To better characterize the roles of metabotropic glutamate receptors (mGluRs) in physiological and pathophysiological processes, there is an important need to learn more about the structural features relevant to the design of novel, high-affinity ligands that are family and subtype specific. To date, many of the biological studies that have been conducted in the area of mGluR research have made use of the agonist (1S,3R)-ACPD. This compound has been shown to act as an agonist at both the group I and group II receptors while showing little selectivity among the four subtypes belonging to these two groups. Moreover, (1S,3S)-ACPD, the cis isomer, shows negligible activity at group I receptors and is a good agonist of mGluR2. Since ACPD is itself somewhat flexible, with four distinctive conformations being identified from molecular modeling studies for the trans isomer and five conformations for the cis isomer, we believed that it would be of interest to examine the activity of an ACPD analogue that has been constrained through the introduction of a single carbon atom bridge. Accordingly, we have prepared an aminobicyclo[2.1.1]hexanedicarboxylic acid (ABHxD-I) analogue of ACPD. The synthesis of this compound was accomplished by use of an intramolecular [2 + 2] photocycloaddition reaction, in which four distinct isomers were isolated. Of these four compounds, only a single isomer, ABHxD-I (6a), was found to be a potent agonist of the mGluRs. This compound, which expresses the fully extended glutamate conformation, was found to be more potent than ACPD at all six of the eight mGluR subtypes that were investigated and to be comparable to or more potent than the endogenous ligand, glutamate, for these receptors. Interestingly, despite its fixed conformation, ABHxD-I, like glutamate, shows little subtype selectivity. Through modeling studies of ABHxD-I (6a), ABHD-VI, LY354740, (1S,3R)-ACPD, (1S, 3S)-ACPD, and l-glutamate, we conclude that the aa conformation of l-glutamate is the active conformation for both group I and group II mGluRs. Moreover, the modeling-based comparisons of these ligands suggest that the selectivity exhibited by LY354740 between the group I and group II mGluRs is not a consequence of different conformations of L-glutamate being required for recognition at these mGluRs but rather is related to certain structural elements within certain regions having a very different impact on the group I and group II mGluR activity. The enhanced potency of ABHxD-I relative to trans-ACPD commends it as a useful starting point in the design of subtype selective mGluR ligands.

A comparative study of the neuroprotective and nootropic activities of two pharmaceutical substances, the HLDF-6 peptide (HLDF-6-OH) and its amide form (HLDF-6-NH2), was conducted. The study was performed in male rats using two models of a neurodegenerative disorder. Cognitive deficit in rats was induced by injection of the beta-amyloid fragment 25-35 (βA 25-35) into the giant-cell nucleus basalis of Meynert or by coinjection of βA 25-35 and ibotenic acid into the hippocampus. To evaluate cognitive functions in animals, three tests were used: the novel object recognition test, the conditioned passive avoidance task and the Morris maze. Comparative analysis of the data demonstrated that the neuroprotective activity of HLDF-6-NH2, evaluated by improvement of cognitive functions in animals, surpassed that of the native HLDF-6-OH peptide. The greater cognitive/ behavioral effects can be attributed to improved kinetic properties of the amide form of the peptide, such as the character of biodegradation and the half-life time. The effects of HLDF-6-NH2 are comparable to, or exceed, those of the reference compounds. Importantly, HLDF-6-NH2 exerts its effects at much lower doses than the reference compounds.

Metabotropic glutamate receptors mGluR1 and mGluR5 stimulate phospholipase C, leading to an increased inositol triphosphate level and to Ca 2+ release from intracellular stores. Cyclothiazide (CTZ), known as a blocker of AMPA receptor desensitization, produced a non-competitive inhibition of [Ca 2+ ] i increases induced by mGluR agonists in HEK 293 cells transfected with rat mGluR1a but had no effect on the [Ca 2+ ] i signals in cells expressing rat mGluR5a. In cells expressing mGluR1, CTZ also inhibited phoshoinositide hydrolysis, as well as cAMP accumulation and arachidonic acid release induced by mGluR1 agonists, indicating a direct inhibition of the receptor and not of a particular signal transduction system. However, CTZ failed to antagonize cAMP inhibition stimulated by rat mGluR2, -3, -4, -6, -7 and -8 receptors confirming its selectivity for mGluR1. The use of chimeric receptors with substituted N-terminal domains showed that CTZ did not interact with the N-terminal mGluR1a domain. Instead, mutation analysis revealed that CTZ interacts with the Thr-815 and Ala-818 residues, located at the 7 th transmembrane domain, similarly as the mGluR1-selective antagonist CPCCOEt. In primary cultures of cerebellar granule neurons, expressing native metabotropic and ionotropic glutamate receptors, the final outcome of CTZ effects depended on its combined ability to potentiate AMPA receptors and inhibit mGluR1a receptors.

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