Affinity competition experiment

Fig. 3.12 The ALIS affinity competition experiment 50% inhibitory concentration (ACE50) method. (A) The warfarin versus warfarin-De ALIS competition data from Fig. 3.9, normalized and plotted on a logarithmic axis, yields the ACE50 value, which is the titrant concentration at which the ligand binding is reduced by 50%. (B) Simulated...

Of course, saturation experiments are only possible when a radiolabeled form of the ligand of interest is available. Competition experiments, on the other hand, are particularly useful in allowing the determination of dissociation constants for unlabeled drugs which compete for the binding sites with a ligand that is available in a labeled form. This approach has been widely adopted by the pharmaceutical industry as a rapid means of determining the affinity of novel compounds for a particular receptor for which a well-characterized radioligand is available. 

The affinity constant of the test compound K can in turn be calculated from the IC50 value, the Kj of the marker and the concentration of M according to Cheng-Prusoff [see Eq. (3)] [22]. Since the concentration of free marker (M) is usually not determined in radioligand binding assays, it should be noted that the calculation of Ki according to Eq. (3) based on Mtot yields only reliable results if the depletion of the marker is negligible (i.e. <5% to 10% [7, 17, 21]). In competition experiments performed under typical conditions (Ttot Mtot this criterion is fulfilled. 

Fig. 10.8 SAR by MS applied to the A1061 construct (see text), (a) Structures of key motifs screened against the RNA target. Compound A is a D-amino acid. Compounds B1 and B2 are quinoxalin-2,3-diones. Compound AB is the rigid biaryl linked compound, (b) Binding affinity for the motifs when screened individually as well as binding affinity for motifs when screened in competition experiments are shown. Binding...

Finally, two non-equilibrium assays allow comparison of relative ligand affinities of PBPs. First, treatment of the PBP with a photoactivatable analog, such as a diazoacetate (e.g. 7a) followed by photoactivation, permits dissection of close contacts between the ligand and the PBP (Du et al., 1994). Competition experiments,... 

The substrate-specific condensation of aromatic carboxylates 52 and 53 having strong affinity for the CD cavity of 54 was realized with in-situ activation by formation of an acyloxytriazine, which undergoes aminolysis to give an amide [43]. Competitive experiments between 52 and 53 showed high selectivity, resulting in the major formation of compound 55 over compound 56 (Scheme 13.13). The observed selectivity can be attributed to differences in the affinity of the substrates for the CD cavity (Scheme 13.13). Most importantly, the CD-based catalyst 54... 

Chemical modification can be used to obtain information on the catalytic mechanism and on the catalytic site of the enzyme of interest. One goal in the design of affinity labels for enzymes is to determine the catalytically important residues. First, the affinity label has to behave as an analogue of the substrate (or of the activator or inhibitor) by competition experiments. 

Since the major driving force of inclusion is hydrophobic interaction, stabilities of ICs depend strongly on the polarity of the polymer. The more hydrophobic the polymer is, the higher is the affinity of CDs towards it. On the other hand, solubility in water decreases with increasing hydrophobicity of the polymer. Since affinity and solubility have to be compromised, an optimum of polarity of the polymer should exist for complexation by CDs. It is difficult to quantify the binding free energies of CD channel inclusion compounds, since they are insoluble in water. Stabilities of these polymeric ICs can be qualitatively compared by competition experiments. For example, PLLA and PCL were competitively included in a-CD. The IC of PCL was... 

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