Peripherally Restricted, Highly Potent, Selective, Aqueous-Soluble EP2 Antagonist with Anti-Inflammatory Properties.

The prostaglandin E2 receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.

The First 50 Years of Molecular Pharmacology.

In this Perspective, former and current editors of Molecular Pharmacology, together with the guest editors for this 50th Anniversary Issue, provide a historical overview of the journal since its founding in 1965. The substantial impact that Molecular Pharmacology has had on the field of pharmacology as well as on biomedical science is discussed, as is the broad scope of the journal. The authors conclude that, true to the original goals for the journal, Molecular Pharmacology today remains an outstanding venue for work that provides a mechanistic understanding of drugs, molecular probes, and their biologic targets.

EP2 receptor signaling pathways regulate classical activation of microglia.

Activation of EP2 receptors by prostaglandin E2 (PGE2) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gαs and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE2 had no effect on expression of CXCL10, TGF-ß1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1ß but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE2 or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.

When and how do seizures kill neurons, and is cell death relevant to epileptogenesis?

The effect of seizures on neuronal death and the role of seizure-induced neuronal death in acquired epileptogenesis have been debated for decades. Isolated brief seizures probably do not kill neurons; however, severe and repetitive seizures (i.e., status epilepticus) certainly do. Because status epilepticus both kills neurons and also leads to chronic epilepsy, neuronal death has been proposed to be an integral part of acquired epileptogenesis. Several studies, particularly in the immature brain, have suggested that neuronal death is not necessary for acquired epileptogenesis; however, the lack of neuronal death is difficult if not impossible to prove, and more recent studies have challenged this concept. Novel mechanisms of cell death, beyond the traditional concepts of necrosis and apoptosis, include autophagy, phagoptosis, necroptosis, and pyroptosis. The traditional proposal for why neuronal death may be necessary for epileptogenesis is based on the recapitulation of development hypothesis, where a loss of synaptic input from the dying neurons is considered a critical signal to induce axonal sprouting and synaptic-circuit reorganization. We propose a second hypothesis - the neuronal death pathway hypothesis, which states that the biochemical pathways causing programmed neurodegeneration, rather than neuronal death per se, are responsible for or contribute to epileptogenesis. The reprogramming of neuronal death pathways - if true - is proposed to derive from necroptosis or pyroptosis. The proposed new hypothesis may inform on why neuronal death seems closely linked to epileptogenesis, but may not always be.

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F.

Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.

Role of prostaglandin receptor EP2 in the regulations of cancer cell proliferation, invasion, and inflammation.

Population studies, preclinical, and clinical trials suggest a role for cyclooxygenase-2 (COX-2, PTGS2) in tumor formation and progression. The downstream prostanoid receptor signaling pathways involved in tumorigenesis are poorly understood, although prostaglandin E2 (PGE(2)), a major COX-2 metabolite which is usually upregulated in the involved tissues, presumably plays important roles in tumor biology. Taking advantage of our recently identified novel selective antagonist for the EP2 (PTGER2) subtype of PGE(2) receptor, we demonstrated that EP2 receptor activation could promote prostate cancer cell growth and invasion in vitro, accompanied by upregulation of the tumor-promoting inflammatory cytokines, such as IL-1ß and IL-6. Our results suggest the involvement of prostaglandin receptor EP2 in cancer cell proliferation and invasion possibly via its inflammatory actions, and indicate that selective blockade of the PGE(2)-EP2 signaling pathway via small molecule antagonists might represent a novel therapy for tumorigenesis.

Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor.

Activation of the Galphas-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 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 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 microM 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.

Glutamate receptor ion channels: structure, regulation, and function.

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

Pharmacological inhibition of the inflammatory receptor CCR2 relieves the early deleterious consequences of status epilepticus.

