HOW DOES ALENDRONATE INHIBIT PROTEIN-TYROSINE PHOSPHATASES

Alendronate (4-amino-1-hydroxybutylidene 1,l-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The in hibition of protein-tyrosine phosphatases (PTPs) by alendronate sugges ts 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 elimin ated by the addition of catalase, These observations suggest that a co mbination of alendronate, metal ions, and hydrogen peroxide is respons ible for the inhibition of PTPs, The individual effects of alendronate , calcium, or hydrogen peroxide on the inactivation of CD45 were deter mined. Electrospray ionization mass spectrometry demonstrated that the mass of PTP1B increased by 34 +/- 2 units after the enzyme was inacti vated with alendronate/calcium, due to the oxidization of the catalyti c cysteine to sulfinic acid (Cys-SO2H). The inhibited PTP1B could be p artially reactivated by treatment with reducing agents such as hydroxy lamine (NH2OH) and N,N'-dimethyl-N,N'-bis-(mercaptoacetyl)hydrazine, i ndicating the presence of other oxidized forms such as sulfenic acid ( Cys-SOH). This further confirms that the inhibition is the result of o xidation of the catalytic cysteine, The relevance of this oxidative in hibition mechanism in a biological system is discussed.