Affinity Specificity

When ESI-MS is used as a primary high throughput screening tool as in the MASS assay [13, 31-33], the goal is to identify ligands with Kd values less than 100 mM and with some specificity relative to the other targets. These constraints ensure that the ligands bind to a unique structural feature of the target and are not just generic RNA binders. Although the ligands are screened only at a single concentration in the high throughput mode, it is possible to estimate a one-point Kd from the mass spectrometry data. A percent complex is calculated for each identified ligand-target combination by calculating the ratio of the integrated peak areas of the complexed and the free target and multiplying by 100. A one-point estimated Kd is then calculated by dividing 100 by the percent complex of the ligand and multiplying by the screening concentration. The one point estimated Kd values can be used to classify compounds as weak, medium, and strong

Fig. 10.7 ESI-FTICR mass spectra of three RNA targets at 2.5 mM each screened against 11 compounds at 25 mM each. The percent complexes and one-point Kd values are shown for each ligand complex. (a) An example of a ligand that specifically binds target 2. (b) An example of a ligand that nonspecifically binds to all targets.

Fig. 10.7 ESI-FTICR mass spectra of three RNA targets at 2.5 mM each screened against 11 compounds at 25 mM each. The percent complexes and one-point Kd values are shown for each ligand complex. (a) An example of a ligand that specifically binds target 2. (b) An example of a ligand that nonspecifically binds to all targets.

binders but cannot be used to accurately rank order compounds within the same classification. As illustrated in Fig. 10.7a, a ligand binds target 2 with a one-point estimated Kd of 37 mM (i.e. estimated Kd = 100/67 * 25 mM). The ligand binds target 2 with 3.4-fold greater specificity than target 1 and with 1.5-fold greater specificity than target 3. This ligand would be a candidate for further structure-activity relationship studies (SAR) by medicinal chemistry to improve both its binding affinity and specificity. An example of a generic RNA binder is shown in Fig. 10.7b. The ligand binds target 1 and target 2 equally well. In addition, complexes formed by binding two ligands to the target are observed for both target 1 and target 2. This result indicates that there are multiple weak binding sites with similar affinities for the ligand on the targets. It would likely be difficult to improve the affinity and specificity of this ligand, and therefore, it would probably not be pursued further as a drug candidate. Thus, MASS can be used to rapidly identify promising compounds and/or structural motifs from large chemical libraries and guide the medicinal chemistry efforts required to create high-value lead compounds [40-42].

Was this article helpful?

0 0

Post a comment