As part of our ongoing research program aimed at the identification of highly potent, selective, and systemically active agonists for group II metabotropic glutamate (mGlu) receptors, we have prepared novel heterobicyclic amino acids (-)-2-oxa-4-aminobicyclo[3.1. 0]hexane-4,6-dicarboxylate (LY379268, (-)-9) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-10). Compounds (-)-9 and (-)-10 are structurally related to our previously described nanomolar potency group II mGlu receptor agonist, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740 monohydrate, 5), with the C4-methylene unit of 5 being replaced with either an oxygen atom (as in (-)-9) or a sulfur atom (as in (-)-10). Compounds (-)-9 and (-)-10 potently and stereospecifically displaced specific binding of the mGlu2/3 receptor antagonist ([3H]LY341495) in rat cerebral cortical homogenates, displaying IC50 values of 15 +/- 4 and 8.4 +/- 0.8 nM, respectively, while having no effect up to 100 000 nM on radioligand binding to the glutamate recognition site on NMDA, AMPA, or kainate receptors. Compounds (-)-9 and (-)-10 also potently displaced [3H]LY341495 binding from membranes expressing recombinant human group II mGlu receptor subtypes: (-)-9, Ki = 14.1 +/- 1.4 nM at mGlu2 and 5.8 +/- 0.64 nM at mGlu3; (-)-10, Ki = 40.6 +/- 3.7 nM at mGlu2 and 4.7 +/- 1.2 nM at mGlu3. Evaluation of the functional effects of (-)-9 and (-)-10 on second-messenger responses in nonneuronal cells expressing human mGlu receptor subtypes demonstrated each to be a highly potent agonist for group II mGlu receptors: (-)-9, EC50 = 2.69 +/- 0.26 nM at mGlu2 and 4.58 +/- 0.04 nM at mGlu3; (-)-10, EC50 = 3.91 +/- 0.81 nM at mGlu2 and 7.63 +/- 2. 08 nM at mGlu3. In contrast, neither compound (up to 10 000 nM) displayed either agonist or antagonist activity in cells expressing recombinant human mGlu1a, mGlu5a, mGlu4a, or mGlu7a receptors. The agonist effects of (-)-9 and (-)-10 at group II mGlu receptors were not totally specific, however, as mGlu6 agonist activity was observed at high nanomolar concentrations for (-)-9 (EC50 = 401 +/- 46 nM) and at micromolar concentrations (EC50 = 2 430 +/- 600 nM) for (-)-10; furthermore, each activated mGlu8 receptors at micromolar concentrations (EC50 = 1 690 +/- 130 and 7 340 +/- 2 720 nM, respectively). Intraperitoneal administration of either (-)-9 or (-)-10 in the mouse resulted in a dose-related blockade of limbic seizure activity produced by the nonselective group I/group II mGluR agonist (1S,3R)-ACPD ((-)-9 ED50 = 19 mg/kg, (-)-10 ED50 = 14 mg/kg), indicating that these molecules effectively cross the blood-brain barrier following systemic administration and suppress group I mGluR-mediated limbic excitation. Thus, heterobicyclic amino acids (-)-9 and (-)-10 are novel pharmacological tools useful for exploring the functions of mGlu receptors in vitro and in vivo.

We report an experimental approach for high-resolution real-time monitoring of transiently formed species occurring during the onset of precipitation of ionic solids from solution. This is made possible by real-time nuclear magnetic resonance (NMR) monitoring using dissolution dynamic nuclear polarization (D-DNP) to amplify signals of functional intermediates and is supported by turbidimetry, cryogenic electron microscopy, and solid-state NMR measurements. D-DNP can provide drastic signal improvements in NMR signal amplitudes, permitting dramatic reductions in acquisition times and thereby enabling us to probe fast interaction kinetics such as those underlying formation of prenucleation species (PNS) that precede solid–liquid phase separation. This experimental strategy allows for investigation of the formation of calcium phosphate (CaP)-based minerals by 31P NMRa process of substantial industrial, geological, and biological interest. Thus far, many aspects of the mechanisms of CaP nucleation remain unclear due to the absence of experimental methods capable of accessing such processes on sufficiently short time scales. The approach reported here aims to address this by an improved characterization of the initial steps of CaP precipitation, permitting detection of PNS by NMR and determination of their formation rates, exchange dynamics, and sizes. Using D-DNP monitoring, we find that under our conditions (i) in the first 2 s after preparation of oversaturated calcium phosphate solutions, PNS with a hydrodynamic radius of R h ≈ 1 nm is formed and (ii) following this rapid initial formation, the entire crystallization processes proceed on considerably longer time scales, requiring >20 s to form the final crystal phase.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.

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