Discovery of a brain penetrant small molecule antagonist targeting LPA1 receptors to reduce neuroinflammation and promote remyelination in multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood-brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.

Identification and In Vivo Evaluation of Myelination Agent PIPE-3297, a Selective Kappa Opioid Receptor Agonist Devoid of ß-Arrestin-2 Recruitment Efficacy.

Structure-activity relationship studies led to the discovery of PIPE-3297, a fully efficacious and selective kappa opioid receptor (KOR) agonist. PIPE-3297, a potent activator of G-protein signaling (GTPγS EC50 = 1.1 nM, 91% Emax), did not elicit a ß-arrestin-2 recruitment functional response (Emax < 10%). Receptor occupancy experiments performed with the novel KOR radiotracer [3H]-PIPE-3113 revealed that subcutaneous (s.c.) administration of PIPE-3297 at 30 mg/kg in mice achieved 90% occupancy of the KOR in the CNS 1 h post dose. A single subcutaneous dose of PIPE-3297 in healthy mice produced a statistically significant increase of mature oligodendrocytes (P < 0.0001) in the KOR-enriched striatum, an effect that was not observed in animals predosed with the selective KOR antagonist norbinaltorphimine. An equivalent dose given to mice in an open-field activity-monitoring system revealed a small KOR-independent decrease in total locomotor activity versus vehicle measured between 60 and 75 min post dose. Daily doses of PIPE-3297 at both 3 and 30 mg/kg s.c. reduced the disease score in the mouse experimental autoimmune encephalomyelitis (EAE) model. Visually evoked potential (VEP) N1 latencies were also significantly improved versus vehicle in both dose groups, and latencies matched those of untreated animals. Taken together, these findings highlight the potential therapeutic value of functionally selective G-protein KOR agonists in demyelinating disease, which may avoid the sedating side effects typically associated with classical nonbiased KOR agonists.

Discovery of PIPE-359, a Brain-Penetrant, Selective M1 Receptor Antagonist with Robust Efficacy in Murine MOG-EAE.

The discovery of PIPE-359, a brain-penetrant and selective antagonist of the muscarinic acetylcholine receptor subtype 1 is described. Starting from a literature-reported M1 antagonist, linker replacement and structure-activity relationship investigations of the eastern 1-(pyridinyl)piperazine led to the identification of a novel, potent, and selective antagonist with good MDCKII-MDR1 permeability. Continued semi-iterative positional scanning facilitated improvements in the metabolic and hERG profiles, which ultimately delivered PIPE-359. This advanced drug candidate exhibited robust efficacy in mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE), a preclinical model for multiple sclerosis.

Functional characterization of the dendritically localized mRNA neuronatin in hippocampal neurons.

Local translation of dendritic mRNAs plays an important role in neuronal development and synaptic plasticity. Although several hundred putative dendritic transcripts have been identified in the hippocampus, relatively few have been verified by in situ hybridization and thus remain uncharacterized. One such transcript encodes the protein neuronatin. Neuronatin has been shown to regulate calcium levels in non-neuronal cells such as pancreatic or embryonic stem cells, but its function in mature neurons remains unclear. Here we report that neuronatin is translated in hippocampal dendrites in response to blockade of action potentials and NMDA-receptor dependent synaptic transmission by TTX and APV. Our study also reveals that neuronatin can adjust dendritic calcium levels by regulating intracellular calcium storage. We propose that neuronatin may impact synaptic plasticity by modulating dendritic calcium levels during homeostatic plasticity, thereby potentially regulating neuronal excitability, receptor trafficking, and calcium dependent signaling.

Retinoic acid-gated sequence-specific translational control by RARalpha.

Retinoic acid (RA) plays important roles in development by modulating gene transcription through nuclear receptor activation. Increasing evidence supports a role for RA and RA receptors (RARs) in synaptic plasticity in the brain. We have recently reported that RA mediates a type of homeostatic synaptic plasticity through activation of dendritic protein synthesis, a process that requires dendritically localized RARalpha and is independent of transcriptional regulation. The molecular basis of this translational regulation by RA/RARalpha signaling, however, is unknown. Here we show that RARalpha is actively exported from the nucleus. Cytoplasmic RARalpha acts as an RNA-binding protein that associates with a subset of mRNAs, including dendritically localized glutamate receptor 1 (GluR1) mRNA. This binding is mediated by the RARalpha carboxyl terminal F-domain and specific sequence motifs in the 5'UTR of the GluR1 mRNA. Moreover, RARalpha association with the GluR1 mRNA directly underlies the translational control of GluR1 by RA: RARalpha represses GluR1 translation, while RA binding to RARalpha reduces its association with the GluR1 mRNA and relieves translational repression. Taken together, our results demonstrate a ligand-gated translational regulation mechanism mediated by a non-genomic function of RA/RARalpha signaling.

Retinoic acid regulates RARalpha-mediated control of translation in dendritic RNA granules during homeostatic synaptic plasticity.

Homeostatic plasticity is thought to play an important role in maintaining the stability of neuronal circuits. During one form of homeostatic plasticity, referred to as synaptic scaling, activity blockade leads to a compensatory increase in synaptic transmission by stimulating in dendrites the local translation and synaptic insertion of the AMPA receptor subunit GluR1. We have previously shown that all-trans retinoic acid (RA) mediates activity blockade-induced synaptic scaling by activating dendritic GluR1 synthesis and that this process requires RARalpha, a member of the nuclear RA receptor family. This result raised the question of where RARalpha is localized in dendrites and whether its localization is regulated by RA and/or activity blockade. Here, we show that activity blockade or RA treatment in neurons enhances the concentration of RARalpha in the dendritic RNA granules and activates local GluR1 synthesis in these RNA granules. Importantly, the same RNA granules that contain RARalpha also exhibit an accumulation of GluR1 protein but with a much slower time course than that of RARalpha, suggesting that the former regulates the latter. Taken together, our results provide a direct link between dendritically localized RARalpha and local GluR1 synthesis in RNA granules during RA-mediated synaptic signaling in homeostatic synaptic plasticity.

Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity.

Normal brain function requires that the overall synaptic activity in neural circuits be kept constant. Long-term alterations of neural activity lead to homeostatic regulation of synaptic strength by a process known as synaptic scaling. The molecular mechanisms underlying synaptic scaling are largely unknown. Here, we report that all-trans retinoic acid (RA), a well-known developmental morphogen, unexpectedly mediates synaptic scaling in response to activity blockade. We show that activity blockade increases RA synthesis in neurons and that acute RA treatment enhances synaptic transmission. The RA-induced increase in synaptic strength is occluded by activity blockade-induced synaptic scaling. Suppression of RA synthesis prevents synaptic scaling. This form of RA signaling operates via a translation-dependent but transcription-independent mechanism, causes an upregulation of postsynaptic glutamate receptor levels, and requires RARalpha receptors. Together, our data suggest that RA functions in homeostatic plasticity as a signaling molecule that increases synaptic strength by a protein synthesis-dependent mechanism.

Identification of process-localized mRNAs from cultured rodent hippocampal neurons.

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapse-specific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 microm pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.