Winger and Bob        Stamp


The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2)


Jonathan A. Winger, Oliver Hantschel, Giulio Superti-Furga and John Kuriyan


BMC Structural Biology 2009, 9:7 doi:10.1186/1472-6807-9-7 (local copy)

Abstract / Figures from the paper / Coordinates


Abstract:

Background

Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.

Results

We examined the inhibition of NQO2 activity by the Abl kinase inhibitors imatinib, nilotinib, and dasatinib, and obtained IC50 values of 80 nM, 380 nM, and >100 μM, respectively. Using electronic absorption spectroscopy, we show that imatinib binding results in a perturbation of the protein environment around the flavin prosthetic group in NQO2. We have determined the crystal structure of the complex of imatinib with human NQO2 at 1.75 Å resolution, which reveals that imatinib binds in the enzyme active site, adjacent to the flavin isoalloxazine ring. We find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.

Conclusion

The structure of the imatinib-NQO2 complex demonstrates that imatinib inhibits NQO2 activity by competing with substrate for the active site. The overall conformation of imatinib when bound to NQO2 resembles the folded conformation observed in some kinase complexes. Interactions made by imatinib with residues at the rim of the active site provide an explanation for the binding selectivity of NQO2 for imatinib, nilotinib, and dasatinib. These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds. Taken together, these studies provide insight into the mechanism of NQO2 inhibition by imatinib, with potential implications for drug design and treatment of chronic myelogenous leukemia in patients.

Illustrations from the paper.

Click on the small image to get a bigger one.


Figure 1. Inhibition of NQO2 by Abl kinase inhibitors. A) Chemical structures of the Abl kinase inhibitors imatinib, nilotinib, and dasatinib. Imatinib consists of a pyridine ring (A, green), an aminopyrimidine ring (B, blue), a methylbenzene ring (C, red), a benzamide ring (D, magenta), and a N-methylpiperazine ring (E, orange). The structurally analogous rings of nilotinib and dasatinib are similarly labeled. B) NQO2 inhibition assays for kinase inhibitors imatinib (black circles), nilotinib (blue squares), dasatinib (green diamonds), and the flavonoid NQO2 inhibitor quercetin (magenta triangles). The data were fit to the concentration-response equation Math, where x is the log of the inhibitor concentration, to yield IC50 values of 42 nM, 82 nM, and 381 nM for quercetin, imatinib, and nilotinib, respectively. Dasatinib was a very poor inhibitor, with an IC50 value > 100 μM.



Figure 2. Binding of imatinib to NQO2. Binding of imatinib to NQO2 causes significant changes in the electronic absorption spectrum of the flavin cofactor. Shown is the spectrum of 18.3 μM NQO2 in the absence (solid line) and presence (dashed line) of 40 μM imatinib at 25°C. Inset, difference spectrum calculated by subtraction of the spectrum of the unbound protein from that of the imatinib-bound protein.



Figure 3. Structure of NQO2 in complex with imatinib. A) Cartoon representation of the NQO2 dimer bound to imatinib. The two monomers (colored green and orange) are related by a crystallographic two-fold axis of rotation. B) Difference electron density (contoured at 3.0 σ) from a map calculated with the imatinib ligand removed is shown over the refined model of the imatinib-bound NQO2. In each panel, the FAD and imatinib molecules are depicted as yellow and blue stick figures, respectively; carbon is colored yellow (FAD) or light blue (imatinib); nitrogen, blue; oxygen, red; phosphorus, orange. Bound zinc ions are shown as grey spheres.




