AMN107 (Novartis Pharma AG), is a new, highly potent and selective inhibitor of the Abl tyrosine kinase activity of the Bcr-Abl oncoprotein, which causes chronic myelogenous leukemia (CML). In addition to inhibiting wild-type Bcr-Abl, AMN107 inhibits the activity of 32/33 mutant forms of this protein which occur in imatinib-resistant patients. Following promising results from Phase I clinical studies in imatinib-resistant CML patients, AMN107 has now entered Phase II clinical trials. In order to better understand the molecular basis for the activity of this compound we have examined the x-ray crystallographic structures of complexes between AMN107 and the Abl kinase domain of wild-type and mutant Bcr-Abl. For these studies, recombinant proteins (residues 229–500) corresponding to the wild type and M351T mutant of the human Abl kinase domain were expressed in baculovirus infected insect cells and purified in the presence of AMN107. In each case the crystals obtained contained four independent copies of the complex in the unit cell and they diffracted to a resolution of 2.2 and 2.7 Å, respectively. As has been reported for the prototype Bcr-Abl inhibitor, imatinib (

Nagar et al.,
Cancer Research
), AMN107 binds to an inactive conformation of Abl, in which the glycine-rich, P-loop folds over the ATP binding site and the activation-loop adopts a conformation in which it occludes the substrate binding site and disrupts the ATP-phosphate binding site to block the catalytic activity of the enzyme. In order to induce the inactive conformation of the protein, AMN107 participates in hydrogen-bond interactions between (i) the pyridine-N and the backbone-C=O of Met318, (ii) the anilino-NH and the side-chain hydroxyl of Thr315, (iii) the carboxamido-C=O and the backbone-NH of Asp381, and (iv) the carboxamido-NH and the side-chain carboxylate of Glu286. These hydrogen-bond interactions are complemented by a large number of lipophilic interactions, surrounding the pyridine and 4-methylimidazole moieties in particular. Furthermore, careful analysis has revealed the presence of interactions between the protein and the trifluoromethyl group of AMN107, in which a fluorine atom is in close contact with the backbone-C=O of Asp381, with a mean F-C distance of 3.02 Å (4 mols/unit cell: 2.95, 2.97, 3.08 and 3.09). This compares with a value of 3.30 Å for the sum of the van der Waals radii of fluorine and carbon. Similar F-C=O interactions (mean F-C distance 3.02 Å) are observed in a complex between AMN107 and M351TAbl. Such interactions have been reported in the literature (
Olsen et al.,
) and are the result of dipolar interactions between the electronegative fluorine and the positively polarised carbon of the carbonyl group. In order to evaluate the contribution of the fluorine interactions to the binding of AMN107 to Abl we compared the effects of AMN107 on Bcr-Abl autophosphorylation in Ba/F3 cells to those of the corresponding analogue of AMN107, possessing a methyl group in place of trifluoromethyl. Whereas AMN107 displayed a mean IC50 value of 17 ± 0.5 nM, the methyl analogue had a mean IC50 of 83 ± 13 nM, which translates into a difference in binding energy in the region of 0.7 kcal/mole. Although Asp381 is therefore important for the binding of AMN107 to Abl through a hydrogen bond and through F-C interactions, resistance-mutations of this residue are unlikely the arise, since it plays a key role in the catalytic activity of the enzyme by interacting with a Mg ion which coordinates the phosphate groups of ATP.

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