Warren J. Porter

Functional gamma‐secretase inhibitors reduce beta‐amyloid peptide levels in brain

Converging lines of evidence implicate the beta‐amyloid peptide (Aβ) as causative in Alzheimers disease. We describe a novel class of compounds that reduce Aβ production by functionally inhibiting γ‐secretase, the activity responsible for the carboxy‐terminal cleavage required for Aβ production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Aβ production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, to mice transgenic for human APPV717F reduces brain levels of Aβ in a dose‐dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain Aβin vivo. Development of such novel functional γ‐secretase inhibitors will enable a clinical examination of the Aβ hypothesis that Aβ peptide drives the neuropathology observed in Alzheimers disease.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8

Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors

&NA; 6‐[(1S)‐1‐[1‐[5‐(2‐hydroxyethoxy)‐2‐pyridyl]pyrazol‐3‐yl]ethyl]‐3H‐1,3‐benzothiazol‐2‐one (LY3130481 or CERC‐611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) &ggr;−8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food‐maintained responding in rats and spontaneous alternation in a Y‐maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal‐dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive‐impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor‐impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non‐rapid‐eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro‐cognitive and wake‐promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5‐HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic. HighlightsLY3130481 (CERC‐611) is a selective antagonist of AMPA receptors TARP &ggr;−8 protein.LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic.LY3130481 produced mixed results in rodent cognition assessments.LY3130481 produced modest increases in wake times in rats and increased wake and cognition‐associated neurotransmitters.LY3130481 attenuated some impairing effects of standard antiepileptic drugs.

Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post‐mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimers) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)‐α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimers brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets.

Targeted Blockade of TARP-γ8-Associated AMPA Receptors: Anticonvulsant Activity with the Selective Antagonist LY3130481 (CERC-611)

BACKGROUND & OBJECTIVE 6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]- 3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise, LY3130481 attenuated convulsions in mice and rats with long-term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP γ-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient. RESULTS & CONCLUSION Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP γ-8 is sufficient for these protective effects.

P4-276: In vitro characterization of gamma-secretase inhibitors on endogenous APP

Background: Abeta is the major proteinaceous component of the amyloid plaques associated with Alzheimer’s disease (AD) and various forms of Abeta, ranging from 38-43 amino acids, are excised from the amyloid precursor protein (APP) by beta amyloid cleaving enzyme (BACE) and gamma-secretase mediated cleavage. BACE cleaves at the N-terminal of Abeta while gamma-secretase is a multimeric proteolytic complex that cleaves APP within the transmembrane domain and creates the C-terminal heterogeneity of Abeta. Both BACE and gamma-secretase are attractive targets for pharmaceutical intervention, with gamma-secretase inhibitors already entering into clinical trials. Objective(s): We sought to determine the pharmacologic effects of BACE and gamma-secretase inhibition in the human neuroblastoma cell line, SH-SY5Y. Methods: We used standard cell culture methods to culture SH-SY5Y cells. Cells were exposed to compound for 24 hours and soluble Abeta levels were measured by ELISA. Results: Pilot studies confirmed that this cell line produced sufficient amounts of Abeta 1, or Abeta 1-40, to assess the effects of various secretase inhibitors on Abeta production in the absence of transient or stable APP over-expression. Treatment of naı̈ve SH-SY5Y cells with functional gamma-secretase inhibitors resulted in a bi-phasic concentration-response curve, with low concentrations of compound producing up to four fold increases in Abeta, while higher concentrations of compound inhibited Abeta production. Similar bi-phasic responses were observed in naı̈ve SH-SY5Y cells treated with functional gamma-secretase inhibitors from other chemical series. Interestingly, transient transfection of SHSY5Y cells with SweAPP695 cDNA produced a marked increase in Abeta production, as expected, but also resulted in a mono-phasic concentrationresponse curve in response to functional gamma-secretase inhibition. Treatment with BACE inhibitors and allosteric gamma-secretase modulators did not show any differences between naı̈ve and transfected SH-SY5Y cells. Conclusions: The mechanism responsible for this pharmacology is unknown but suggests that in this transformed cell line, APP substrate availability influences how the gamma-secretase multimeric complex responds to functional inhibitors. Whether similar effects would be observed with active site inhibitors or other cell lines is under investigation.