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Kinetic characterization of the inhibition of protein tyrosine phosphatase-1B by Vanadyl (VO2+) chelates

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Abstract

Protein tyrosine phosphatases (PTPases) are a prominent focus of drug design studies because of their roles in homeostasis and disorders of metabolism. These studies have met with little success because (1) virtually all inhibitors hitherto exhibit only competitive behavior and (2) a consensus sequence H/V-C-X5-R-S/T characterizes the active sites of PTPases, leading to low specificity of active site directed inhibitors. With protein tyrosine phosphatase-1B (PTP1B) identifed as the target enzyme of the vanadyl (VO2+) chelate bis(acetylacetonato)oxidovanadium(IV) [VO(acac)2] in 3T3-L1 adipocytes [Ou et al. J Biol Inorg Chem 10: 874–886, 2005], we compared the inhibition of PTP1B by VO(acac)2 with other VO2+-chelates, namely, bis(2-ethyl-maltolato)oxidovanadium(IV) [VO(Et-malto)2] and bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV) [VO(mpp)2] under steady-state conditions, using the soluble portion of the recombinant human enzyme (residues 1–321). Our results differed from those of previous investigations because we compared inhibition in the presence of the nonspecific substrate p-nitrophenylphosphate and the phosphotyrosine-containing undecapeptide DADEpYLIPQQG mimicking residues 988–998 of the epidermal growth factor receptor, a relevant, natural substrate. While VO(Et-malto)2 acts only as a noncompetitive inhibitor in the presence of either subtrate, VO(acac)2 exhibits classical uncompetitive inhibition in the presence of DADEpYLIPQQG but only apparent competitive inhibition with p-nitrophenylphosphate as substrate. Because uncompetitive inhibitors are more potent pharmacologically than competitive inhibitors, structural characterization of the site of uncompetitive binding of VO(acac)2 may provide a new direction for design of inhibitors for therapeutic purposes. Our results suggest also that the true behavior of other inhibitors may have been masked when assayed with only p-nitrophenylphosphate as substrate.

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Abbreviations

Ac:

Acetate

CM:

Carboxymethyl

DMSO:

Dimethyl sulfoxide

EDTA:

N,N,N′,N′-ethylene diamine-tetraacetic acid

EGFR:

Epidermal growth factor receptor

EGFR988−998 :

The phosphotyrosine containing undecapeptide DADEpYLIPQQG simulating residues 988–998 of phosphorylated EGFR

ENDOR:

Electron nuclear double resonance

EPR:

Electron paramagnetic resonance

IPTG:

Isopropyl-β-D-1-thiogalactopyranoside

IR:

Insulin receptor

IRS-1:

Insulin receptor substrate-1

MES:

2-(N-morpholino)-ethanesulfonic acid

pNPP:

para-nitrophenylphosphate

PTPases:

Protein tyrosine phosphatases

PTP1B:

Protein tyrosine phosphatase-1B

pY:

Phosphotyrosine

UV:

Ultraviolet

VO(acac)2 :

Bis(acetylacetonato)oxidovanadium(IV)

VO(Et-malto)2 :

Bis(2-ethyl-maltolato)oxidovanadium(IV)

VO(Me-malto)2 :

Bis(2-methyl-maltolato)oxidovanadium(IV)

VO(mpp)2 :

Bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV)

TCEP:

Tris(2-carboxyethyl)phosphine

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgements

We thank Professor Z. Y. Zhang for providing the DNA for overexpression of human PTP1B. IJR was supported by an NIH Roadmap Training Program (T90 DK070076). PBB was supported by a training Grant of the National Institutes of Health at the Interface of Chemistry and Biology (T32GM008720). This research was supported by Grants from the National Institutes of Health (P50 CA125183 and P30 DK020595) and by the Department of Biochemistry and Molecular Biology at The University of Chicago.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology Center for Integrative Science, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA

    Jason Hon, Michelle S. Hwang, Meara A. Charnetzki, Issra J. Rashed, Patrick B. Brady, Sarah Quillin & Marvin W. Makinen

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  1. Jason Hon
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  2. Michelle S. Hwang
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  7. Marvin W. Makinen
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Contributions

PBB and SQ synthesized VO(mpp)2 and VO(Et-malto)2, respectively; MAC, JHH, MSH, and IJR overexpressed and purified the enzyme and collected and analyzed preliminary steady-state kinetic data; JHH and MWM conducted the final analysis of kinetic data and wrote the manuscript.

Corresponding author

Correspondence to Marvin W. Makinen.

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Hon, J., Hwang, M.S., Charnetzki, M.A. et al. Kinetic characterization of the inhibition of protein tyrosine phosphatase-1B by Vanadyl (VO2+) chelates. J Biol Inorg Chem 22, 1267–1279 (2017). https://doi.org/10.1007/s00775-017-1500-1

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