Discovery of BI-9508, a Brain-Penetrant GPR88-Receptor-Agonist Tool Compound for In Vivo Mouse Studies.

Decreased activity and expression of the G-protein coupled receptor GPR88 is linked to many behavior-linked neurological disorders. Published preclinical GPR88 allosteric agonists all have in vivo pharmacokinetic properties that preclude their progression to the clinic, including high lipophilicity and poor brain penetration. Here, we describe our attempts to improve GPR88 agonists' drug-like properties and our analysis of the trade-offs required to successfully target GPR88's allosteric pocket. We discovered two new GPR88 agonists: One that reduced morphine-induced locomotor activity in a murine proof-of-concept study, and the atropoisomeric BI-9508, which is a brain penetrant and has improved pharmacokinetic properties and dosing that recommend it for future in vivo studies in rodents. BI-9508 still suffers from high lipophilicity, and research on this series was halted. Because of its utility as a tool compound, we now offer researchers access to BI-9508 and a negative control free of charge via Boehringer Ingelheim's open innovation portal opnMe.com.

Evaluation of a 5-HT2B receptor agonist in a murine model of amyotrophic lateral sclerosis.

Degeneration of brainstem serotonin neurons has been demonstrated in ALS patients and mouse models and was found responsible for the development of spasticity. Consistent with involvement of central serotonin pathways, 5-HT2B receptor (5-HT2BR) was upregulated in microglia of ALS mice. Its deletion worsened disease outcome in the Sod1G86R mouse model and led to microglial degeneration. In ALS patients, a polymorphism in HTR2B gene leading to higher receptor expression in CNS, was associated with increased survival in patients as well as prevention of microglial degeneration. Thus, the aim of our study was to determine the effect of a 5-HT2BR agonist : BW723C86 (BW), in the Sod1G86R mouse model. Despite good pharmacokinetic and pharmacological profiles, BW did not ameliorate disease outcome or motor neuron degeneration in a fast progressing mouse model of ALS despite evidence of modulation of microglial gene expression.

Discovery, Structure-Activity Relationship, and Antiparkinsonian Effect of a Potent and Brain-Penetrant Chemical Series of Positive Allosteric Modulators of Metabotropic Glutamate Receptor 4.

The metabotropic glutamate receptor 4 (mGluR4) is an emerging target for the treatment of Parkinson's disease (PD). However, since the discovery of its therapeutic potential, no ligand has been successfully developed enough to be tested in the clinic. In the present paper, we report for the first time the medicinal chemistry efforts conducted around the pharmacological tool (-)-PHCCC. This work led to the identification of compound 40, a potent and selective mGluR4 positive allosteric modulator (PAM) with good water solubility and demonstrating consistent activity across validated preclinical rodent models of PD motor symptoms after intraperitoneal administration: haloperidol-induced catalepsy in mouse and the rat 6-hydroxydopamine (6-OHDA) lesion model. Moreover, we also describe the identification of compound 60 a close analogue of compound 40 with improved pharmacokinetic profile after oral administration. On the basis of its favorable and unique preclinical profile, compound 60 (PXT002331, now foliglurax) was nominated as a candidate for clinical development.

Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors.

Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries.

Exploring the Technology Landscape of 7TMR Drug Signaling Profiling.

Seven transmembrane domain receptors (7TMRs) constitute the largest family of transmembrane proteins in vertebrates and are the targets of more than 40% of currently marketed drugs. It is now accepted that these receptors are highly dynamic "microprocessors" that adopt a continuum of functionally distinct active conformations. The novel concept of biased agonism (or functional selectivity) posits that different ligands stabilize unique receptor conformations with each conformation imparting distinct signaling, and thus biological attributes, to a given receptor. The pharmacotherapeutic potential of biased agonism lies in possibility to develop molecules that selectively engage beneficial pathways while inhibiting or remaining inert towards those producing deleterious outcomes. Various strategies are now applied for the discovery of biased ligands. Many assays use second messenger levels (i.e., calcium, inositol trisphosphate, cAMP) as a quantitative readout of G-protein subtype-specific activity. However, due to complex cross-regulation between the various G-protein pathways, second messenger levels alone are not directly reflective of a ligand's activity on a specific pathway. Consequently, direct measurements of receptor-proximal events (such as G-protein activation and ß-arrestin coupling) are required for a more accurate quantification of ligand's efficacy (or bias) towards different pathways. The discovery that various ligands of the same receptor can display different efficacies and potencies towards different receptor-downstream signaling pathways has not only revitalized the process of 7TMR drug discovery, but has significantly transformed the field of pharmacology as a whole. This review will showcase the current pharmacological toolbox available for the discovery and validation of biased ligands.

