Assay activity

In Table 2, results are given comparing the three above FNAA techniques for total nitrogen and corresponding HMX content of three synthetic Octol samples containing 70, 60 and 80% HMX respectively. Chemical analysis by extraction is also included as another basis for comparison. An 80/20 Octol (80% HMX) was used as the reference standard for the activation assays... 

Many more papers deal with rhizosphere phosphatase activity (63-83) in the presence of a number of different plant species this will partly be due to the simplicity of the enzyme activity assay (85,86) and the generally reported, well-correlated variation trends among organic and inorganic phosphorus content and phosphatase activity. More precisely, closer to the roots, the inorganic P depletion zone in comparison with bulk soil is more pronounced in addition, organic and inorganic P contents are inversely correlated, and the mineralization rate of or-... 

Examples of such systems include the reactions of kinases, phosphatases, hydroxylases, acetylases, ubiquitin transferases, and many other enzyme classes that represent attractive targets for drug discovery. There are several mechanisms by which an enzyme can catalyze these types of reactions, and the details of the mechanism are important in determining the best approach to designing activity assays for the enzyme and for proper evaluation of inhibitors that are identified through those activity assays. 

Achieving this goal depends on use of a well designed activity assay for the target enzyme. 

Figure 4.6 Reaction velocity as a function of enzyme concentration for a non-ideal enzymatic activity assay. Note the deviations from the expected linear relationship at low and at high enzyme concentration.

Thus recombinant enzyme constructs for use in activity assays should be designed to faithfully reflect the physiological state of the enzyme to the extent that is practical in vitro. 

Figure 5.8 Dilution scheme for testing the reversibility of an enzyme inhibition The enzyme and inhibitor are pre-incubated at a concentration of enzyme equal to 100-fold that needed for activity assay, and at a concentration of inhibitor equal to 10-fold the IC50 value. The sample is then rapidly diluted 100-fold into an assay solution. The inhibitor concentration thus goes from 10-fold above die IC50 (corresponding to 91% inhibition) to 10-fold below the IC50 (corresponding to 9% inhibition).

The ultimate goal of lead optimization is to produce compounds that will elicit the desired cellular and organismal phenotype when dosed at appropriate concentrations. During the course of lead optimization activities it is common for pharmacologists to evaluate compounds not only using in vitro enzyme activity assays but also in cell-based assays as well. A question that often arises at this stage of drug discov-... 

Figure 5.12 Diagramatic illustration of the possible correlation between compound potency in cellular and enzymatic activity assays when the cellular phenotype is a direct result of inhibition of the target enzyme. Compounds that fall into the lower left and upper right quadrants demonstrate a correlation of rank-order potency between the cellular and cell-free assays. Compounds in the upper left quadrant may represent potent enzyme inhibitors that for some reason do not achieve adequate intracellular concentrations, as described in the text. Note the absence of any compound points in the lower right quadrant. Population of this quadrant would usually be inconsistent with enzyme inhibition being the direct cause of the observed cellular phenotype.

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

Addition of the L-732,531 FKBP binary complex to a calcineurin activity assay resulted in increasingly nonlinear progress curves with increasing binary complex concentration. The htting of the data to Equation (6.3) revealed an inhibitor concentration effect on v-, as well as on vs and obs, consistent with a two-step mechanism of inhibition as in scheme C of Figure 6.3. Salowe and Hermes analyzed the concentration-response effects of the binary complex on v, and determined an IC50 of 0.90 pM that, after correction for I.S I/A (assuming competitive inhibition), yielded a A) value for the inhibitor encounter complex of 625 nM. 

As stated earlier, the velocity terms are dependent on the concentration of substrate, relative to KM, used in the activity assay. Likewise in an activity assay the free fraction of enzyme is also in equilibrium with the ES complex, and potentially with an ESI complex, depending on the inhibition modality of the compound. To account for this, we must replace the thermodynamic dissociation constant Kt with the experimental value K-pp. Making this change, and substituting Equations (7.4) and (7.6) into Equation (7.7), we obtain (after canceling the common E T term in the numerator and denominator)... 

In vitro activity assay (for pharmacological activity Activity of the metabolite toward the therapeutic target is determined in vitro with a purified sample. Sometimes, the 0.1-10 mg Mammalian or Microbial bioreactors... 

P-gp-ATPase Activation Assays H-Bonding Determines Activation Rate... 

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