Manzamenones Inhibit T-Cell Protein Tyrosine Phosphatase

Manzamenones A~C (1~3) and E~F (5~6), unique oxylipin metabolites isolated from a marine sponge Plakortis sp., have been found to exhibit inhibitory activity against T-cell protein tyrosine phosphatase (TCPTP). The inhibitory activity of 2 and 5 against TCPTP was 4 times more potent than that against protein tyrosine phosphatase-1B (PTP1B).


Introduction
Protein tyrosine phosphatases (PTPs) are responsible for selective dephosphorylation of tyrosine residues, and regulate a wide variety of cellular processes including cell growth, differentiation, metabolism, cell-cell communication, cell migration, the immune response, and apoptosis/survival decisions [1][2][3]. Since recent studies have demonstrated that loss of protein tyrosine phosphatase-1B (PTP1B) activity resulted in enhancement of insulin sensitivity in addition to decrease in susceptibility to diet-induced obesity [4][5], specific PTP1B inhibitors have been developed as therapeutic agents in type-2 diabetes and obesity [6][7]. On the other hand, little attention has been paid to other PTPs such as T-cell PTP (TCPTP), CD45, SHP-1, and SHP-2. Among them TCPTP is the most homologous phosphatase to PTP1B [8], and recent biochemical analysis revealed that TCPTP regulates hematopoietic development, cytokine response, and insulin signaling in vitro [9][10]. In our screening of a number of sponge metabolites against TCPTP and PTP1B, manzamenones B (2) and E (5) [11][12] were found to be relatively potent inhibitors, and more potent against TCPTP rather than PTP1B. In this paper we describe inhibitory activity of manzamenones A~F (1~6) [11][12] against TCPTP and PTP1B, and show manzamenones B (2) and E (5) to be relatively potent inhibitors against TCPTP. CO

Results and Discussion
The inhibitory effects against TCPTP of manzamenones A~F (1~6) were examined as follows. The  Table 1. The IC 50 values of relatively potent compounds 2 and 5, possessing a carboxylic acid functionality in the side chain, were 2.5 and 3.2 µg/ml, respectively, while compound 1, the C-5 epimer of 2, and compounds 3 and 6, the ethyl or n-butyl ester of 1, respectively, showed less inhibitory activity than 2. On the other hand, compound 4, a simple amide at C-5 of 1, did not show such activity. Thus, the presence of a carboxylic acid or ester functionality and the stereochemistry at C-5 may be important for the inhibitory activity.
Next, inhibitory activities of manzamenones A~F (1~6) against PTP1B were examined (Table1).   12.6 >20 --Since manzamenone B (2) showed relatively potent inhibitory activity against TCPTP, its inhibition kinetics were analyzed (Fig. 2). The inhibition mode of 2 was a mixed type one as shown in Fig. 2A, suggesting that 2 might interact with the catalytic center and peripheral site(s) of TCPTP. The Ki value for inhibition of TCPTP by 2 was calculated to be 2.2 µM (Fig.2B).
In this study, manzamenones B (2) and E (5) were found to be relatively potent inhibitors against TCPTP rather than TCP1B. Compounds 2 and 5 may be useful bioprobes for studies of TCPTP functions as well as drug leads of selective TCPTP inhibitors.

Inhibitory activity against protein tyrosine phosphatases
The inhibitory activity of sponge metabolites against TCPTP and PTP1B was examined using pnitrophenyl phosphate (pNPP) as a substrate. An assay mixture (100 µl) containing 100 mM sodium acetate (pH5.0)/1.6 mM dithiothreitol/1 mM pNPP, and enzyme was incubated at 30 °C for 10min. Reactions were terminated by addition of 125 µl of 1M NaOH and 225 µl of each quenched reaction mixture was then added to a well of a 96-well titer plate where the absorbance was read at 405 nm using a microplate spectrophotometer (TECAN). IC 50 values of inhibitors were obtained on the basis of the data at the concentrations of 1, 2, 5 and 10 µM of compounds 1~6. Activities for PP1, PP2A and A-kinase were analyzed as previously described [14].

Determination of Ki values
The initial rate at 10 different substrate concentrations (0.60, 0.70, 0.75, 0.85, 0.90, 1.0, 1.25, 1.5, 2.0 and 2.5 mM) was measured at three different inhibitor concentrations (2, 4 and 6 µM). The inhibition pattern was evaluated using Lineweaver-Burk plot, and the Ki values were obtained as the X-intercept by linear regression of the plot of Km versus inhibitor concentration.