Chemical switch of a metabotropic glutamate receptor 2 silent allosteric modulator into dual metabotropic glutamate receptor 2/3 negative/positive allosteric modulators.

Using an mGluR2 FRET-based binding assay, binders of the transmembrane region devoid of functional activity were identified. It is reported that slight chemical modifications of these SAMs can dramatically change activity of the resulting analogues without altering their affinities. Starting from compound 1, three mGluR2 NAMs showing also mGluR3 PAM activities were obtained. SAMs therefore represent a useful approach to explore the chemical space for GPCR allosteric modulator identification.

Amino-pyrrolidine tricarboxylic acids give new insight into group III metabotropic glutamate receptor activation mechanism.

Like most class C G-protein-coupled receptors, metabotropic glutamate (mGlu) receptors possess a large extracellular domain where orthosteric ligands bind. Crystal structures revealed that this domain, called Venus FlyTrap (VFT), adopts a closed or open conformation upon agonist or antagonist binding, respectively. We have described amino-pyrrolidine tricarboxylic acids (APTCs), including (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (FP0429), as new selective group III mGlu agonists. Whereas FP0429 is an almost full mGlu4 agonist, it is a weak and partial agonist of the closely related mGlu8 subtype. To get more insight into the activation mechanism of mGlu receptors, we aimed to elucidate why FP0429 behaves differently at these two highly homologous receptors by focusing on two residues within the binding site that differ between mGlu4 and mGlu8. Site-directed mutagenesis of Ser157 and Gly158 of mGlu4 into their mGlu8 homologs (Ala) turned FP0429 into a weak partial agonist. Conversely, introduction of Ser and Gly residues into mGlu8 increased FP0429 efficacy. Docking of FP0429 in mGlu4 VFT 3D model helped us characterize the role of each residue. Indeed, mGlu4 Ser157 seems to have an important role in FP0429 binding, whereas Gly158 may allow a deeper positioning of this agonist in the cavity of lobe I, thereby ensuring optimal interactions with lobe II residues in the fully closed state of the VFT. In contrast, the presence of a methyl group in mGlu8 (Ala instead of Gly) weakens the interactions with the lobe II residues. This probably results in a less stable or a partially closed form of the mGlu8 VFT, leading to partial receptor activation.

Design and synthesis of APTCs (aminopyrrolidinetricarboxylic acids): identification of a new group III metabotropic glutamate receptor selective agonist.

A new family of mGlu receptor orthosteric ligands called APTCs was designed and synthesized using a parallel chemistry approach. Amongst 65 molecules tested on mGlu4, mGlu6 and mGlu8 subtypes, (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (8a06-FP0429) has been shown to be a full mGlu4 agonist and a partial mGlu8 agonist. In addition, 8a06 was shown to be selective versus group I and II mGlu subtypes. A possible explanation for this efficacy difference is proposed by docking experiment performed with molecular model of the receptor.

Adenoviral-mediated skeletal muscle transcriptional targeting using chimeric tissue-specific promoters.

BACKGROUND: Adenoviral vectors are promising tools to achieve skeletal muscle gene transfer for the treatment of peripheral ischemia. However, the use of ubiquitous viral promoters represents a major safety issue that could limit their use. Cellular regulatory sequences that allow strong and tissue-specific expression could circumvent this problem. MATERIAL/METHODS: Adenoviral vectors encoding the firefly luciferase under the control of the human skeletal a-actin promoter, alone or combined with the b-enolase or creatine kinase enhancer, were studied in vitro in murine C2C12 cells and in vivo in C57BL/6 mice. The expression of the reporter gene was measured in cell lysates and animal tissue homogenates. Adenoviral distribution was evaluated by PCR on DNA extracted from liver, spleen, heart and lungs. RESULTS: Skeletal a-actin promoter-based expression cassettes follow the regulation of the endogenous skeletal a-actin gene in vitro as luciferase expression strongly increases with myoblast differentiation into myotubes. The addition of the cellular b enolase or the creatine kinase enhancer improves the specificity of the skeletal a-actin promoter in vitro as well as in vivo. When adenoviral vectors are locally injected into skeletal muscles, the chimeric promoters drive a relatively strong gene expression, ranging from 16 to 28% of the Rous sarcoma virus promoter-related expression. CONCLUSIONS: Chimeric regulatory sequences based on the skeletal a-actin promoter are highly specific and allow transgene expression in vivo at high levels. These results indicate that expression cassettes designed for the treatment of peripheral ischemia by gene therapy can efficiently target gene expression to skeletal muscle.

Nicalin and its binding partner Nomo are novel Nodal signaling antagonists.

Nodals are signaling factors of the transforming growth factor-beta (TGFbeta) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated gamma-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.