Christopher F. Bigge

Potent and stereospecific anticonvulsant activity of 3-isobutyl GABA relates to in vitro binding at a novel site labeled by tritiated gabapentin.

3-Isobutyl GABA is a derivative of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and is also structurally related to the novel anticonvulsant gabapentin. The S(+) enantiomer of 3-isobutyl GABA blocks maximal electroshock seizures in mice and also potently displaces tritiated gabapentin from a novel high-affinity binding site in rat brain membrane fractions. The R(-) enantiomer is much less active in both assays, suggesting that the gabapentin binding site is involved in the anticonvulsant activity of 3-isobutyl GABA.

Ionotropic glutamate receptors

The glutamate-binding sites of ionotropic glutamate receptors are formed from two extracellular domains of a single subunit. Conformational changes induced by agonist binding produce mechanical processes that are translated into ion gating and receptor desensitization. The interactions between macromolecular assemblies of synaptic proteins and ionotropic glutamate receptors, and their subsequent roles in receptor clustering and specificity are being elucidated. Kainate receptor pharmacology is finally revealing its secrets as a result of the availability of selective pharmacological agents.

Mechanisms of neuronal cell injury/death and targets for drug intervention

The loss of neurons is responsible for many acute neurological disorders, as well as many chronic neurodegenerative diseases. Drug discovery research has concentrated on blocking necrosis produced by activation of one or more excitatory amino acid (EAA) receptors. Both receptor antagonists and compounds reducing EAA release are currently in clinical trials. With the recent advances in the understanding of apoptosis new strategies for neuroprotection are also emerging. It is anticipated that clinically useful neuroprotective drugs will soon be available for treatment of a range of CNS disorders. This article reviews some of the mechanisms responsible for neuronal cell death and outlines strategies to block calcium overload and free radical production.

New preparations of the N-methyl-D-aspartate receptor antagonist, 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid (CPP)

Two new methods to ensure selective alkylation of the N4 of 2-piperazinecarboxylic acid to give 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid (CPP, 1) are reported. CPP can be conveniently prepared using a copper chelate to selectively protect the N1 position during alkylation. A second procedure uses methyl-4-BOC-1-CBZ-2-piperazinecarboxylate 5 as a versatile intermediate, which was further elaborated to CPP.

Synthesis and NMDA receptor binding of 2-amino-7,7-difluoro-7-phosphonoheptanoic acid

Abstract In an NMDA specific receptor binding assay, 1 has lower affinity than its parent, APH. This result suggests that for competitive antagonists, diionization of the phosphonic acid moiety may be detrimental to receptor affinity.

Evidence for Overlapping Substrate Specificity Between Large Neutral Amino Acid (LNAA) and Dipeptide (hPEPTl) Transporters for PD 158473, an NMDA Antagonist

AbstractPurpose. The objective of this research was to investigate the substrate specificity of large neutral amino acid carrier (LNAA) and di/tripeptide (hPEPTl) transporters with respect to PD 158473, an NMDA antagonist.nMethods. Cellular uptake studies were carried out using two types of Chinese Hamster Ovary (CHO). CHO-K1 cells represent the wild type with inherent large neutral amino acid (LNAA) activity. CHO-PEPT1 cells were generated by stable transfection of hPEPTl gene into CHO cells. Therefore, these cells possess both LNAA activity and di/tripeptide transporter activities as a result of the transfection. Cellular uptake of PD 158473 was quantified using a HPLC method previously developed in our laboratory.nResults. The utility of the CHO-PEPT1 cell model was demonstrated by determining the uptake kinetics of Gly-Sar, a prototypical dipeptide transporter substrate. Uptake kinetics of PD 158473 displayed two carrier-mediated transport components in CHO-PEPT1 cells, while in CHO-K1 cells the relationship was consistent with classic one component Michaelis-Menten kinetics. These results confirmed the affinity of PD 158473 for both LNAA and di/tripeptide transporters. Further, results from inhibition experiments using these two cell types indicate that the high affinity-low capacity system was the LNAA carrier and the low affinity-high capacity carrier was the di/tripeptide transporter.nConclusions. This study demonstrates overlapping substrate specificity between LNAA carrier and di/tripeptide transporter (hPEPTl) for PD 158473, an amino acid analog. Establishing Structure Transport Relationship (STR) for this overlap will aid in a design strategy for increasing oral absorption or targeting specific drugs to selected tissues.

