Michael J. Gresser

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) andN,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.

Vanadates as Phosphate Analogs in Biochemistry

There are two general kinds of ways in which vanadates can serve as phosphate analogs in biochemical systems. In the case of enzymes or other proteins which do not catalyse cleavage of a bond to the phosphorus atom, and for which phosphates are the natural ligands, vanadates can serve as alternate ligands or substrates. Inorganic vanadate (Vi) itself can function as an analog of inorganic phosphate (Pi) in these systems, and spontaneously formed vanadate complexes can act as analogs of phosphate esters. The vanadate can in some systems be as “good” a ligand as the corresponding phosphate.

Bis(N,N-dimethylhydroxamido)hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate.

We have shown previously that bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHV) is an excellent reversible inhibitor of protein tyrosine phosphatase (PTP) in vitro. DMHV does not carry a charge under physiological pH conditions and is anticipated to permeate cell membranes more easily than vanadate. In the present study, the efficacy of DMHV as a PTP inhibitor in intact cells was compared with that of vanadate by measuring phosphotyrosine levels in various cells treated with these compounds. DMHV was more effective in increasing both the phosphotyrosine levels of various proteins in 3T3L1 fibroblasts and the level of insulin-receptor phosphorylation in CHO cells overexpressing the human insulin receptor. DMHV was about 10- to 20-fold more effective than vanadate in increasing glucose transport and glycogen synthesis in 3T3L1 adipocytes. DMHV, unlike vanadate, also inhibited PTP in Jurkat cells. The implications of these observations are discussed.

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state

Abstract Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells.

In vitro PKA phosphorylation-mediated human PDE4A4 activation

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg2+ dose–response (EC50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA‐catalytic subunit increases the enzymes sensitivity to Mg2+, leading to 4‐fold increased cAMP hydrolysis without affecting its K m. The phosphorylation also increases the potencies of (R)‐ and (S)‐rolipram without affecting CDP‐840 and SB‐207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.

Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases.

We have characterized some novel caged fluorescein diphosphates as photoactivatable, cell-permeable substrates for protein tyrosine phosphatases and explored their usefulness in identifying inhibitors of tyrosine phosphatases. 1-(2-Nitrophenyl)ethyl protected fluorescein diphosphate (NPE-FDP) undergoes rapid photolysis to release FDP upon irradiation with a 450-W UV immersion lamp and its by-product does not inactivate protein tyrosine phosphatase 1B (PTP1B) or alters the viability of cells. The generated FDP from photolysis of NPE-FDP was shown to have exactly the same properties as FDP, which can be used as a PTP substrate in pure enzyme assays. We have also demonstrated that the PTP activity can be measured using NPE-FDP in small droplets. Its advantage as an inert substrate before photolysis allows the possibility of applying nanospray technology in screening and optimizing PTP inhibitors through a large chemical library. Like other caged bioeffectors such as nucleotide and inositol trisphosphate, NPE-FDP is cell-permeable. The NPE-FDP can be photolyzed to generate FDP inside cells, and then can be hydrolyzed by phosphatases to produce fluorescein monophosphate and subsequently to fluorescein. Although Jurkat cells contain high concentrations of CD45, it has not been possible to use FDP as a substrate to measure CD45 activity in the intact cell. This is due to the hydrolysis of FDP by several other cellular phosphatases. However, NPE-FDP can be useful as a cell-permeable substrate for overexpressed phosphatases such as alkaline phosphatase.

A general method for the rapid characterization of tyrosine‐phosphorylated proteins by mini two‐dimensional gel electrophoresis

Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3‐L1 using a mini two‐dimensional gel electrophoresis approach. The minigel format, 8 × 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two‐dimensional gels. The accurate localization and relative quantification of tyrosine‐phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone‐40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity.

Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase.

The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-end inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the initial velocities of LTC4 formation in the absence of the inhibitor using non-linear regression fits of various models to the data favoured a random, rapid equilibrium mechanism, with strong substrate inhibition by LTA4, over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be Vmax = 14 +/- 4 microM/min, KLTA4 = 40 +/- 18 microM, KGSH = 0.4 +/- 0.2 mM, and a KiLTA4 = 2.3 +/- 1.7 microM for the rapid equilibrium random model. Inhibition of enzymatic activity by L-699,333 was found to be reversible as assessed by the ability of the enzyme to restore its activity by 95% upon dilution. L-699,333 was found to be a competitive inhibitor against GSH and non-competitive against LTA4. Non-linear least squares regression analysis yielded estimated parameters of Km = 0.7 +/- 0.1 mM, Vmax = 2.5 +/- 0.1 microM/min, and Ki = 0.7 +/- 0.1 microM for GSH at a fixed LTA4 concentration of 20 microM, and Km = 45 +/- 3 microM, Vmax = 4.9 +/- 0.2 microM/min, and a Ki = 5.8+/-0.4 microM for LTA4 at a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed for L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation.

Cyclooxygenase-2 inhibitors

Publisher Summary This chapter deals with aspects of the discovery and development of Cyclooxygenase-2 (Cox-2) inhibitors, which, in the case of rofecoxib, resulted from efforts that were initiated in July of 1992 and culminated in the launch of the drug in the United States in June of 1999. Nonsteroidal antiinflammatory drugs (NSAIDs) have long been used to treat pain, fever, and inflammation. Gastropathy caused by NSAIDs, thus constitutes a major public health problem and is one of the most prevalent serious adverse drug effects in industrialized societies. The biochemical target of NSAIDs is the enzyme Cox, which catalyzes the synthesis of prostaglandin G2 (PGG2) from arachidonic acid and its conversion to prostaglandin H2 (PGH2), which is the precursor of all the prostanoids. The availability of Cox-1 to provide a convenient counterscreen predictive of the main adverse effects to be avoided provided a reliable guide for the medicinal chemistry program. The availability of numerous structural leads is a major factor in the rapid progress of the Cox-2 inhibitor drug discovery programs.

Catalytic Properties and Reaction Mechanism of 5-Lipoxygenase

The 5-lipoxygenase from leukocytes catalyzes the oxidation of arachidonic acid to 5hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 (LTA4) as the first two steps of the leukotriene biosynthesis pathway. The reaction catalyzed by 5-lipoxygenase is similar to that of other mammalian and plant lipoxygenases, showing activation by the hydroperoxide product, kinetic lag phases and turnover-dependent inactivation, but with additional requirements for ATP, Ca2+1 and for a leukocyte protein (FLAP) presumably involved in the translocation of the enzyme to the membrane during cellular leukotriene production2.