Protein-Ligand Dissociation Rate Measurement

The biological efficacy of a drug candidate depends critically on the rate at which it dissociates from its therapeutically relevant target biomolecule. As described in 

very slow dissociation kinetics can contribute to slow clearing of a drug, which can be problematic in the event of adverse reactions such as an undesired allergic response. Therefore, methods to accurately determine the dissociation kinetics for protein-ligand interactions are of great value to the drug discovery process. 

---

Fig. 3.14 Simulated ALIS-based dissociation rate measurements. See text for details. (A) Quench experiments modeled at varying inhibitor association rates. Even with a very slow-binding inhibitor, the decay curve resembles pure first-order dissociation kinetics. (B) Data in (A), shown on a log axis. (C) Simulated ALIS quench experiment with varying protein-ligand dissociation rates,...

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. 

Figure 3.14D shows the degree of correlation for the rate of decay of the protein-ligand complex in a modeled ALIS quench experiment and the theoretical decay curve expected from infinite dilution. The modeled decay curve is shown for ks-off = 0.01 s and theoretical curves are shown for dissociation rates +10% of this value. The results indicate that the measured dissociation rate is well within 10% of the actual value, a very good approximation of the actual dissociation rate given the simplicity of this experimental method. 

Measurement of the rates of dissociation of enzyme-substrate and protein-ligand complexes, usually promoted by dilution. The utility of dissociation kinetics is well illustrated in the report of Dunn and Raftery who examined the kinetics of pH]acetylcholine and [ H]sub-eryldicholine dissociation from the membrane-bound... 

Fig. 9.3 Kinetics of ligand dissociation for LdisPBP2. Data were obtained by equilibrating isolated binding-protein-ligand complex (BP.L) in fresh buffer and separating protein-bound ligand from dissociated ligand at various time points (see text). Ligands tested were +) disparlure 7R, 8S) c/a -7,8-epoxy-2-methyloctadecane) and the enantiomer, (-) disparlure. The dissociation is exponential and biphasic, with a rapid phase (koff i) and a slow phase (koff 2)- The association rates given were calculated from the measured dissociation constants, Ka, and the rapid off rate (koff 1). The half-life for each rate dissociation is given as ti/2 (calculated from the initial value measured and the individual exponential decay).

The behavior of protein-ligand complexes in NMR measurements depends largely on their thermodynamic and kinetic properties (i.e., dissociation constants and on/off rates). The characteristics of the system under study and the kind of information desired determine the adequate approach for an NMR investigation. 

---