CAMP assays

There are cAMP assays that can be measured using a variety of techniques [35], including HTRF (CisBio) [36], bioluminescence (cAMP-Glo, Promega) [37], TR-FRET (CisBio) [38], LANCE [39], Alphascreen (Perkin Elmer) [40] and Enzyme Fragment Complementation (EFC) [41] (Hithunter, DiscoverX). This review will focus on the LANCE, the TR-FRET and ECF assays as they are the most used in our laboratories. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Fusion of a barbituric acid motif and a pyrone ring afforded compounds containing a novel pyranopyrimidine core (L) that were discovered as GPR109A agonists [92,93]. This core appears to be distinct from other fused bicyclic cores such as xanthine and anthranilide based on their poor overlap. Furthermore, several compounds, exemplified by 39 and 40, provided remarkable potency in the cAMP assay. The critical acidic functional group is present as the N-PI of barbituric acid motif which has a calculated pKa of 8 [82]. 

Weber, M., Ferrer, M., Zheng, W., Inglese, J., Strulovici, B., and Kunapuli, P., A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation, Assay Drug Dev. Technol., 2, 39, 2004. 

The selected 4-bismethanesulfonylamino-2-phenyl-l,2,4-triazolo[4,3-a] quinoxalin-l-one, which shows high hA3 affinity (13, K. = 5.5 nM) and selectivity versus hA1( hA2A (both selectivity ratios > 1,800) and hA2B (cAMP assay, IC50 > 10,000 nM) receptors, recently showed protective effect on cerebral ischemia induced by oxygen and glucose deprivation (Colotta et al. 2008). 

For BSA assay, to 1.5 ml of buffer-BSA solution, typically 40 fi of a 1 100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40 /aI of a 1 10 dilution of urease-BSA conjugate were added. For cAMP assay, 30 ju.1 of a 1 10 dilution of the (NH4)2S04 fraction of cAMP antiserum (0.5 mg/ml) and 30/al of a 1 10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.0 ml of buffer or buffer-nucleotide standard. 

The cyclic adenosine monophosphate (cAMP) assay, in which the ability to inhibit forskolin-induced cAMP production is measured. 

We thank Dr. H. Hashemzadeh for his assistance in performing some of the cAMP assays, and Mrs. L. Caesar for her technical assistance. Supported in part by NIH training grant T32 ES07039. 

G Protein Binding (pS]-GTPyS Binding) Nonselective Assay G Protein Signaling (cAMP Assay/SPA-IPs Assay) Selective Assays p-Arrestin Recruitment Assay... 

G Protein Signaling (cAMP Assay/SPA-IPs Assay) Selective Assays... 

Provided in DiscovePoc HitHunter cAMP Assay for small molecules or biologies (cat. no. 90-0075) ... 

Urinary cAMP was analyzed by mean of cAMP assay kit (Radiochemical Centre Amersham England). Samples were added to a liquid scintillant mixture (Instagel, Packard) and counted in a liquid scintillation system Unilux I Nuclear Chicago C 6850- (Normal range in our laboratory is 1440 - 4500 nmoles/24 hours) (8). 

Representative compounds from 10 clusters were tested in a rat GPR81 cAMP assay. Surprisingly, none of the cluster 200 compounds showed any activity in the rat assay, while cluster 86 showed a clear 1 1 correlation between human and rat data. As for the human receptor, most cluster 86 compounds behaved as full agonists on the rat GPR81 receptor. 

None of the cluster 200 compounds showed any activity in the rat antihpolysis assay. This was expected since no compound showed any agonistic activity on the rat GPR81 receptor in the rat cAMP assay. In human adipocytes, only the adamantyi-substituted compound 4 had an antihpolytic effect up to 80% with an ECso aroimd 5 pM. 

Compound 4, ECso = 120nM and a top effect of 60% in the cAMP assay, was the most potent and efficacious compound of the series. The compound showed bell-shaped behavior in the human cAMP assay at 30 pM concentration. It was unclear if the partial antilipolytic effect of the compound seen in human adipocytes was due to its partial agonism in the GPR81 cAMP assay or due to low solubility (1 pM) and/or toxicity at high concentrations. The next most promising compounds had EC50 values between 200 and 300 nM with a top efficacy at 45-50% and no drop-off in efficacy at high concentrations (Scheme 21.2). 

Figure 21.26 There is an excellent correlation the top effect in the GPR81 cAMP assay black between GPR81 and antilipolytic unbound 100% and white 72%. potency with a = 0.961. The shades indicate...

Table 21.6 -values resulting from the straight line fit between the various GPCR cAMP assays. 

---