Energetic dissection of Gleevec's selectivity toward human tyrosine kinases

Abstract

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abl's high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.

Figure 1

Monitoring the Gleevec binding process to Src and Abl by NMR at 25 °C. (a) Zoom of [¹H, ¹⁵N]-HSQC spectra showing an unusual pattern of chemical shifts perturbations (shifting of free peak position coinciding with the reappearance of Gleevec-bound peak) upon the titration of Src with Gleevec. (b) Biochemical scheme that can explain the observed titration patterns (see also Supplementary Fig. 1). E and E.I correspond to free and inhibitor bound kinase, E*.I corresponds to inhibitor bound kinase in a distinct conformational state. (c) Abl titration shows a simpler pattern with disappearance and reappearance of the peaks indicative of tight binding. (d) Residues with chemical shift changes upon Gleevec binding are plotted onto the crystal structures (pdb ids: 1OPJ, 2OIQ^,) in magenta, Gleevec is shown in orange. The mesh representation of the Gleevec binding pocket illustrates how part of the drug is covered by the protein suggesting a conformational change after binding (step 2 in Fig. 1b).

Figure 2

Kinetics of Gleevec binding to Abl and Src at 25 °C. Trp fluorescence change after mixing of Abl (a) or Src (b) with increasing amounts of Gleevec. Both Abl and Src kinetics are monoexponential. (c,d) The observed rates (k_on^obs) for Gleevec binding to Abl (c) and Src (d) do not show the expected linear dependence on the Gleevec concentration, but a curvature approaching a plateau in agreement with the proposed binding scheme in Fig. 1b. (n=3 experiments, mean ± s. e. m.) (e,f) Dissociation kinetics of Gleevec from Abl and Src measured by dilution of Abl.Gleevec and Src.Gleevec complexes. (g) Binding scheme highlighting a 1000-fold tighter affinity for Abl caused by a newly identified conformational step after binding (red). E_in and E_out defines apo kinase with the DFG-loop in “in” and “out” position respectively, E_out.I and E_out^*.I is kinase with DFG-loop in the “out” position and Gleevec bound, and the relevant rate constants are defined.

Figure 3

Kinetics of Gleevec binding measured at 5 °C allows dissection of energetics of individual steps. (a) Gleevec binding to Abl at 5 °C is biphasic. Double-exponential fit (red) gives an excellent fit, but not a mono-exponential fit (cyan). (b) Gleevec binding to Src at 5 °C is monophasic. Mono-exponential fit (cyan) is as good as double-exponential fit (red). (c,d) Dissociation of Gleevec from Abl and Src. (e,f) Concentration dependence of the fast (e) and slow (f) phases of Gleevec binding to Abl identifies them as the binding and conformational step, respectively. (n=3 experiments, mean ± s. e. m.) (g) Gleevec’s overall observed K_d^obs to Abl at 5 °C determined independently via steady-state measurement of Trp fluorescence quench by Gleevec. (n=3 experiments, mean ± s. e. m.)

Figure 4

Global fit of Abl binding and dissociation kinetics data measured by Trp fluorescence at 5 °C. Black circles – experimental data, red lines - global numerical simulation of the whole dataset using Kintek Explorer software (Kintek Corp)^,. The induced fit model (E⇔E.I⇔E*.I) was used and the global fitting results (k_on=1.3 ± 0.3 µM⁻¹s⁻¹, k_off = 23 ± 5 s⁻¹, k_conf+ = 1.3 ± 0.2 s⁻¹, k_conf− = (6±2)•10⁻⁴ s⁻¹) were in excellent agreement with individual fits (Table 1) validating the robustness of the model.

Figure 5

Direct observation of the conformational exchange step in the Src.Gleevec complex by NMR. [¹H,¹⁵N]-HSQC spectrum of Src at saturating Gleevec concentrations recorded for 16 h shows presence of major and minor peaks corresponding to the Src*.I and Src.I complexes respectively. The minor peaks appear exactly where predicted from the Gleevec titrations (Fig. 1a, Supplementary Fig. 5).