Figure 4. Interactions of imatinib with NQO2. A) Schematic of imatinib-NQO2 interactions. Van der Waals interactions of imatinib with NQO2 residues are indicated as semi-circles, and hydrogen bonds are represented by dotted lines. Contacts from each NQO2 monomer are colored green or orange. B) Residues involved in contacts between imatinib and NQO2 are primarily hydrophobic interactions. One water-mediated hydrogen bond is formed between imatinib and the side chain of Asn 161, which is positioned by a hydrogen-bonding interaction with Tyr 132. Residues involved in contacts between imatinib and NQO2 are shown as stick figures, and water molecules are shown as red spheres. Not shown: Gly 149, Gly 150, Thr 151, and Ile 194. C) Surface representation of the active site of NQO2 in complex with the kinase inhibitor imatinib. In each panel, the FAD and imatinib molecules are depicted as yellow and blue stick figures, respectively; nitrogen is colored blue and oxygen, red. Water molecules are shown as red spheres. The imatinib rings are lettered as in Figure 1A.




Figure 5. Comparison between binding of imatinib and other small molecules to NQO2. A) Overlay of the structures of several substrate- and inhibitor-NQO2 complexes with the imatinib-NQO2 complex. The loop containing Asn 161 has been removed for clarity. All of the bound molecules contain aromatic rings that stack above the flavin isoalloxazine group. B) The same overlay as in A), rotated to show interactions of the imatinib methylbenzene, benzamide, and N-methylpiperazine rings with hydrophobic residues (shown as CPK models) around the rim of the NQO2 active site. In each panel, imatinib (blue), the FAD cofactor (yellow), and several residues important for inhibitor binding are shown as stick figures, while the other overlaid NQO2-bound molecules are shown as line figures. The imatinib rings are lettered as in Figure 1A. The other molecules are menadione (magenta), resveratrol (pink), adrenochrome (grey), dopamine (green), melatonin (orange), and CB1954 (teal), from PDB ID 2QR2[31], 1SG0[26], 2QMY[37], 2QMZ[37], 2QWX[35], and 1XI2[36], respectively.




Figure 6. NQO1 active site is incompatible with imatinib binding. A) Model of imatinib in the active site of NQO1. The model was generated by superimposing the structure of human NQO1 (PDB ID 1D4A) onto the structure of the NQO2-imatinib complex. Imatinib (blue) is shown as a CPK model, while NQO1 is shown in surface and cartoon representations, with the FAD cofactor and selected residues depicted as stick figures. The imatinib rings are lettered as in Figure 1A. Potential clashes between NQO1 residues and imatinib are highlighted in yellow.




Figure 7. Comparison between imatinib-NQO2 and imatinib-kinase binding modes. A) The structure of imatinib bound to the kinase domain of Abl (PDB ID 1IEP) [6]. Imatinib binds in an extended conformation, with the pyridylpyrimidine moiety (rings A and B) trans to the methylbenzene and benzamide rings (rings C and D). B) The structure of imatinib bound to the kinase domain of Syk (PDB ID 1XBB) [44]. Imatinib binds in a compact conformation, with the pyridylpyrimidine moiety cis to the methylbenzene and benzamide rings. C) The conformation of imatinib bound to NQO2 most resembles the cis kinase-binding conformation. Shown are the structures of imatinib from the Syk complex 1XBB (yellow), the Abl complex (green), and the NQO2 complex (blue), with their pyridylpyrimidine moieties superimposed. In all panels, proteins are shown in ribbon representation and imatinib is shown as a stick model. The imatinib rings are lettered as in Figure 1A.




Figure 8. Analysis of potential NQO2 phosphorylation sites. A) Relative NQO2 activities of putative phosphorylation site mutants. Mutation of either Ser 16 or Ser 20 results in diminished activity, with the phosphorylation-mimicking S16D mutation having the most drastic effect. B) Ser 16 and Ser 20 are located next to the binding site for adenine and diphosphate moieties of the FAD cofactor. Ser 20 is solvent-exposed and involved in recognition of the FAD adenine moiety, while Ser 16 is mostly buried and involved in interactions that help form the adenine binding site. The FAD cofactor (yellow) and selected residues (green) are shown as stick models. Hydrogen bonds are depicted as dashed lines.



Table 1. Crystallographic data and refinement statistics




Coordinates


Coordinates in the Protein Data Bank: 3FW1