Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site’s location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.
Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the 'gatekeeper' threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-α and -β, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions-the hydrophobic spinecharacteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors.Deregulated protein kinases have been linked to numerous diseases including cancer and diabetes as well as inflammation. The targeted inhibition of protein tyrosine kinases is now well established as an effective therapeutic regimen for chronic myeloid leukemia (CML) and several solid tumors1-4. Many small-molecule kinase inhibitors have exploited a conserved threonine residue within the ATP binding site for binding specificity5. This threonine controls access of the inhibitors to a hydrophobic pocket deep in the active site that is not contacted by ATP, hence leading to its designation as a 'gatekeeper' residue6. Substitution of the gatekeeper threonine residue with bulky side chains is a common mechanism of resistance to pharmacological ATP-competitive kinase inhibitors7,8.Imatinib has been used successfully to inhibit BCR-ABL in CML9, c-KIT in gastrointestinal stromal tumor (GIST)10 and platelet-derived growth factor receptor-α (PDGFRA) in hypereosinophilic syndrome (HES)11,12. The first imatinib-resistant mutation described in CML patients was an isoleucine substitution at the gatekeeper residue Thr315 (numbered according to the sequence for the type Ia isoform of c-ABL)13. The T315I mutation has been detected in imatinib-naïve CML patients and accounts for ∼20% of the total burden of clinical resistance14. Mutation at the analogous position to Thr315 in other imatinib targets such as c-KIT (Thr670) and PDGFRA (Thr674) have been linked to imatinib resistance in patients with GIST and HES, respectively15,16. Similarly, the gatekeeper mutation T790M in EGFR causes resistance to gefitinib and erlotinib, and has been...
The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl*imatinib and c-Kit*imatinib, and differs significantly from the inactive "Src/CDK" conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.
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