Explaining why Gleevec is a specific and potent inhibitor of Abl kinase Yen-Lin Lina,1, Yilin Menga,1, Wei Jiangb, and Benoît Rouxa,b,2 aDepartment of Biochemistry and Molecular Biology, Gordon Center for Integrative Science, The University of Chicago, Chicago, IL 60637; and bBiosciences Division, Argonne National Laboratory, Argonne, IL 60439 Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved December 12, 2012 (received for review August 20, 2012) Tyrosine kinases present attractive drug targets for specifictypes inhibition. Because the DFG-out conformation had never been of cancers. Gleevec, a well-known therapeutic agent against chronic observed previously with other related tyrosine kinases of the Src myelogenous leukemia, is an effective inhibitor of Abl tyrosine family, it was initially thought that this inactive conformation was kinase. However, Gleevec fails to inhibit closely homologous tyrosine only accessible to Abl kinase, leading to the suggestion that the low kinases, such as c-Src. Because many structural features of the binding affinity of Gleevec to c-Src might reflect its inability to adopt binding site are conserved, the molecular determinants responsible a DFG-out conformation. However, the latter was subsequently for binding specificity are not immediately apparent. Some have observed in an X-ray structure of c-Src in the DFG-out inactive attributed the difference in binding specificity of Gleevec to subtle conformation in complex with Gleevec (7), thus ruling out this variations in ligand–protein interactions (binding affinity control), possibility. Therefore, despite the extensive experimental studies and whereas others have proposed that it is the conformation of the the X-ray structures of Abl and c-Src, the basic question remains DFG motif, in which ligand binding is only accessible to Abl and not unanswered: Why is Gleevec a specific and potent inhibitor of Abl to c-Src (conformational selection control). To address this issue, the but not of c-Src? Two distinct mechanisms, binding affinity and absolute binding free energy was computed using all-atom molecu- conformational selection, have been invoked to explain the differ- lar dynamics simulations with explicit solvent. The results of the free ences in the binding specificity of Gleevec. In the first, it is hypoth- energy simulations are in good agreement with experiments, esized that specificity arises primarily from differences in binding thereby enabling a meaningful decomposition of the binding free affinities caused by subtle variations in the residue sequence of the ’ energy to elucidate the factors controlling Gleevec s binding speci- binding pocket presented by Abl and c-Src in the DFG-out confor- fi city. The latter is shown to be controlled by a conformational selec- mation. In the second, it is proposed that the DFG-out conforma- tion mechanism and also by differences in key van der Waals tion, although being accessible to both kinases, incurs a higher free interactions responsible for the stabilization of Gleevec in the bind- energy penalty in c-Src compared with Abl. ing pocket of Abl. Although a contrast can be drawn between the binding affinity and conformational selection mechanisms, both are necessarily thermodynamics | alchemical free energy perturbation | sampling oversimplified. Nevertheless, it is difficult to achieve a deeper un- derstanding of the underlying mechanism of the binding specificity yrosine kinases are crucial to cellular signaling pathways regu- of Gleevec without a detailed dissection of the equilibrium associ- ( ) Tlating cell growth, and for this reason, they represent attractive ation constant or absolute (standard) binding free energy, ΔGeq ° , drug targets for curing certain types of cancers. The development of in terms of all the thermodynamic contributions associated with the kinase inhibitors is, however, challenging because of the high se- protein conformational change and ligand binding. Achieving this quence conservation of the kinase ATP-binding site, the major site directly by experimental means is difficult because critical con- targeted by these small molecules. The difficulties encountered by tributions remain essentially “hidden” from direct experimental these efforts are displayed most clearly by Gleevec (Novartis), measurements. Computations based on atomic models offer a vir- a potent inhibitor of Abl tyrosine kinase (1). Gleevec is one of the tual, albeit approximate, route to address these issues. The binding most successful drugs against chronic myelogenous leukemia (2), of tyrosine-kinase inhibitors to target kinases has been the object of a pathological condition that is caused by mutations leading to a number of computational studies (8–14). So far, two computa- a constitutively activated Abl kinase (3, 4). The remarkable effec- tional studies have presented evidence supporting the view that tiveness of Gleevec raised the hope that one might be able to de- Gleevec binding specificity to Abl and c-Src is controlled by con- velop