Functional characterisation of A2A receptor thermostable mutants using a yeast signalling assay

G-protein-coupled receptors (GPCR) are transmembrane proteins that play a crucial role in the communication of cells with their external environment. In the last few years, several GPCR crystal structures have been solved using genetically engineered protein. The turkey β1-adrenergic receptor, the human neutrotensin 1 receptor and the adenosine A2A receptor (A2AR) structures involved the introduction of stabilizing mutations. The engineered mutant can be stabilized in an agonist or an antagonist bound conformation making the GPCR less flexible and therefore easier to crystallize. The aim of this study was to use functional characterization of the key thermostabilising mutants of the A2AR in order to understand the molecular basis of the thermostabilisation. The different mutants were characterized using a yeast-based growth assay, which measures down-stream signaling in response to agonist and radioligand binding analysis using both an agonist and an antagonist. Point mutations leading to a reduction/loss of constitutive receptor activity have been identified. In addition, a single point mutation abolishing the ability of receptor to bind the agonist NECA has also been identified. Conformational stabilization of the receptor is thus achieved by reducing basal activity along with modifying the ligand-binding pocket leading to inability to bind agonist. Such markers can be used to screen for stable mutants for structural characterization. Since thermostabilising mutations are not directly transferable across receptors, the yeast based growth assay could serve as a quick and inexpensive way to screen for mutations for a wide range of GPCRs.Open Acces

Compounds which potentiate AMPA receptor and uses thereof in medicine

A compound of formula (I) and salts thereof are provided wherein Y is defined in the specification. Processes for preparation, pharmaceutical compositions, and uses thereof as a medicament, for example in the treatment of a disease or condition mediated by a reduction or imbalance in glutamate receptor function, such as schizophrenia or cognition impairment, are also disclosed

Compounds which potentiate AMPA receptor and uses thereof in medicine

A compound of formula (I) and salts thereof are provided wherein Y is defined in the specification. Processes for preparation, pharmaceutical compositions, and uses thereof as a medicament, for example in the treatment of a disease or condition mediated by a reduction or imbalance in glutamate receptor function, such as schizophrenia or cognition impairment, are also disclosed

Compounds which potentiate the AMPA receptor and uses thereof in medicine

Compounds of formula (I) and salts thereof are provided: wherein n is 0, 1, 2 or 3; R1 is selected from phenyl and pyridyl, each of which is optionally substituted by one or two groups independently selected from C1-4alkyl and halogen; and R2 is selected from H and CH3 when n is 1 and R2 is H when n is 2 or 3. Processes for preparation, pharmaceutical compositions, and uses thereof as a medicament, for example in the treatment of a disease or condition mediated by a reduction or imbalance in glutamate receptor function, such as schizophrenia or cognition impairment, are also disclosed

Functional profiles of constructs illustrating the major effects of the R199A mutation.

<p>The R199A mutation reduces constitutive activity and potency, both alone (Rag23.28) and in combination with other mutations (Rag23.6, Rag23.17, Rag23.21, Rag23.24 and Rag23.28). The curves (A) are the average of two experiments performed in triplicate. The table (B) shows the pharmacological profile of each mutant characterized. The colours of the curves correspond to those in the table. The data for the WT (pink) are also shown for comparison.</p

Tetrazole compounds as calcium channel blockers

The present invention relates to novel tetrazole compounds; to processes for their preparation; to pharmaceutical compositions containing the compounds; and to the use of the compounds in therapy to treat diseases for which blocking the Cav2.2 calciu