The formation and growth of our bones and cartilages are tightly controlled by several growth factors called “bone morphogenetic proteins” (BMP). Aberrant activation of BMP signaling results in developmental defects or cancers. Recently several small molecules with pyrazolo[1,5-α]pyrimidine scaffold have been developed to block BMP signaling by targeting the serine/threonine kinase BMP type-I receptors (BMPR-I). However, most of the small molecules have off-target effects against the tyrosine kinase VEGFR2 and/or the structurally closely related TGF-β type-I receptor ALK5. There is no clear structure-activity relationship among existing analogs, and the mechanism of the binding specificity remains poorly understood.<br/>Here we use molecular dynamics and free energy calculation to investigate the binding selectivity of a model compound DMH1, which specifically targets BMPR-I subtype ALK2, but not VEGFR2 and ALK5. We have docked DMH1 into the kinase domain of ALK2, VEGFR2 and ALK5 X-ray structures. Instead of ranking the binding poses using docking score, we clustered all possible docking poses using RMSD criteria and calculated the absolute binding free energy for each cluster. We carried out alchemical free energy perturbation coupled with Hamiltonian replica exchange (FEP/REMD) to determine the absolute binding free energies. Each FEP/REMD simulation consist of 128 replicas, and the attempted neighboring λ-swap frequency is 0.2 ps<sup>-1</sup>. Even though, the docking scores suggested DMH1 binds equally well to all three receptors, rigorous free energy calculation confirmed that DMH1 does not bind to VEGFR2 and ALK5 (△G &gt; -2 kcal/mol). We ruled out multiple binding poses and captured a single binding pose of DMH1 in ALK2 that gives binding affinity (-8.1 kcal/mol) in agreement with kinase assay (-9.6 kcal/mol). Detailed and quantitative analysis of the trajectories revealed the distinct binding feature of DMH1, which is different from the crystal structures of its analogs. The key structure features and various thermodynamic contributions affecting the binding specificity of DMH1 to examined kinases will guide for developing the next generation of specific BMP inhibitors.

Binding specificity of bone morphogenetic protein (BMP) receptors: Insight from free energy simulations

249th National Meeting (2015)

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