Generalized status epilepticus (SE) triggers a robust neuroinflammatory response involving reactive astrocytosis, activation of brain-resident microglia, and brain infiltration of CCR2+ monocytes. Multiple lines of evidence indicate that quenching SE-induced neuroinflammation can alleviate the adverse consequences of SE, including neuronal damage and cognitive impairments. Our recent findings show that blocking monocyte brain entry after SE, via global Ccr2 KO, rescues several SE-induced adverse effects including blood-brain barrier (BBB) erosion, microgliosis and neuronal damage while enhancing weight regain. The goals of the present study were to determine if CCR2 antagonism with a small molecule after SE replicates the effects of the CCR2 knockout. Male Ccr2+/rfp heterozygous mice were subject to intraperitoneal injection of kainic acid, scored for seizure severity, weight recovery, and nest building capability. Surviving mice were randomized into CCR2 antagonist and vehicle groups. The CCR2 antagonist, or vehicle, was administered 24- and 48-h post-SE via oral gavage, and mice were sacrificed three days post-SE. Mice subject to the CCR2 antagonist displayed faster weight recovery between one- and three-days post-SE and modestly enhanced ability to build a nest on the third day after SE when compared to vehicle-treated controls. CCR2 antagonism limited monocyte recruitment to the hippocampus and reduced numbers of Iba1+ macrophages. The mRNA levels of inflammatory mediators were depressed by 47%, and glial markers were reduced by 30% in mice treated with the CCR2 antagonist compared to controls. Astrocytosis was reduced in four brain regions. Neuroprotection was observed in the hippocampus, and erosion of the BBB was lessened in mice subject to the antagonist. Our findings provide proof-of-concept that brief CCR2 antagonism beginning one day after SE can alleviate multiple adverse SE-induced effects, including functional impairment, and identify circulating CCR2+ monocytes as a viable therapeutic target.

Phase 1 Clinical Results for NP10679, a pH-sensitive GluN2B-selective N-methyl-d-aspartate Receptor Inhibitor.

NP10679 is a context-dependent and subunit-selective negative allosteric modulator of N-methyl-d-aspartate (NMDA) receptors. It is a more potent inhibitor of GluN2B-containing NMDA receptors at the acidic levels of extracellular pH (eg, 6.9) found in the penumbral regions associated with cerebral ischemia than at physiological pH. This property allows NP10679 to act selectively in ischemic tissue while minimizing the nonselective blockade of NMDA receptors in healthy brain, thereby reducing on-target adverse effects. We report the results of a first-in-human pharmacokinetic and safety phase 1 clinical trial in healthy volunteers receiving single or multiple doses of NP10679 (NCT04007263). We found that NP10679 was well-tolerated and with a half-life of 20 hours, which is amenable to once per day dosing. The only notable side effect in this clinical trial was modest somnolence at higher doses, atypical in that the subject could easily be aroused. The overall results suggest that NP10679 is a candidate for further development for use in acute brain injury, such as ischemic stroke or aneurysmal subarachnoid hemorrhage, as well as for use in neuropsychiatric indications.

Novel GluN2B-Selective NMDA Receptor Negative Allosteric Modulator Possesses Intrinsic Analgesic Properties and Enhances Analgesia of Morphine in a Rodent Tail Flick Pain Model.

Many cases of accidental death associated with drug overdose are due to chronic opioid use, tolerance, and addiction. Analgesic tolerance is characterized by a decreased response to the analgesic effects of opioids, requiring increasingly higher doses to maintain the desired level of pain relief. Overactivation of GluN2B-containing N-methyl-d-Aspartate receptors is thought to play a key role in mechanisms underlying cellular adaptation that takes place in the development of analgesic tolerance. Herein, we describe a novel GluN2B-selective negative allosteric modulator, EU93-108, that shows high potency and brain penetrance. We describe the structural basis for binding at atomic resolution. This compound possesses intrinsic analgesic properties in the rodent tail immersion test. EU93-108 has an acute and significant anodyne effect, whereby morphine when combined with EU93-108 produces a higher tail flick latency compared to that of morphine alone. These data suggest that engagement of GluN2B as a target has utility in the treatment of pain, and EU93-108 could serve as an appropriate tool compound to interrogate this hypothesis. Future structure-activity relationship work around this scaffold could give rise to compounds that can be co-administered with opioids to diminish the onset of tolerance due to chronic opioid use, thereby modifying their utility.

Molecular cascades that mediate the influence of inflammation on epilepsy.

