Robert E. Petroski

Characterization of γ-Aminobutyric Acid Receptor GABAB(1e), a GABAB(1) Splice Variant Encoding a Truncated Receptor

We have identified a splice variant encoding only the extracellular ligand-binding domain of the γ-aminobutyric acid B (GABAB) receptor subunit GABAB(1a). This isoform, which we have named GABAB(1e), is detected in both rats and humans. While GABAB(1e) is a minor component of the total pool of GABAB(1) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABAB(1e) lacks the ability to bind the radiolabeled antagonist [3H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABAB(2). Interestingly, when co-expressed with GABAB(2), not only does the truncated receptor heterodimerize with GABAB(2), the association is of sufficient avidity to disrupt the normal GABAB(1a)/GABAB(2) association. Despite this strong interaction, GABAB(1e) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABAB(1a)/GABAB(2).

Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies

Peripheral corticotropin‐releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C‐terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1‐ and CRF2‐transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti‐CRF1 antiserum (4467a‐CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a‐CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a‐CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a‐CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre‐absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co‐localization of 4392a‐CRF1&2 but not 4467a‐CRF1 immunoreactivity with H/K‐ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a‐CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co‐localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.

Characterization of Novel Selective H1-Antihistamines for Clinical Evaluation in the Treatment of Insomnia

Analogues of the known H(1)-antihistamine R-dimethindene were profiled as potential agents for the treatment of insomnia. Several highly selective compounds were efficacious in rodent sleep models. On the basis of overall profile, indene 1d and benzothiophene 2a had pharmacokinetic properties suitable for evaluation in night time dosing. Compound 2a did not show an in vivo cardiovascular effect from weak hERG channel inhibition.

Role of the GLT‐1 subtype of glutamate transporter in glutamate homeostasis: the GLT‐1‐preferring inhibitor WAY‐855 produces marginal neurotoxicity in the rat hippocampus

Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock‐out animals and antisense techniques have been used to study their specific roles. Here we used WAY‐855, a GLT‐1‐preferring blocker, to assess the role of GLT‐1 in rat hippocampus. GLT‐1 was the most abundant transporter in the hippocampus at the mRNA level. According to [3H]‐l‐glutamate uptake data, GLT‐1 was responsible for approximately 80% of the GLAST‐, GLT‐1‐, and EAAC1‐mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY‐855 (100 µm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad‐spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 µm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY‐855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY‐855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1–4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT‐1 is insufficient to provoke glutamate build‐up, leading to NMDA receptor‐mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.

WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake.

The pharmacological profile of a novel glutamate transport inhibitor, WAY‐855 (3‐amino‐tricyclo[2.2.1.02.6]heptane‐1,3‐dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY‐855 inhibited glutamate uptake mediated by all three subtypes in a concentration‐dependent manner, with preferential inhibition of the CNS‐predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 μM, respectively, while EAAT1 activity was inhibited by 50% at 100 μM (IC50 values determined in oocytes were 1.3 μM (EAAT2), 52.5 μM (EAAT3) and 125.9 μM (EAAT1)). Application of WAY‐855 to EAAT‐expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]D‐aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY‐855 inhibited D‐aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY‐855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY‐855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes.

Lead Optimization of 4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines as A2A Adenosine Receptor Antagonists for the Treatment of Parkinson’s Disease

4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinsons disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems.

The glutamate analog (±) threo‐3‐methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high‐affinity, Na+‐dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate‐elicited currents mediated by EAATs 1–4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1–3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of d‐[3H]‐aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate‐like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded d‐[3H]‐aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 µm T3MG were required to elicit significant NMDA receptor‐mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.

N-[6-amino-2-(heteroaryl)pyrimidin-4-yl]acetamides as A2A receptor antagonists with improved drug like properties and in vivo efficacy.

In the present article, we report on a strategy to improve the physical properties of a series of small molecule human adenosine 2A (hA2A) antagonists. One of the aromatic rings typical of this series of antagonists is replaced with a series of aliphatic groups, with the aim of disrupting crystal packing of the molecule to lower the melting point and in turn to improve the solubility. Herein, we describe the SAR of a new series of water-soluble 2,4,6-trisubstituted pyrimidines where R1 is an aromatic heterocycle, R2 is a short-chain alkyl amide, and the typical R3 aromatic heterocyclic substituent is replaced with an aliphatic amino substituent. This approach significantly enhanced aqueous solubility and lowered the log P of the system to provide molecules without significant hERG or CYP liabilities and robust in vivo efficacy.

Brain-penetrating 2-aminobenzimidazole H1-antihistamines for the treatment of insomnia

The benzimidazole core of the selective non-brain-penetrating H(1)-antihistamine mizolastine was used to identify a series of brain-penetrating H(1)-antihistamines for the potential treatment of insomnia. Using cassette PK studies, brain-penetrating H(1)-antihistamines were identified and in vivo efficacy was demonstrated in a rat EEG/EMG model. Further optimization focused on strategies to attenuate an identified hERG liability, leading to the discovery of 4i with a promising in vitro profile.

2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.