Biological Effect Assay

Compounds from all clusters were submitted to both human and rat antilipolytic assays in adipocytes. The assays were based on forskolin-induced lipolysis and glycerol release in the presence of GPR81 compounds. The release of glycerol was calculated with a standard curve. Compounds were tested in a 10-point concentration response using triplicate plates. 

This was one of the most important steps in the HTS evaluation. The overarching working hypothesis to run an HTS was not simply to identify new chemical entities activating GPR81. The main objective was to demonstrate that activation of GPR81 leads to an antilipolytic effect on the most relevant cell line, the adipocyte. We knew from earlier studies that such an effect should translate into an in vivo antilipolytic effect with lowering of 

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. 

Hu GPR81 CAMP pECso 7.7 Top effect hu GPR81 cAMP 108% 

Hu GTPyS pECso 7.6 Hu antilipolysis unbound pICso 7.4 Activity in counter screen No Hu Ghrelin binding piCso 8.0 (inverse agonist) 

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We have already stressed the potential importance of lipid-rich membranes in the skin as potential targets for ROS-induced damage and ageing of human skin is morphologically identical to changes found by peroxidative processes (Serri et al., 1977). The involvement of AA metabolites in skin disease, and in particular psoriasis, has been the subject of much recent interest. Studies have included topical and intradermal administrations of AA metabolites, and assay of such products in clinical specimens. Results show that concentration of AA, 12-hydroxy-eicosatetraenoic acid (12-HETE), PG and leu-kotrienes are increased in psoriatic lesions (Hammarstrom etal., 1975 Camp etal., 1983 Brain etal., 1984 Duell et al., 1988) and also that full-thickness epidermis from normal and diseased skin has the enzymatic capacity to convert AA to some of the same metabolites (Hammarstrom etal., 1975, 1979 Camp etal., 1983 Brain etal., 1984 Ziboh et al., 1984 DueU et al., 1988). The biological effect of both 12-HETE and leukotrienes was confirmed by both topical application and intradermal injection, which caused epidermal inflammation and... 

Molecules which exhibit optical activity are molecules which have a handedness in their structure. They are chiral . Chemists often have reasons to obtain chemical pure aliquots of particular molecules. Since the chirality of molecules can influence biological effect in pharmaceuticals, the chiral purity of a drug substance can pose a challenge both in terms of obtaining the molecules and in assaying the chiral purity by instrumental methods. While diastereomers can have different physical properties including solubility, enantiomers have the same physical properties and the same chemical composition. How then to separate optically active molecules ... 

Laboratory methods for detecting marine toxins (Table 7.2) can be characterized into two types of analyses indirect assays and direct measurement analyses. Indirect assays, or bioassays, measure the biological effect of a toxin on a system and can implicate, but not verify, the presence of a particular toxin. By contrast, direct measurements can both confirm and quantify the amount of a specific toxin in a food or biological specimen. 

Preference is obviously for a simple chemical assay for PSP. Unfortunately the more specific the chemical test, the narrower is the window of compounds it can assay. The Paralytic Shellfish Poison is not just Saxitoxin (STX) as originally believed, but is a mixture of compounds closely related to STX Q) and the mix varies widely with location and with time ( ). It would seem, therefore that a chemical assay should determine at least the ratios of the several compounds, and that the relative toxicity of each of the compounds must be known. An effective assay must evaluate the actual biological toxicity of the shellfish being tested. For the chemical assay this requires the summated toxicity of all the... 

Collins, A.R., Assays for oxidative stress and antioxidant status applications to research into the biological effectiveness of polyphenols. Am. J. Clin. Nutr., 81, 261S, 2005. 

For proteins that are not enzymes, other quantification methods are required. Transport proteins can be assayed by their binding to the molecule they transport, and hormones and toxins by the biological effect they produce for example, growth hormones will stimulate the growth of certain cultured cells. Some structural proteins represent such a large fraction of a tissue mass that they can be readily extracted and purified without a functional assay. The approaches are as varied as the proteins themselves. 

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