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DOI 10.1007/s11418-016-1061-6
NOTE
123
J Nat Med
PTPs are key regulators of various cellular functions and Prior to performing cell-based experiments on 1 and 2,
consist of more than 100 members, including PTP1B [2]. cell viability was tested in three human cancer cell lines,
Therefore, selective activity against PTP1B over other A549 (lung adenocarcinoma), MCF-7 (breast adenocarci-
PTPs is one of the important properties used in the noma) and K562 (erythroleukemia), in addition to Huh-7
development of clinically useful PTP1B inhibitors. The (hepatoma) and EJ-1 (bladder) cells, which were evaluated
inhibitory effects of 1 and 2 on T-cell protein tyrosine previously [6]. Cancer cells were treated with compounds 1
phosphatase (TCPTP), one of the non-transmembrane PTPs and 2 at 10 or 50 lM for 48 h, and cell proliferation was
[7], and vaccinia H-1-related phosphatase (VHR), one of measured using the WST-1 assay [14]. Similar to the
the dual-specificity phosphatases [8], were evaluated. effects observed against Huh-7 and EJ-1 cells, compounds
The IC50 values of compounds 1 and 2 against TCPTP 1 and 2 did not appear to exhibit cytotoxicity against A549,
were similar to those against PTP1B. On the other hand, MCF-7 or K562 cells (Table 1).
compounds 1 and 2 were less active to VHR than PTP1B PTP1B is mainly expressed in the liver and controls the
(Fig. 2; Table 1). Compound 1 showed threefold greater insulin signal transduction pathway [1]. Thus, the phos-
selectivity against PTP1B than VHR. phorylation levels of Akt, a key downstream effector of the
T-cell protein tyrosine phosphatase shares 72% insulin signaling cascade, were evaluated by Western
sequence similarity in its catalytic domain with PTP1B [7] blotting using Huh-7 cells [15]. As shown in Fig. 3, com-
and is also a negative regulator of insulin and leptin sig- pounds 1 and 2 did not enhance insulin-stimulated p-Akt
naling pathways [3]. Although improved insulin resistance levels in Huh-7 cells up to 50 lM, and did not affect cell
and glucose homeostasis have been reported in ptp1B -/- proliferation.
mice [1, 9, 10], tcptp -/- mice exhibited severe inflam- Although the IC50 value of 1 (0.4 lM) against PTP1B
matory phenotypes [11, 12]. However, recent studies have was more potent than that of oleanolic acid as a positive
indicated that ptp1B ?/- and tcptp ?/- mice with deletions control (1.1 lM) in the PTP1B enzyme assay (Fig. 2;
of single copies of PTP1B and TCPTP displayed no Table 1), phosphorylated Akt levels in Huh-7 cells were
abnormalities [13]. Thus, dual inhibitors against PTP1B not enhanced by 1 (Fig. 3). This discrepancy between the
and TCPTP are still expected to be drug candidates without results obtained in enzyme- and cell-based experiments
major side effects. Compound 1 may become a candidate may be due to low selectivity against PTP1B over other
lead compound in the development of drugs for the treat- PTPs and/or the permeability of 1 in intact cell membranes.
ment of type-2 diabetes and obesity. Therefore, structural optimizations are needed in order for
these compounds to become candidate lead compounds in
therapeutic agents for the treatment of type-2 diabetes.
COOH
H Materials and methods
R
Materials
HOOC
1: R = COOH
(24E)-3,4-Seco-cucurbita-4,24-diene-3,26,29-trioic acid
2: R = CH2OH (1) and (24E)-3,4-seco-cucurbita-4,24-diene-3-hydroxy-
26,29-dioic acid (2) were isolated from the fruiting bodies
Fig. 1 Structures of compounds 1 and 2 isolated from fruiting bodies
of Russula lepida
of R. lepida as described previously [6]. Human
Fig. 2 Inhibitory activities of
compounds 1 (a) and 2
(b) against PTP1B, TCPTP and
VHR
123
J Nat Med
123
J Nat Med
Western blotting 3. Zhang ZY, Dodd GT, Tiganis T (2015) Protein tyrosine phos-
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5 min with 1 nM insulin, washed with phosphate-buffered Opin Ther Pat 24:1101–1115
saline (PBS), and lysed in CelLytic MÒ (Sigma) in order to 6. Lee JS, Maarisit W, Abdjul DB, Yamazaki H, Takahashi O,
collect whole cell lysates, according to the manufacturer’s Kirikoshi R, Kanno S, Namikoshi M (2016) Structures and bio-
instructions. Protein concentrations were measured using a logical activities of triterpenes and sesquiterpenes obtained from
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Acknowledgements This work was supported in part by a Grant-in- 15. Abdjul DB, Kanno S, Yamazaki H, Ukai K, Namikoshi M (2016)
Aid for Scientific Research (25870660 and 16K21310) from the A dimeric urea of the bisabolene sesquiterpene from the Oki-
Ministry of Education, Culture, Sports, Science, and Technology nawan marine sponge Axinyssa sp. inhibits protein tyrosine
(MEXT) of Japan to H.Y. and the Takeda Science Foundation to H.Y. phosphatase 1B activity in Huh-7 human hepatoma cells. Bioorg
We are grateful to the Center for Biomedical Research, Institute of Med Chem Lett 26:315–317
Development, Aging and Cancer, Tohoku University for providing 16. Yamazaki H, Sumilat DA, Kanno S, Ukai K, Rotinsulu H,
the human cancer cell lines. Wewengkang DS, Ishikawa M, Mangindaan REP, Namikoshi M
(2013) A polybromodiphenyl ether from an Indonesian marine
sponge Lamellodysidea herbacea and its chemical derivatives
inhibit protein tyrosine phosphatase 1B, an important target for
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