 61 ng/g and a brain/plasma (b/p) ratio of 0.03. Attempts to identify a more suitable analogue identified the des-nitrogen analogue 1s with a reduced polar surface area of 76.7 Å(2) and an improved b/p ratio of 2.8. The whole brain exposure remained low at 95 ng/g due to a low plasma exposure.

Abnormal visual gain control in a Parkinson's disease model

Our understanding of Parkinsons disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor ‘BMPPB-32’. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK− null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.

Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson’s disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50’s equivalent to IC50’s on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

Asc-1 Transporter Regulation of Synaptic Activity via the Tonic Release of d-Serine in the Forebrain

Abstract d‐Serine is a co‐agonist of NMDA receptors (NMDARs) whose activity is potentially regulated by Asc‐1 (SLC7A10), a transporter that displays high affinity for d‐serine and glycine. Asc‐1 operates as a facilitative transporter and as an antiporter, though the preferred direction of d‐serine transport is uncertain. We developed a selective Asc‐1 blocker, Lu AE00527, that blocks d‐serine release mediated by all the transport modes of Asc‐1 in primary cultures and neocortical slices. Furthermore, d‐serine release is reduced in slices from Asc‐1 knockout (KO) mice, indicating that d‐serine efflux is the preferred direction of Asc‐1. The selectivity of Lu AE00527 is assured by the lack of effect on slices from Asc‐1‐KO mice, and the lack of interaction with the co‐agonist site of NMDARs. Moreover, in vivo injection of Lu AE00527 in P‐glycoprotein‐deficient mice recapitulates a hyperekplexia‐like phenotype similar to that in Asc‐1‐KO mice. In slices, Lu AE00527 decreases the long‐term potentiation at the Schaffer collateral‐CA1 synapses, but does not affect the long‐term depression. Lu AE00527 blocks NMDAR synaptic potentials when typical Asc‐1 extracellular substrates are present, but it does not affect AMPAR transmission. Our data demonstrate that Asc‐1 mediates tonic co‐agonist release, which is required for optimal NMDAR activation and synaptic plasticity.

Synthesis and Application of a New Fluorous-Tagged Ammonia Equivalent

A novel fluorous-tagged ammonia equivalent has been developed. It is based on a nitrogen-oxygen bond, which can be cleaved in a traceless manner by a molybdenum complex or samarium diiodide. The application in the synthesis of ureas, amides, sulfonamides, and carbamates is described. The scope of the fluorous N-O linker is exemplified by the synthesis of itopride, a drug used for the treatment of functional dyspepsia. Itopride was synthesized with the aid of fluorous purification methods and the product was isolated in good overall yield, with high purity.

High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139

Aim:To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules.Methods:Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies.Results:Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds.Conclusion:The findings provide important tools for further study of this orphan G-protein coupled receptor.

Protection of Primary Dopaminergic Midbrain Neurons by GPR139 Agonists Supports Different Mechanisms of MPP+ and Rotenone Toxicity

The G-protein coupled receptor 139 (GPR139) is expressed specifically in the brain in areas of relevance for motor control. GPR139 function and signal transduction pathways are elusive, and results in the literature are even contradictory. Here, we examined the potential neuroprotective effect of GPR139 agonism in primary culture models of dopaminergic (DA) neuronal degeneration. We find that in vitro GPR139 agonists protected primary mesencephalic DA neurons against 1-methyl-4-phenylpyridinium (MPP+)-mediated degeneration. Protection was concentration-dependent and could be blocked by a GPR139 antagonist. However, the protection of DA neurons was not found against rotenone or 6-hydroxydopamine (6-OHDA) mediated degeneration. Our results support differential mechanisms of toxicity for those substances commonly used in Parkinson’s disease (PD) models and potential for GPR139 agonists in neuroprotection.

Development of LRRK2 Inhibitors for the Treatment of Parkinson's Disease

Linkage and genome-wide association studies have identified a genetic risk locus for late-onset Parkinsons disease in chromosome 12, originally identified as PARK6. The causative gene was identified to code for a large multifunctional protein, LRRK2 (leucine-rich repeat kinase 2). The combined genetic and biochemical evidence supports a hypothesis in which the LRRK2 kinase function is causally involved in the pathogenesis of sporadic and familial forms of PD, and therefore that LRRK2 kinase inhibitors could be useful for treatment. Although LRRK2 has so far not been crystallised, the use of homology modelling and crystallographic surrogates has allowed the optimisation of chemical structures such that compounds of high selectivity with good brain penetration and appropriate pharmacokinetic properties are now available for understanding the biology of LRRK2 in vitro and in vivo. This chapter reviews LRRK2 biology, the structural biology of LRRK2 and gives an overview of inhibitors of LRRK2.