Douglas Linn Gernert

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.

Transmembrane AMPA receptor regulatory proteins and cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [3H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [3H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [3H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [3H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.

Discovery of a potent, dual serotonin and norepinephrine reuptake inhibitor.

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug-drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.

Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist

The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

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.