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their optimization as drug leads, including pharmacokinetic, metabolism, and excretion profiles.

The dissociation rates of certain protein-ligand complexes can be affected by binding of allosteric ligands. This effect is especially well known for GPCRs, where the dissociation of active site antagonists can be retarded by binding of an allosteric ligand. In GPCRs, this effect is attributed to blocking the channel in which the antagonists bind, thus inhibiting their escape from their binding site. The ALIS quench method can be used to evaluate the effect of an allosteric ligand on the dissociation rate of another ligand [49]. The M2 receptor ligands N-methyl scopolamine (NMS) and W-84 bind allosterically with respect to one another, with binding by one reducing the affinity of binding by the other. Figure 3.17 shows the result of quenching an equilibrated mixture of the M2 receptor plus NMS by

Time, min

Fig. 3.17 (A) ALIS-MS results from quenching an equilibrated mixture of 2.0 mM M2 receptor plus 1.5 mM NMS by 200 mM of the isosteric ligand NMS-D3, in the presence and absence of the known allosteric ligand W-84 at 50 mM concentration. Binding by the allosteric ligand W-84 decreases the off-rate of NMS. (B) Compound structures.

Time, min

Fig. 3.17 (A) ALIS-MS results from quenching an equilibrated mixture of 2.0 mM M2 receptor plus 1.5 mM NMS by 200 mM of the isosteric ligand NMS-D3, in the presence and absence of the known allosteric ligand W-84 at 50 mM concentration. Binding by the allosteric ligand W-84 decreases the off-rate of NMS. (B) Compound structures.

the isosteric ligand NMS-D3, both in the presence and absence of the known allosteric ligand W-84. These ALIS experiments show that the presence of the allosteric ligand decreases the off-rate of NMS, which is consistent with similar experiments performed by radioligand binding analysis [52].

The ALIS ''quench'' method for dissociation rate measurement uses little protein and requires no biochemical assay for its implementation, yet the method readily yields quantitative values for the dissociation rates of the protein-ligand complexes. The technique can be used with pools of ligands to provide a quantitative rank ordering of the dissociation rates of all the components of the mixture. Since it is not necessary to know the exact concentrations of the ligands under study, the dissociation rate assessment can be performed using impure compounds, such as unpurified compound mixtures derived from combinatorial chemistry synthesis. The method does not require a foreknowledge of active protein concentration to measure and rank ligands based on their rates of dissociation. As such, the technique is self-contained and does not rely upon an external measure of protein activity as one of its input parameters.

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