Chapter 2. Recent Advances In Excitatory Amino Acid Research

Publisher Summary This chapter discusses the recent advances across the spectrum of excitatory amino acid (EAA) research. The chapter also discusses the structure and mechanism of action of new EAA based therapeutic agents. EAAs have been shown to mediate a range of physiological functions, including nociception, vision, cardiovascular reflex, respiration, and motor control. EAAs have been suggested to play a role in the etiology of epilepsy. Competitive and non-competitive N-methyl-D-aspartic acid (NMDA) antagonists also have been shown to exert a protective action in animal models of anxiety. A central role for EAAs in stroke has been firmly established. The second messenger pathways, associated with EAA receptor activation, have been discussed in this chapter. NMDA receptor activation has been shown to stimulate the arachidonic acid cascade and induced proteolysis of brain spectrin. The AP4 (or APB) receptor is the least well characterized of the EAA receptors. But the NMDA receptor is the best characterized among them. The nature of interaction of the NMDA and phencyclidine (PCP) receptors has been defined, further using both receptor binding and electrophysiology. Binding of PCP site ligands are reported to be dependent on both NMDA receptor activation and membrane potential. From the high level of pharmaceutical interest in NMDA antagonists, increasing numbers of new antagonist structures have appeared. However, no deviations from the amino acid-spacer-phosphonic acid structural type have been reported. Dextromethorphan and MK-801 have been patented as an antineurotoxic agent. In addition, other opioids and nonopioids, including naloxone, have also been reported to protect against neurotoxicity in vitro . The stoichiometry of the glycine modulation of the NMDA receptor has been determined. It is clear that the foundation now exists to support rapid future progress in the area of excitatory amino acid research.

Chapter 2. Neuronal Cell Death and Strategies for Neuroprotection

Publisher Summary Necrotic cell death, prominent in acute neurological conditions, such as stroke, is characterized by swelling of the cell and disruption of the internal and external membranes and cell lysis. Acute neuronal death is easily modeled in primary neuronal cell culture and brain slices. In apoptosis, cells undergo nuclear condensation, fragmentation, and nuclear fragments, along with intracellular organelles. The apoptotic process is energy dependent, and in some instances requires ongoing RNA and protein synthesis. High levels of intracellular calcium [Ca 2+ ] i is thought to be the primary causative event in mediating necrotic neuronal death. Calcium enters the neuron, through a variety of mechanisms, including ligand-gated ion channels, such as the N-methyl-D-aspartate (NMDA) glutamate receptor, voltage-gated calcium channels, leak channels, and reversal of the Na + -Ca 2+ anti-porter. Neurons also have inositol trisphosphate receptors (IPS) on intracellular organelles that can liberate calcium from intracellular stores. Alterations in the mosaic of phosphorylated products, with concomitants, increase and decrease in the activity of cellular enzymes, and ion channels, influence the expression of immediate early genes (IEGs) that is involved in programmed cell death. Sporadic energy supplies disrupt ion homeostasis as in focal ischemia, where depolarization in the penumbra is initially moderate and/or intermittent although adenosine triphosphate (ATP) is produced. Important clues, regarding apoptotic cell death, have come from genetic studies in the nematode, caenorhabditis elegans.

Excitatory amino acids: 6-phosphonomethyltetrahydro-4-pyrimidinecarboxylic acids and their acyclic analogues are competitive N-methyl-D-aspartic acid receptor antagonists.

Abstract 6-Phosphonoalkyltetrahydro-4-pyrimidinecarboxylic acids and their acyclic equivalents were determined to be competitive N-methyl-D-aspartic acid (NMDA) receptor antagonists using a [3H]-CPP binding assay. Results suggest that internal hydrogen bonding interactions may exert either a positive or negative influence on NMDA receptor affinity depending on whether an appropriate receptor active configuration is stabilized.

Synthesis and anticonvulsant activity of the (+)- and (−)-enantiomers of 1,2,3,4-tetrahydro-5-(2-phosphonoethyl)-3-isoquinolinecarboxylic acid, a competitive NMDA antagonist

Abstract The (+)- and (−)-enantiomers of 1,2,3,4-tetrahydro-5-(2-phosphonoethyl)-3-isoquinoline-carboxylic acid ( 12 and 13 ) were prepared. The anticonvulsant activity of 12 and 13 and their parent ( 14 ) was compared to the reference agent, CPP, in the maximal electroshock assay in mouse. 12 was similarly potent to CPP, three-fold more potent than the racemate and twenty-fold more potent than 13 in this assay.