mGlu5 positive allosteric modulation normalizes synaptic plasticity defects and motor phenotypes in a mouse model of Rett syndrome.

Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.

Acyl dihydropyrazolo[1,5-a]pyrimidinones as metabotropic glutamate receptor 5 positive allosteric modulators.

We report the optimization of a series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from an acyl dihydropyrazolo[1,5-a]pyrimidinone class. Investigation of exocyclic amide transpositions with this unique 5,6-bicyclic core were conducted in attempt to modulate physicochemical properties and identify a suitable backup candidate with a reduced half-life. A potent and selective PAM, 1-(2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrimidin-4(5H)-yl)ethanone (9a, VU0462807), was identified with superior solubility and efficacy in the acute amphetamine-induced hyperlocomotion (AHL) rat model with a minimum effective dose of 3mg/kg. Attempts to mitigate oxidative metabolism of the western phenoxy of 9a through extensive modification and profiling are described.

Chemical inhibition of fatty acid absorption and cellular uptake limits lipotoxic cell death.

Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic ß-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC50s between 3 and 6µM for all cell lines except human adipocytes (39µM). Inhibition was specific for long and very long chain fatty acids but had no effect on medium chain fatty acids (C6-C10), which are transported by passive diffusion. Derivatives of Lipofermata were evaluated to understand structural contributions to activity. Lipofermata prevented palmitate-mediated oxidative stress, induction of BiP and CHOP, and cell death in a dose-dependent manner in hsHepG2 and rnINS-1E cells, suggesting it will prevent induction of fatty acid-mediated cell death pathways and lipotoxic disease by channeling excess fatty acids to adipose tissue and away from liver and pancreas. Importantly, mice dosed orally with Lipofermata were not able to absorb (13)C-oleate demonstrating utility as an inhibitor of fatty acid absorption from the gut.

Further optimization of the mGlu5 PAM clinical candidate VU0409551/JNJ-46778212: Progress and challenges towards a back-up compound.

This Letter describes the progress and challenges in the continued optimization of the mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212. While many analogs addressed key areas for improvement, no one compound possessed the amalgamation of improvements needed within the (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanone scaffold to advance as a back-up clinical candidate. However, many analogs displayed excellent solubility and physiochemical properties, and were active in the amphetamine-induced hyperlocomotion (AHL) model. Moreover, the SAR was robust for this series of PAMs, and both polar and hydrogen-bond donors were found to be tolerated, leading to analogs with overall attractive profiles and good ligand efficiencies.

Tetrahydronaphthyridine and dihydronaphthyridinone ethers as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5).

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Starting from an acetylene-based lead from high throughput screening, an evolved bicyclic dihydronaphthyridinone was identified. We describe further refinements leading to both dihydronaphthyridinone and tetrahydronaphthyridine mGlu5 PAMs containing an alkoxy-based linkage as an acetylene replacement. Exploration of several structural features including western pyridine ring isomers, positional amides, linker connectivity/position, and combinations thereof, reveal that these bicyclic modulators generally exhibit steep SAR and within specific subseries display a propensity for pharmacological mode switching at mGlu5 as well as antagonist activity at mGlu3. Structure-activity relationships within a dihydronaphthyridinone subseries uncovered 12c (VU0405372), a selective mGlu5 PAM with good in vitro potency, low glutamate fold-shift, acceptable DMPK properties, and in vivo efficacy in an amphetamine-based model of psychosis.

Identification of specific ligand-receptor interactions that govern binding and cooperativity of diverse modulators to a common metabotropic glutamate receptor 5 allosteric site.

A common metabotropic glutamate receptor 5 (mGlu5) allosteric site is known to accommodate diverse chemotypes. However, the structural relationship between compounds from different scaffolds and mGlu5 is not well understood. In an effort to better understand the molecular determinants that govern allosteric modulator interactions with mGlu5, we employed a combination of site-directed mutagenesis and computational modeling. With few exceptions, six residues (P654, Y658, T780, W784, S808, and A809) were identified as key affinity determinants across all seven allosteric modulator scaffolds. To improve our interpretation of how diverse allosteric modulators occupy the common allosteric site, we sampled the wealth of mGlu5 structure-activity relationship (SAR) data available by docking 60 ligands (actives and inactives) representing seven chemical scaffolds into our mGlu5 comparative model. To spatially and chemically compare binding modes of ligands from diverse scaffolds, the ChargeRMSD measure was developed. We found a common binding mode for the modulators that placed the long axes of the ligands parallel to the transmembrane helices 3 and 7. W784 in TM6 not only was identified as a key NAM cooperativity determinant across multiple scaffolds, but also caused a NAM to PAM switch for two different scaffolds. Moreover, a single point mutation in TM5, G747V, altered the architecture of the common allosteric site such that 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (VU29) was noncompetitive with the common allosteric site. Our findings highlight the subtleties of allosteric modulator binding to mGlu5 and demonstrate the utility in incorporating SAR information to strengthen the interpretation and analyses of docking and mutational data.

Catalytic cross-coupling of alkylzinc halides with alpha-chloroketones.

The cross-coupling of alkylzinc halides with alpha-chloroketones catalyzed by Cu(acac)2 is described. Using this method, primary and secondary alkyl groups are introduced adjacent to a ketone carbonyl under mild reaction conditions and in good yield. Cyclic, acyclic, aromatic, and aliphatic alpha-chloroketones are suitable substrates. Optically active alpha-chloroketones are converted to optically active products. The reaction was found to proceed stereospecifically with inversion of stereochemistry. The reaction is proposed to occur by direct substitution of the chloride with the alkyl group of an organocopper, -magnesium, or -zinc species.