Experimental evidence strongly indicates a significant role for inflammatory and immune mediators in initiation of seizures and epileptogenesis. Here we will summarize data supporting the involvement of IL-1ß, TNF-α and toll-like receptor 4 in seizure generation and the process of epileptogenesis. The physiological homeostasis and control over brain immune response depends on the integrity of the blood-brain barrier, transforming growth factor (TGF)-ß signaling and leukocyte migration. To what extent targeting the immune system is successful in preventing epileptogenesis, and which signaling pathway should be beleaguered is still under intensive research.

Region-specific changes in gene expression in rat brain after chronic treatment with levetiracetam or phenytoin.

PURPOSE: It is commonly assumed that antiepileptic drugs (AEDs) act similarly in the various parts of the brain as long as their molecular targets are present. A few experimental studies on metabolic effects of vigabatrin, levetiracetam, valproate, and lamotrigine have shown that these drugs may act differently in different brain regions. We examined effects of chronic treatment with levetiracetam or phenytoin on mRNA levels to detect regional drug effects in a broad, nonbiased manner. METHODS: mRNA levels were monitored in three brain regions with oligonucleotide-based microarrays. RESULTS: Levetiracetam (150 mg/kg for 90 days) changed the expression of 65 genes in pons/medulla oblongata, two in hippocampus, and one in frontal cortex. Phenytoin (75 mg/kg), in contrast, changed the expression of only three genes in pons/medulla oblongata, but 64 genes in hippocampus, and 327 genes in frontal cortex. Very little overlap between regions or drug treatments was observed with respect to effects on gene expression. DISCUSSION: We conclude that chronic treatment with levetiracetam or phenytoin causes region-specific and highly differential effects on gene expression in the brain. Regional effects on gene expression could reflect regional differences in molecular targets of AEDs, and they could influence the clinical profiles of AEDs.

Inhibition of the prostaglandin EP2 receptor prevents long-term cognitive impairment in a model of systemic inflammation.

Long-term cognitive and affective impairments are common problems in the survivors of sepsis, which weakens their vocational and daily life ability. Neuroinflammation has been reported to exert a key role in the development of cognitive deficit in different disorders including epilepsy, Alzheimer's disease (AD) and stroke. Mice treated with lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, show a robust but short-lived neuroinflammation and develop long-term memory and affective problems. In this study, we test the hypothesis that pharmacological blockade of the EP2 receptor for prostaglandin E2 reduces neuroinflammation and prevents long-term affective and memory deficits in a mouse model of LPS-induced, sepsis-associated encephalopathy (SAE). Our results show that an EP2 antagonist, TG6-10-1, promotes the recovery of body weight, mitigates neuroinflammation as judged by inflammatory cytokines and microgliosis, prevents the loss of synaptic proteins, and ameliorates depression-like behavior in the sucrose preference test as well as memory loss in the novel object recognition test. Our results point to a new avenue to ameliorate neuroinflammation and long-term affective and cognition problems of sepsis survivors.

An Agonist Dependent Allosteric Antagonist of Prostaglandin EP2 Receptors.

All reported prostaglandin EP2 receptor antagonists have a purely orthosteric, competitive mode of action. Herein, we report the characterization of compound 1 (pubchem CID 664888) as the first EP2 antagonist that features a reversible, agonist dependent allosteric mode of action. Compound 1 displayed an unsurmountable inhibition of cAMP accumulation stimulated by different EP2 agonists in C6 glioma cells overexpressing human EP2 (C6G-hEP2). The degree of reduction of agonist potency and efficacy depended on the agonist employed. Negative allosteric modulation was not observed in C6G cells overexpressing human EP4, IP, or DP1 receptors. Moreover, in the murine microglial cell line that stably expresses human EP2 receptors (BV2-hEP2), compound 1 reduced the EP2 agonist-induced elevation of interleukin 6 (IL-6), IL-1ß, and hEP2 mRNA levels and increased that of tumor necrosis factor (TNF)-α. Compound 1 was docked into a homology model of hEP2. The predicted binding site on the cytoplasmic receptor surface was similar to that of allosteric inhibitors of the ß2-adrenergic, CC chemokine receptor 9 (CCR9), and CC chemokine receptor 2 (CCR2) receptors, which supports the notion of a conserved G-protein-coupled receptor (GPCR) binding pocket for allosteric inhibitors. As the first agonist dependent negative allosteric modulator of EP2 receptor, the structure of this compound may provide a basis for developing improved allosteric modulators of EP2 receptors.

