Simulations and Experimental Results

Figure 3.6 shows the simulated titration of a receptor at a fixed concentration by increasing concentrations of a ligand that binds a single site. Because the receptor concentration is fixed, the ligands saturate the receptor at high concentrations, and the amount of receptor-ligand complex present asymptotically approaches the total receptor concentration. Importantly, the rate at which saturation occurs - the steepness of the hyperbolic portion of the binding curve - depends on the binding affinity.

Such a titration using ALIS is operationally simple to execute. First, samples of varying ligand concentration are generated by serial dilution (for example, 80, 40, 20, 10... mM), and then each sample is incubated with a fixed concentration of the receptor, and subsequently injected on the SEC-RPC-MS system for analysis. Figure 3.7 shows the results of such a titration experiment for the small molecule ligand warfarin binding to human serum albumin (HSA, 5 mM) as its protein target. The x-axis of this plot is the total warfarin concentration, which includes both bound and unbound ligand. Fitting the raw data to the equation above yields a Kd value of 5.6 + 1.0 mM, which is consistent with literature values determined by frontal affinity chromatography and other methods [47]. The sigmoidal representation of this data and plots of the residuals of the curve fit demonstrate how well the model fits the raw data.

Further examples of the ALIS-based Kd measurement are shown in Fig. 3.8. Here, in Fig. 3.8A, titration of Akt-1 kinase (PKB-a) by the known ligand Merck-1 yields a Kd value of 0.3 + 0.1 mM, which correlates with its reported IC50 value of 0.4 mM [48]. Figure 3.8B shows binding of Staurosporine to Jun N-terminal kinase 1 (JNK1), yielding a Kd value of 1.0 + 0.4 mM, which corresponds well to its IC50 value of 0.5 mM. As a further example, Figure 3.8C shows that NGD-3350 binds its GPCR target, the M2 muscarinic acetylcholine receptor, with a Kd of 0.7 + 0.1 mM, which compares to its Ki value of 0.2 mM [49]. Finally, the DHFR ligand NGD-157, described in the preceding section, yields a Kd value of 3.5 + 1.7 mM by ALIS titration shown in Fig. 3.8D. Independent isothermal calorimetry experiments indicate that NGD-157 binds DHFR with a Kd of 5.9 mM [50].

Titration experiments in the presence of allosteric-binding proteins, peptides, and cofactors can indicate whether a ligand's binding affinity is positively or negatively affected by binding of the allosteric ligand. The next section of this chapter describes a method of determining ligand-ligand binding cooperativity where two ligands are detectable by ALIS.

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