How does alendronate inhibit protein-tyrosine phosphatases?
Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of protein-tyrosine phosphatases (PTPs) by alendronate suggests that PTPs may be molecular targets. As a clear understanding of the inhibition mechanism is lacking, our aim was to analyze the mechanism to provide further insight into its therapeutic effect. We show here that the inhibition of PTPs by alendronate in the presence of calcium followed first-order kinetic behavior, and kinetic parameters for the process were determined. Evidence is presented that the inhibition by alendronate/calcium is active site-directed. However, this process was very sensitive to assay constituents such as EDTA and dithiothreitol. Furthermore, the inhibition of PTPs by alendronate/calcium was eliminated by the addition of catalase. These observations suggest that a combination of alendronate, metal ions, and hydrogen peroxide is responsible for the inhibition of PTPs. The individual effects of alendronate, calcium, or hydrogen peroxide on the inactivation of CD45 were determined. Electrospray ionization mass spectrometry demonstrated that the mass of PTP1B increased by 34 +/- 2 units after the enzyme was inactivated with alendronate/calcium, due to the oxidization of the catalytic cysteine to sulfinic acid (Cys-SO2H). The inhibited PTP1B could be partially reactivated by treatment with reducing agents such as hydroxylamine (NH2OH) and N,N'-dimethyl-N, N'-bis(mercaptoacetyl)hydrazine, indicating the presence of other oxidized forms such as sulfenic acid (Cys-SOH). This further confirms that the inhibition is the result of oxidation of the catalytic cysteine. The relevance of this oxidative inhibition mechanism in a biological system is discussed.
Skorey
K
K
Department of Biochemistry and Molecular Biology, Merck-Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec H9R 4P8, Canada.
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H D
HD
Kelly
J
J
Hammond
M
M
Ramachandran
C
C
Huang
Z
Z
Gresser
M J
MJ
Wang
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Q
D019386:Alendronate / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004793:Enzyme Reactivators / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D006861:Hydrogen Peroxide; D002374:Catalase; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine; D002118:Calcium; D019386:Alendronate