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

Winger JA, Hantschel O, Superti-Furga G, Kuriyan J - BMC Struct. Biol. (2009)

Bottom Line: 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 find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds.

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Affiliation: Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, Howard Hughes Medical Institute, University of California, Berkeley, USA. wingerj@berkeley.edu

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 microM, 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 A 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.

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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.
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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.

Mentions: NQO2 is closely related to another quinone reductase, NQO1. Despite catalyzing the same reaction, namely, the two-electron reduction of quinones, and sharing 49% identity at the amino acid level [33], NQO1 is not inhibited by imatinib [22]. A comparison of the structures of human NQO1 [38] with the structure of imatinib-bound NQO2 described here provides an explanation for this observation. While the structures of NQO1 and NQO2 superimpose well, with a r. m. s. deviation of 0.770 Å over 220 Cα atoms, NQO2 lacks a C-terminal domain of 43 amino acids. The C-terminal domain of NQO1 is involved in binding of the adenosine and diphosphate moieties of the cosubstrate NAD(P)H, which is not used by NQO2 [28,39]. When the two structures are superimposed, the side chain of Phe 232 in the C-terminal domain of NQO1 occupies the space in which the imatinib N-methylpiperazine ring (ring E, Figure 6) is found in the NQO2 structure. In addition, the side chains of Tyr 128 and Pro 68 at the rim of the NQO1 active site occlude the space that is occupied in the NQO2 structure by the imatinib benzamide and methylbenzenes rings (rings D and C), respectively, and the side chain hydroxyl group of Tyr 126 clashes with the imatinib aminopyrimidine ring (ring B). Thus, steric hindrance by residues in the C-terminal domain unique to NQO1, and by residues in the active site that differ between NQO1 and NQO2, prevents imatinib binding in the NQO1 active site.


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

Winger JA, Hantschel O, Superti-Furga G, Kuriyan J - BMC Struct. Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2655291&req=5

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.
Mentions: NQO2 is closely related to another quinone reductase, NQO1. Despite catalyzing the same reaction, namely, the two-electron reduction of quinones, and sharing 49% identity at the amino acid level [33], NQO1 is not inhibited by imatinib [22]. A comparison of the structures of human NQO1 [38] with the structure of imatinib-bound NQO2 described here provides an explanation for this observation. While the structures of NQO1 and NQO2 superimpose well, with a r. m. s. deviation of 0.770 Å over 220 Cα atoms, NQO2 lacks a C-terminal domain of 43 amino acids. The C-terminal domain of NQO1 is involved in binding of the adenosine and diphosphate moieties of the cosubstrate NAD(P)H, which is not used by NQO2 [28,39]. When the two structures are superimposed, the side chain of Phe 232 in the C-terminal domain of NQO1 occupies the space in which the imatinib N-methylpiperazine ring (ring E, Figure 6) is found in the NQO2 structure. In addition, the side chains of Tyr 128 and Pro 68 at the rim of the NQO1 active site occlude the space that is occupied in the NQO2 structure by the imatinib benzamide and methylbenzenes rings (rings D and C), respectively, and the side chain hydroxyl group of Tyr 126 clashes with the imatinib aminopyrimidine ring (ring B). Thus, steric hindrance by residues in the C-terminal domain unique to NQO1, and by residues in the active site that differ between NQO1 and NQO2, prevents imatinib binding in the NQO1 active site.

Bottom Line: 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 find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, Howard Hughes Medical Institute, University of California, Berkeley, USA. wingerj@berkeley.edu

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 microM, 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 A 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.

Show MeSH
Related in: MedlinePlus