Functional assays receptor activation

The majority of functional assays involve primary signaling. In the case of GPCRs, this involves activation of G-proteins. However, receptors have other behaviors— some of which can be monitored to detect ligand activity. For example, upon stimulation many receptors are desensitized through phosphorylation and subsequently taken into the cell and either recycled back to the cell surface or digested. This process can be monitored by observing ligand-mediated receptor internalization. For... 

A series of 3,5-diarylisoxazole and 3,5-diaryl-l,2,4-oxadiazole derivatives, novel classes of small molecule interleukin-8 (IL-8) receptor antagonists, 456 (Ar = 4-FC6H4), have been identified as IL-8 inhibitors. These compounds exhibit activity in an IL-8 binding assay as well as in a functional assay of IL-8 induced... 

Affinities/functional activities at relevant molecular targets Pharmacological profiling — binding or functional assays at receptors, enzymes, ion channels, transporters Wakefield et al.,-83 Bowes et al.20... 

Interestingly, the two diastereomers 18 and 19 are equipotent to All in binding to the ATj receptor. In a functional assay, using contraction of rabbit aorta rings, one of these diastereomers is a full agonist, but has approximately 300-fold less potency that All. Both diastereomers 20 and 21 were devoid of activity. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

Interestingly, the conformational changes observed in these biophysical assays were correlated with biological responses in functional assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine... 

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