novel kinase inhibitors for specific cancers. The situation is formational selection (11, 14). However, the absolute binding free nonetheless complicated by the fact that Gleevec displays a much ( ) energy of Gleevec, ΔGeq ° , has never been calculated within lower inhibitory effect on c-Src for unclear reasons, even though a single and consistent computational model and methodology, and these two tyrosine kinases display a high degree of sequence the origin of binding specificity of Gleevec ultimately remains un- identity (47%) and similar structural scaffolds (Fig. 1). From this resolved despite previous efforts. In the present study, we report perspective, understanding the molecular basis for the binding complete computations of the absolute binding free energies of fi speci city of Gleevec is likely to hold important lessons for the Gleevec to the catalytic domain of Abl and c-Src kinases using de- rational design of kinase inhibitors in general. tailed all-atom molecular dynamics (MD) simulations with explicit The X-ray crystallographic structure of the Abl kinase in com- solvent molecules. The computations allow the identification of the plex with Gleevec revealed an important clue to understand the molecular basis of binding specificity explaining the binding spec- fi i city of Gleevec to Abl (5). A short motif composed of the resi- Author contributions: Y.-L.L., Y.M., W.J., and B.R. designed research; Y.-L.L. and Y.M. dues Asp-Phe-Gly near the N-terminal region of the activation performed research; Y.-L.L., Y.M., W.J., and B.R. analyzed data; and Y.-L.L., Y.M., W.J., loop (A-loop) adopted an unusual conformation referred to as and B.R. wrote the paper. “DFG-out,” giving rise to a previously unobserved inactive con- The authors declare no conflict of interest. formational state of the catalytic domain. This binding mode This article is a PNAS Direct Submission. contributing to the selectivity of Gleevec to Abl has been further 1Y.-L.L. and Y.M. contributed equally to this work. confirmed by a solution NMR study (6). The binding of Gleevec to 2To whom correspondence should be addressed. E-mail: [email protected]. the DFG-out conformation appears to prevent the transition to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the conformationally active form of the kinase, hence causing 1073/pnas.1214330110/-/DCSupplemental. 1664–1669 | PNAS | January 29, 2013 | vol. 110 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1214330110 Downloaded by guest on October 2, 2021 Abl:Gleevec c-Src:Gleevec was obtained by integration of the Boltzmann factor of the 2D- PMF over the variable associated with the side-chain rotation of P-loop P-loop C-helix the aspartate residue (Fig. S1 shows the calculated 2D-PMFs for the DFG-flip in Abl and c-Src). The overall stability of the out vs. in C-helix ΔG = G − G A-loop conformation of the DFG motif, in→out [ out in], is esti- DFG mated to be 1.4 kcal/mol for Abl and 5.4 kcal/mol for c-Src. This motif shows that there is an inherent conformational selection phe- nomenon and that the inactive DFG-out conformation is more A-loop stable in Abl than in c-Src. Previous computational results based on metadynamics found a ΔGin→out of 4.0 kcal/mol and 6.0 kcal/mol for Abl and c-Src, respectively (14), confirming that the DFG flip is Fig. 1. Structural comparison of Abl and c-Src in the Gleevec-bound more costly in c-Src than in Abl. kinase domain. Binding Free Energy of Gleevec to the DFG-Out Conformation. The second task is to determine the absolute binding free energy of molecular determinants of ligand-protein recognition controlling Gleevec from a bulk solution to the binding pockets of Abl and fi the binding speci city of Gleevec, thereby permitting an objective c-Src with the DFG motif in the out configuration. Table 1 evaluation of the previously proposed mechanisms. The study summarizes the various free energy contributions to the binding demonstrates that the MD simulations, when combined with a rig- affinity of the ligand with the kinases [progression of the free orous step-by-step formulation of absolute binding free energy, energy during successive FEP/λ-replica exchange molecular dy- together with extensive sampling methodologies, can provide criti- namics (REMD) cycles is shown in Fig. S2]. When the DFG cal information underlying protein-ligand binding to help guide motif is in the out conformation, the absolute binding free en- rational de novo drug design. ergy of Gleevec to Abl kinase and c-Src is estimated be to −10.8 kcal/mol and −6.8 kcal/mol, respectively (these numbers include Results and Discussion the contribution for restricting the ligand conformations calcu- All the free energy contributions must be accounted for to draw lated from the rmsd PMF). This shows that when the two kinases fi de nitive conclusions about the mechanism underlying the bind- are in the DFG-out conformation, Abl provides a more favorable fi fl ing speci city of Gleevec to Abl and c-Src kinases (i.e., DFG- ip binding pocket than c-Src.