Crystal Structures of Anaplastic Lymphoma Kinase in Complex with ATP Competitive Inhibitors

Abstract

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the development of several human cancers and, as a result, is a recognized target for the development of small-molecule inhibitors for the treatment of ALK-positive malignancies. Here, we present the crystal structures of the unphosphorylated human ALK kinase domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684. Analysis of these structures provides valuable information concerning the specific characteristics of the ALK active site as well as giving indications about how to obtain selective ALK inhibitors. In addition, the ALK-KD-PHA-E429 structure led to the identification of a potential regulatory mechanism involving a link made between a short helical segment immediately following the DFG motif and an N-terminal two-stranded beta-sheet. Finally, mapping of the activating mutations associated with neuroblastoma onto our structures may explain the roles these residues have in the activation process.

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

“…3 Although the F1174L muta- Other aspects of the ALK F1174L kinase domain structure also resembled features observed in the wild-type protein, notably the conformation of the DFG motif at the beginning of the A-loop, the position of the ␣C-helix, and the relative degree of closure between the N-and C-terminal lobes of the kinase. These features, together with the positioning of the C-terminal portion of the A-loop that sterically blocks the substrate binding site, have all been discussed previously as contributing to an overall inactive kinase conformation (38,39). In contrast to the F1174L ALK crystal structure, the structure of the R1275Q ALK kinase domain showed a dramatic difference in the activation loop conformation compared with the wild-type protein (r.m.s.…”

“…Several structural features of the published, unphosphorylated ALK kinase domain differ from the structural template provided by the IRK ternary structure and interestingly, ALK also differs from the unphosphorylated, inactive form of IRK kinase domain (43). These differences have been described elsewhere (38,39).…”

“…A structural understanding of inhibitor binding to ALK was recently enabled by the publication of crystal structures of the ALK kinase domain both alone and in complex with ATP-competitive inhibitors (38,39). The structures revealed that the ALK kinase domain adopts the canonical kinase-fold, but that it also contains two notable features.…”

“…All of the structures described in this work use ALK(1084 -1410) C1097S. By way of comparison, ALK kinase domain structures have also been determined using constructs with the boundaries 1072-1410, 1094 -1407, 1093-1411, and 1069 -1411 (19,38,39,52).…”

“…All of the mutations cluster around the active site of ALK. One of the most commonly mutated residues, Phe 1174 , sits in a hydrophobic pocket at the base of the ␣C-helix, where it is involved in hydrophobic stacking interactions with other phenylalanine residues from the N-terminal ␤-turn region (Phe 1098 ), the activation loop (Phe 1271 ), and C-terminal kinase domain (Phe 1245 ) as described by others (35,38,39). The second commonly mutated residue, Arg 1275 , is found in the activation loop where it is part of the ␣-helix formed in the unphosphorylated ALK kinase domain structures.…”