Initial assay definition

In our study we compare two diversity-driven design methods (uniform cell coverage and clustering), two analysis methods motivated by similarity (cell-based analysis and cluster-classification), and two descriptor sets (BCUT and constitutional). Thus, our study addresses some of the many questions arising in a sequential screen how to choose the initial screen, how to analyze the structure-activity data, and what molecular descriptor set to use. The study is limited to one assay and thus cannot be definitive, but it at least provides preliminary insights and reveals some trends. 

Calculation of the Endocellulase Activity from the Intrinsic Viscosity Values. The enzymic degradation of polymeric substrates can occur at different bonds in the same substrate molecule, and the enzymic activity has to be defined here as the initial number of moles of glyco-sidic bonds split per second (53). This definition corresponds to the definition of the katal, symbolyzed kat. This unit is defined as the catalytic amount of any catalyst (including any enzyme) that catalyzes a reaction rate of one mole per second in an assay system (54), and it is recommended by the International Union of Pure and Applied Chemistry (55) for the quantitative evaluation of catalytic activities. 

The definition of an antioxidant suggests a functional assay of antioxidants by measuring inhibition of appropriate (easy to study) oxidation reactions. Such assays can be called inhibition assays for antioxidants (Fig. 2). Various oxidants are used in TAC assays. In many cases, thermal decomposition of 2,2 -azobis (2-amidopropane) (ABAP) is the source of oxidizing radicals. ABAP undergoes temperature-dependent homolysis. The primary radicals produced by thermal decomposition of the initiator react with oxygen to produce peroxyl and alkoxyl radicals, which are oxidizing species in the system (Fig. 3). The amount of free radicals formed in an aqueous medium by decomposition of ABAP at pH 7.4 and at 37°C equals 1.36 x 10-6 [ABAP] x t, where t is time in seconds and [ABAP] is in mol L-1 (N8). 

Once the assay and assay format have been decided upon, the next step in the discovery process is to initiate compound screening for the purpose of identifying hits or lead compounds. The fundamental requirement is that the assay results identify a collection of actives or hits. The definition of hit varies between organizations, but most accept the definition that the compound shows a confirmed structure, shows a confirmed dose response, exhibits an IC50 < lOpM potency, and is a member of a chemotype that is amenable to analoging and fast follow-on synthesis. 

The similarity of ecdysteroids and brassinosteroids may cause some difficulties in their definition. Ecdysteroids were initially defined as "all compounds structurally related to ecdysone" (3). This definition would include brassinosteroids. Recently an attempt has been made by Lafont and Horn to redefine ecdysteroids (6). According to their definition one may "distinguish between true ecdysteroids as compounds where the steroid bears a cis-fused A/B ring junction, a 7-ene-6-one chromophore and a 14a-OH (irrespective of activity in a moulting hormone assay) and ecdysteroid-related compounds which do not fulfil all the above criteria (either they lack the 14a-OH, or the 7-ene, or they present one additional 4- or 14-ene double bond, etc.)" (6). The latter group would include brassinosteroids. Unfortunately, comparable definitions of brassinosteroids are not available so far. 

Having established a spectrophotometric assay for enzyme activity (see below), it is vital before setting off to do any detailed measurements of enzyme activity to check two factors. The first of these is that true initial rates are being measured. A plot of product (P) appearance (in this case some spectral change) against time (t) must be linear over the period of measurement to measure true initial rates (the slope of the [P]/t curve is, by definition the initial rate v). There are a number of reasons that such [P]/t curves may deviate from linearity ... 

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