Direct Method for Discovering and Ranking Multiple Ligands

The above discussion presents the utility of the FAC-MS method as a supportive tool for higher-throughput discovery initiatives, in which hits might well be carried forward after their binding parameters are carefully re-measured and validated, and related to the data from the other hits. When incorporating MS detection, this evaluation may be done all at once. It is easy to see how a single li-gand can be detected in a large pool of nonligands, but what if there exists a competitive multi-ligand environment?

Let us first consider the nature of the breakthrough curves when multiple li-gands are present, and do so through the presentation of an example. Figure 6.6 shows the breakthrough curves for three isobaric compounds (m/z 359) and a void marker, generated in a sorbitol dehydrogenase FAC assay. Two different MS/MS transitions were monitored, to resolve the individual breakthrough curves for the three ligands. What is immediately obvious from this example is that the breakthrough curves deviate from simple sigmoidal shapes. Most biomolecular interactions are modeled with a nonlinear binding isotherm reflective of saturable binding events, thus one ligand can actively compete with another [18]. When this is established in flowing system as encountered in a FAC assay, ligand displacement occurs in a predicable fashion, assuming equilibrium conditions. This produces a displacement of the weaker ligand(s) and represents the only condition under which ligand enrichment can occur in a constant-infusion system.

The shape of this displacement feature is dictated by the kinetics of binding and the overall efficiency of the cartridge. Notice that the first breakthrough (359 ! 162 transition) is peak-like in nature, while the second breakthrough (359 ! 188 transition) appears as a regular sigmoidal curve. The third breakthrough (359 ! 162 transition) is also a sigmoid curve. So in this example, while it is not clear which stereoisomer is the strongest or weakest, it is clear that the positional isomer is intermediate in binding strength between two stereoisomers. This indicates that stereochemistry is significant for this interaction, and most likely that a common binding site is accessed. Under ideal, infinitely fast condi-

Fig. 6.6 Demonstration of the displacement phenomenon that occurs when FAC experiments are conducted in the nonlinear region of the binding isotherm. Three isobaric ligands (m/z 359) are infused at micromolar concentration through a sorbitol dehydro

genase FAC assay, as monitored by a triple quadrupole mass spectrometer in MRM mode. The solid trace represents two stereoisomers and the dashed trace a positional isomer. The dot-dash trace represents a nonbinding void marker (m/z 639).

Void marker

Void marker

Time (min)

Fig. 6.7 A FAC-MS experiment for ligand ranking. Eight ligands for sorbitol dehydrogenase were infused, spanning a Kd range of 2 mM to 8 nM [10]. All ligands were applied at 1 mM concentration, ensuring operation in the nonlinear region of the binding isotherm. Adapted with permission from Elsevier.

Time (min)

Fig. 6.7 A FAC-MS experiment for ligand ranking. Eight ligands for sorbitol dehydrogenase were infused, spanning a Kd range of 2 mM to 8 nM [10]. All ligands were applied at 1 mM concentration, ensuring operation in the nonlinear region of the binding isotherm. Adapted with permission from Elsevier.

Table 6.1 The order of breakthrough for the example of Fig. 6.7 closely parallels the IC50 values from independent determinations using plate-based activity assays [10]. Kd values were measured for the strongest and weakest ligands in separate FAC-MS experiments.

Compound FAC-MS RANK ORDER IC50 (nM) Kd (nM)

Was this article helpful?

0 0

Post a comment