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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.

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.

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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.
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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.

Mentions: NQO2 is phosphorylated on either Ser 16 or Ser 20 in the Bcr-Abl-positive cell line K562 [22]. To examine the potential role of this modification in regulation of NQO2 activity, we mutated each residue to Ala or to phosphoserine-mimicking Asp, purified the resulting proteins, and measured their activities. As shown in Figure 8A, the S16A, S20A, and S20D mutants exhibited ~70% of the activity of the wild-type enzyme, while the activity of the S16D mutant was reduced to ~10% of wild-type enzyme activity. Additionally, the S16D mutant was colorless as purified, as opposed to the yellow color displayed by the other mutants and the wild-type protein, and was found to be a mixture of monomer and dimer by analytical gel filtration (data not shown).


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)

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

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

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.
Mentions: NQO2 is phosphorylated on either Ser 16 or Ser 20 in the Bcr-Abl-positive cell line K562 [22]. To examine the potential role of this modification in regulation of NQO2 activity, we mutated each residue to Ala or to phosphoserine-mimicking Asp, purified the resulting proteins, and measured their activities. As shown in Figure 8A, the S16A, S20A, and S20D mutants exhibited ~70% of the activity of the wild-type enzyme, while the activity of the S16D mutant was reduced to ~10% of wild-type enzyme activity. Additionally, the S16D mutant was colorless as purified, as opposed to the yellow color displayed by the other mutants and the wild-type protein, and was found to be a mixture of monomer and dimer by analytical gel filtration (data not shown).

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