How Does Alendronate Inhibit Protein-tyrosine Phosphatases?

Abstract

Alendronate(4-amino-1-hydroxybutylidene1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of proteintyrosine 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-SO 2 H). The inhibited PTP1B could be partially reactivated by treatment with reducing agents such as hydroxylamine (NH 2 OH) 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.Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) has been successfully used as a therapeutic agent for the treatment of bone disorders such as osteoporosis (1-4) and Paget's disease (5, 6). The rationale for the therapeutic effect at the cellular level is that alendronate interferes with osteoclast resorption, resulting in reduced bone turnover and a net gain in bone mineral density (7). However, at the molecular level, the precise target and mechanism of action of alendronate are not yet clear. The evidence that vanadate, which is a potent and nonselective protein-tyrosine phosphatase (PTP) 1 inhibitor (8), inhibits bone resorption induced by parathyroid hormone in organ culture (9) suggests that PTP is possibly one of the targets.Protein-tyrosine phosphatases are important enzymes in living systems, acting in opposition to protein-tyrosine kinases to control the tyrosine phosphorylation level of proteins. Tyrosine phosphorylation plays a critical role in the transduction of signals that control many diverse cell processes (10 -12). PTPs are divided into two main groups: the receptor-like forms, such as CD45 (leukocyte common antigen); and the cytoplasmic forms, such as PTP1B. Each of the PTPs contains at least one conserved segment...

Alendronate induces anti-migratory effects and inhibition of neutral phosphatases in UMR106 osteosarcoma cells

“…Our results further suggest that this effect was mainly mediated by tyrosine phosphatases since vanadate showed a similar and non-additive effect to alendronate. These results are in agreement with previous observations demonstrating that alendronate inhibits protein tyrosine phosphatases Opas et al, 1997;Schmidt et al, 1996;Skorey et al, 1997). Previous data suggest that the Rho family of GTPases activate kinases that are important in cytoskeletal-mediated changes affecting motility (Mackesky and Hall, 1997;Schmitz et al, 2000).…”

2-(Oxalylamino)-Benzoic Acid Is a General, Competitive Inhibitor of Protein-tyrosine Phosphatases

“…As an example, in a recent, thorough study it was shown that addition of EDTA or catalase prevented alendronate from inhibiting PTP1B and CD45, suggesting that a combination of alendronate, trace metal ions, and H 2 O 2 was responsible for the observed timedependent PTP inhibition of alendronate (14). We, therefore, analyzed if OBA inhibited PTP1B in a time-dependent manner.…”

“…Bisphosphonates, such as alendronate, have been shown to inhibit PTPs, but their inherent affinity for bone is likely to prevent their general use in other target tissues (12). Furthermore, several of these inhibitors are time-dependent and seem to act through covalent modification of the catalytic cysteine in PTPs (13,14). The most specific inhibitors produced so far have either been phosphonates (9,15) or those based on peptides that are not suited for clinical use due to their lack of oral bioavailability and metabolic instability (reviewed in Ref.…”

Structure-based Design of Selective and Potent Inhibitors of Protein-tyrosine Phosphatase β

“…Indeed, when searching for PTP inhibitors, it is a common experience among laboratories that perform high throughput screenings to get high hit rates, which in many cases are caused by oxidation or alkylation of the active site cysteine (16,24). 2 Although compounds that irreversibly and/or more broadly in a time-dependent manner modify PTPs may still be useful in the clinic (25)(26)(27), structurebased optimization is often difficult, if not impossible. Hence, there is a need for development of tools that will allow structure-based design of selective inhibitors of other members of the PTP family.…”