Potent, Selective, Water Soluble, Brain-Permeable EP2 Receptor Antagonist for Use in Central Nervous System Disease Models.

Activation of prostanoid EP2 receptor exacerbates neuroinflammatory and neurodegenerative pathology in central nervous system diseases such as epilepsy, Alzheimer's disease, and cerebral aneurysms. A selective and brain-permeable EP2 antagonist will be useful to attenuate the inflammatory consequences of EP2 activation and to reduce the severity of these chronic diseases. We recently developed a brain-permeable EP2 antagonist 1 (TG6-10-1), which displayed anti-inflammatory and neuroprotective actions in rodent models of status epilepticus. However, this compound exhibited moderate selectivity to EP2, a short plasma half-life in rodents (1.7 h) and low aqueous solubility (27 µM), limiting its use in animal models of chronic disease. With lead-optimization studies, we have developed several novel EP2 antagonists with improved water solubility, brain penetration, high EP2 potency, and selectivity. These novel inhibitors suppress inflammatory gene expression induced by EP2 receptor activation in a microglial cell line, reinforcing the use of EP2 antagonists as anti-inflammatory agents.

Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-D-aspartate receptors.

The synthesis and structure-activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modulated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and alpha(1)-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood-brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency.

Suppressing pro-inflammatory prostaglandin signaling attenuates excitotoxicity-associated neuronal inflammation and injury.

Glutamate receptor-mediated excitotoxicity is a common pathogenic process in many neurological conditions including epilepsy. Prolonged seizures induce elevations in extracellular glutamate that contribute to excitotoxic damage, which in turn can trigger chronic neuroinflammatory reactions, leading to secondary damage to the brain. Blocking key inflammatory pathways could prevent such secondary brain injury following the initial excitotoxic insults. Prostaglandin E2 (PGE2) has emerged as an important mediator of neuroinflammation-associated injury, in large part via activating its EP2 receptor subtype. Herein, we investigated the effects of EP2 receptor inhibition on excitotoxicity-associated neuronal inflammation and injury in vivo. Utilizing a bioavailable and brain-permeant compound, TG6-10-1, we found that pharmacological inhibition of EP2 receptor after a one-hour episode of kainate-induced status epilepticus (SE) in mice reduced seizure-promoted functional deficits, cytokine induction, reactive gliosis, blood-brain barrier impairment, and hippocampal damage. Our preclinical findings endorse the feasibility of blocking PGE2/EP2 signaling as an adjunctive strategy to treat prolonged seizures. The promising benefits from EP2 receptor inhibition should also be relevant to other neurological conditions in which excitotoxicity-associated secondary damage to the brain represents a pathogenic event.

Why Is It so Hard to Do Good Science?

"Good science" means answering important questions convincingly, a challenging endeavor under the best of circumstances. Our inability to replicate many biomedical studies has been the subject of numerous commentaries both in the scientific and lay press. In response, statistics has re-emerged as a necessary tool to improve the objectivity of study conclusions. However, psychological aspects of decision making introduce preconceived preferences into scientific judgment that cannot be eliminated by any statistical method. The psychology of decision making, expounded by Kahneman, Tversky, and Thaler, is well known in the field of economics, but the underlying concepts of cognitive psychology are also relevant to scientific judgments. I repeated experiments carried out on undergraduates by Kahneman and colleagues four to five decades ago, but with scientists, and obtained essentially the same results. The experiments were in the form of written reactions to scenarios, and participants were scientists at all career stages. The findings reinforce the roles that two inherent intuitions play in scientific decision making: our drive to create a coherent narrative from new data regardless of its quality or relevance and our inclination to seek patterns in data whether they exist or not. Moreover, we do not always consider how likely a result is regardless of its p value. Low statistical power and inattention to principles underpinning Bayesian statistics reduce experimental rigor, but mitigating skills can be learned. Overcoming our natural human tendency to make quick decisions and jump to conclusions is a deeper obstacle to doing good science; this too can be learned.