B

We have used this approach in the FAC-MS analysis of a number of challenging receptor-ligand systems [10]; overcoming nonspecific binding effects and slow on-rates is a particular strength of this method. As discussed in the previous section, nafoxidine (an estrogen antagonist used in the treatment of breast cancer) is a slow-tight binder targeting the ligand binding domain of estrogen receptor-b [27]. By using a weak-binding steroid as an indicator ligand, the equilibrium K¿ value was measured to be 23 nM, comparable with other methods. We find this

Fig. 6.10 Breakthrough behavior for a weak indicator ligand (norethindrone) in the presence of an increasing concentration of the strong ligand b-estradiol [10]. These ligands access the ligand-binding domain of estrogen receptor b. Displayed is an overlay of four chromatograms showing breakthrough curves of norethindrone after successive equilibrations with b-estradiol. Corresponding void marker traces at each concentration of b-estradiol are also shown (overlaid at a breakthrough time of @1 min). A Kd of 1.3 nM was determined by this method. Adapted with permission from Elsevier.

Fig. 6.10 Breakthrough behavior for a weak indicator ligand (norethindrone) in the presence of an increasing concentration of the strong ligand b-estradiol [10]. These ligands access the ligand-binding domain of estrogen receptor b. Displayed is an overlay of four chromatograms showing breakthrough curves of norethindrone after successive equilibrations with b-estradiol. Corresponding void marker traces at each concentration of b-estradiol are also shown (overlaid at a breakthrough time of @1 min). A Kd of 1.3 nM was determined by this method. Adapted with permission from Elsevier.

method to be extremely useful for Kd measurements of single ligands in general: weak ligands are easy to obtain for most biomolecular interactions and they usually present rapid kinetics. For our FAC-MS work, we typically select indicators in the range of 10 mM to 1 mM Kd, and apply them at low micromolar concentrations. However, the Kd of the indicator ligand can be much lower than 10 mM, as long as equilibrium conditions can be met.

These analyses are rapid, usually limited by the incubation time for the test compound on the cartridge. Figure 6.10 shows the breakthrough curves for an indicator analysis of b-estradiol, demonstrating that each ''probe'' of binding capacity with the indicator can be achieved in approximately five minutes. Full equilibration with low concentration, high-affinity test ligands can require large volumes, however. For example, the 4 nM estradiol infusion concentration was applied at 50 mL min-1 for 100 min (5 mL). This is unavoidable, as full cartridge equilibration is required. A lower-capacity cartridge can always be implemented if this is an issue, as the equilibration volume is directly proportional to column capacity.

The displacement-based rollup feature described above can also be used to detect test-ligand binding. Under equilibrium conditions, integrating the rollup provides a means of measuring the Kd of the stronger ligand, but this is a less reliable method than that described above; resistance to mass transfer makes the rollup hard to quantitate with accuracy, especially under low occupancy condi

Fig. 6.11 Using rollups to efficiently pre-screen mixtures for the presence of "hits". In this example, six mixtures of approximately 90 compounds each (A-E) were screened in a dual protein FAC assay (b-galactosidase, GS1B4). The dashed red and blue curves in each chromatogram represent the breakthroughs of the b-galactosidase and GS1B4 indicators, respectively, in the absence of the

Fig. 6.11 Using rollups to efficiently pre-screen mixtures for the presence of "hits". In this example, six mixtures of approximately 90 compounds each (A-E) were screened in a dual protein FAC assay (b-galactosidase, GS1B4). The dashed red and blue curves in each chromatogram represent the breakthroughs of the b-galactosidase and GS1B4 indicators, respectively, in the absence of the mixtures. The solid red and blue curves in each chromatogram represent the breakthroughs of the b-galactosidase and GS1B4 indicators, respectively, in the presence of the mixtures. In this example, mixture C was quickly determined to be the only mixture with a hit against one of the proteins (b-galactosidase). Adapted with permission from Elsevier.

tions ([ligand] < Kd). It is far more useful as a quick test for the presence of stronger ligands in a mixture. Figure 6.11 illustrates that this mode of indicator analysis can readily pinpoint mixtures containing hits of greater affinity than the indicator, and opens the door to multiplexed "pre-screening", where mixtures can be interrogated against multiple proteins in one experiment [10]. This mode of analysis works best when the indicator is present at a concentration equivalent to its Kd value. The example in Fig. 6.11 involves screening mixtures of approxi mately 90 compounds from the Optiverse (Tripos) library against a dual-target column containing immobilized b-galactosidase and GSIB42. Monitoring only the indicators allows a quick survey to determine which mixture should proceed to deconvolution, offering a rapid means to reduce the screening burden. In this example, a rollup in mixture three indicates the presence of a ligand for b-galactosidase stronger than the indicator.

This mode successfully avoids false positives arising from mixtures containing many weak ligands - a classic problem when conducting mixture screening. Again, a method such as this would find its ideal application in the screening of single-pot syntheses and/or natural product extracts where deconvolution is particularly time-consuming. Note that in these pre-screening applications, a fluores-cently labelled indicator ligand may offer a simpler instrumental solution, although multiplexing the analysis would require differential, multi-color labelling.

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