Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor

Abstract

Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE 2 ) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE 2 by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE 2 on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE 2 on EP2 receptors 4-to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE 2 receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE 2 released in a cell-injury setting is neuroprotective.excitotoxicity | neuronal injury | prostaglandin E2 | time-resolved fluorescence resonance energy transfer | ultra high-throughput screening C yclooxygenase-2 (COX2), which is constitutively expressed at low to moderate levels in both neuronal cell bodies and dendritic spines in the hippocampus, is regulated by synaptic activity (1) and is rapidly induced in neurons after a seizure (2) or cerebral ischemia (3,4). Studies in rodents have demonstrated that COX2 activation by ischemia or status epilepticus generally contributes to neuronal injury (5-8), but multiple downstream COX2 signaling pathways suggest that the mechanisms promoting and opposing brain injury are complex. Among the enzymatic products of COX2 is prostaglandin E2 (PGE 2 ), which can activate four G protein-coupled receptors (GPCRs): EP1, EP2, EP3, and EP4. Activation of the EP2 receptor by PGE 2 appears to be neuroprotective after ischemia (3, 4), whereas EP1 activation promotes neurodegeneration (8). The yin-yang nature of the PGE 2 receptor family demonstrates the value of a neuroprotection strategy involving modulation of a specific prostanoid receptor rather than generic block of the entire COX2 signaling cascade.EP2 is a Gαs-coupled receptor that, when activated by PGE 2 , stimulates adenylate cyclase, resulting in elevation of cytoplasmic cAMP (cAMP) level. In addition to its suspected neuroprotective role, EP2 activation has also been shown to promote spatial learning (9), to improve survival of epithelial cells after radiation injury (10), to accelerate bone healing after fracture (11), and to improve renal function in a HgCl 2 model of chronic renal failure (12). On the other hand, EP2 activation in microglia could promote inflammation and neurotoxicity in some neurodegenerative disease models, such as Alzheimer's disease and amyotrophic late...

Small molecule antagonist reveals seizure-induced mediation of neuronal injury by prostaglandin E2 receptor subtype EP2

“…We hypothesize that after seizures neuronal COX-2 produces PGE 2 , which activates EP2 receptors on microglia, accelerating the innate immune response after SE and triggering secondary neurodegeneration. At the same time, activation of EP2 receptors on neurons, via a PKAdependent pathway, appears to be neuroprotective in acute models of NMDA-induced excitotoxicity and ischemia (3,11,12). The dual role of EP2 receptors, mediating both neuroprotection and neurodegeneration perhaps by different cell types and different pathways (32), complicates exploitation of EP2 as a therapeutic target.…”

“…This work was reported in preliminary form (22,23 (Fig. 1A) and showed robust inhibition of PGE 2 (1 μM)-induced cAMP accumulation (i.e., reduction of the TR-FRET signal) in human EP2-overexpressing C6 glioma (C6G-EP2) cells (11), without affecting prostaglandin EP4 or β2-adrenergic receptors under agonist-saturated conditions (SI Results and Fig. S1).…”

“…Neuronal EP2 activation appears to mediate some beneficial effects, such as PKA-dependent neuroprotection in acute models of ischemia and excitotoxicity (3,11,12), early neuroprotection following seizures (13), and promotion of spatial learning (14). Conversely, on the basis of the phenotype of EP2 knockout mice, EP2 activation is thought to promote inflammation and neurotoxicity in animal models of neurodegenerative diseases including Alzheimer's disease (15), Parkinson's disease (16), and amyotrophic lateral sclerosis (10).…”

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

“…The novel EP2 potentiator TG3-95-1 (referred to as compound 1 in Ref. 27) and EP2 antagonist TG4-155 were synthesized in our laboratory (27)(28)(29). All plasticware and reagents were endotoxin-free.…”

“…To explore EP2 pharmacology in microglia, we examined the effects of an EP2 receptor antagonist (28) and allosteric potentiator (27). Rat primary microglia cultures were preincubated with vehicle or 1 M competitive EP2 antagonist TG4-155 (28) for 20 min followed by addition of the EP2 agonist butaprost at different concentrations.…”