Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing receptor desensitization and/or deactivation. Here we describe the synthesis and pharmacological testing at GluA2 of a new generation of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. The most potent modulator 3 in complex with GluA2-LBD-L483Y-N754S was subjected to structural analysis by X-ray crystallography, and the thermodynamics of binding was studied by isothermal titration calorimetry. Compound 3 binds to GluA2-LBD-L483Y-N754S with a Kd of 0.35 μM (ΔH = -7.5 kcal/mol and -TΔS = -1.3 kcal/mol). This is the first time that submicromolar binding affinity has been achieved for this type of positive allosteric modulator. The major structural factor increasing the binding affinity of 3 seems to be interactions between the cyclopropyl group of 3 and the backbone of Phe495 and Met496.

Background: Molecular features governing ␣4␤2 nAChRs efficacy have remained elusive. Results: Binding studies, electrophysiology, and structural data from co-crystallization with Ls-AChBP are reported for a series of ␣4␤2 agonists. Conclusion: Direct halogen bonds and an invariant Loop-C suggest that intersubunit bridge formation governs efficacy. Significance: The data provide a structural basis for understanding of efficacy levels at nAChRs.

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6 μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1 kcal≈4.187 kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46 mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Expression of phosphate starvation inducible genes was studied during recovery from phosphate starvation of Arabidopsis thaliana. Genes analysed were ACP5 (encoding an acid phosphatase), RNS1 (encoding an RNase), At4 and IPS1 (both of unknown function), Pht1;7 and Pht2;1 (encoding phosphate transporters). Plants grown at limiting phosphate exhibited reduced growth rate and accumulated anthocyanins, soluble sugars, and starch. Re-supply of phosphate resulted in dramatic uptake of phosphate, increased growth rate and decreased levels of anthocyanins, soluble sugars, and starch in leaf tissue. In both shoots and roots re-supplied with P i , transcript levels decreased rapidly, and first changes were observed within 30 min. These alterations in gene expression occurred before the content of carbohydrates decreased, indicating that transcriptional regulation was due to phosphate sensing, and not a secondary effect of carbon accumulation. The data reveal different response rates for individual genes and demonstrate that roots and shoots can differ with respect to both timing and genes responding. In general, the changes in transcriptional activity in roots preceded the changes in shoots. Furthermore, transcriptional regulation was observed in isolated roots and shoots. This implies that roots do not strictly require a signal from the shoot, and vice versa, in order to respond to phosphate starvation.