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PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Thus, small molecule inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes and cancer diseases. In a continuing search for new PTP1B inhibitors, a new tetramic acid possessing a rare pyrrolidinedione skeleton named fumosorinone A (1), together with five known ones 2–6 were isolated from the entomogenous fungus Isaria fumosorosea. The structures of 2–6 were elucidated by extensive spectroscopic analysis. Fumosorinone A (1) and beauvericin (6) showed significant PTP1B inhibitory activity with IC50 value of 3.24 μM and 0.59 μM.


Introduction
Tyrosine phosphorylation and dephosphorylation are crucial elements in eukaryotic signal transduction. Phosphatases can be subdivided based on structure and substrate specificity into the protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSP) classes [1]. More recent evidence has suggested protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling pathway [2]. As so far, several 'classical' protein tyrosine phosphatases are attractive therapeutic targets, including PTP1B for obesity and type II diabetes; as well as SHP2 for cancer and Lyp for rheumatoid arthritis [3]. Despite the research efforts in academia and industry over the past decade, there are very few PTPase inhibitors that have been advanced into clinical trials [4]. Thus, inhibition of PTP1B also can be considered as an attractive approach for the design of new therapeutic agents for the treatment of type II diabetes and for new antitumor drugs. To date, very few inhibitors have been isolated from microorganisms, particularly insect pathogenic fungi [3]. Therefore, insect pathogenic fungi have been considered as an untapped source of small molecules PTP inhibitors. In our previous study, a PTP1B inhibitor fumosorinone has been reported [5], which was found to improve insulin resistance in type II diabetes [6]. As part of our continuing search for bioactive constituents, a PTP1B inhibitory alkaloid fumosorinone A together with another five known constituents 2-6 were isolated from Isaria fumosorosea. Herein, the isolation, structural elucidation, and PTP1B inhibitory activity evaluation of these compounds are described.  Figure S2). The NMR data of 1 were similar to those of militarinone C [7], suggesting that 1 ( Figure 1) had a same pyrrolidinedione skeleton deduced from the HMBC correlations (H-5/C-2, C-3, and C-4) and a p-hydroxybenzyl group supported by the 1 H-1 H COSY cross-peaks (H-2 /H-3 and H-5 /H-6 ) and HMBC correlations (H-6/C-2 , C-6 ; H-2 , H-6 /C-4 ) ( Figure 2). The linkage of the p-hydroxybenzyl group with C-5 was established by the 1 H-1 H COSY cross-peak of H-5/H-6 and HMBC correlation from H-6 to C-4. The difference between militarinone C and compound 1 was that the side chain of compound 1 had one more propene group than militarinone C. A proton spin system of an olefinic chain, consisting of two doublets (H-9 and H-13) and three doublets of doublets (H-10 to H-12), was readily detected by 1 H-1 H COSY signals. The connectivity from C-15 to C-22 was also supported by the 1 H-1 H COSY correlations ( Figure 2). Two methyls of C-23 and C-24 were unambiguously assigned to C-8 and C-14, respectively, by HMBC correlations from H 3 -24 (δ H 2.06, s) to C-7, C-8, and C-9 and from H 3 -23 (δ H 1.84, s) to C-13, C-14, and C-15. Therefore, the planar structure of 1 was determined as shown in Figure 1. The relative configuration of the olefinic moiety in fumosorinone A was determined by analysis of the 1 H-1 H coupling constants and NOESY data. The C-10, C-11, and C-12, C-13 double bonds were all assigned E-geometry based on a large coupling constant of 15.2 Hz observed for corresponding olefinic protons, and the same assignment was made for C-14, C-15, and C-8, C-9. The relative configurations of C-16 and C-18 were deduced by the 13 C-NMR shift values of C-21 and C-22 methyl groups, and a difference of 2.2 ppm revealed their syn-configuration [7][8][9]. Thus, compound 1 was elucidated as shown in Figure 1 and named fumosorinone A.

PTP1B Inhibitory Activity of Compounds 1-6
Since fumosorinone have been reported to possess PTP1B inhibitory activity [5], compounds 1-6 were evaluated for inhibition of PTP1B in vitro. Results indicated that 1 and 6 exhibited significant PTP1B inhibitory activity with IC50 value 3.24 and 0.59 μM compared with the positive control Sodium orthovanadate (11.3 μM) ( Table 2).

PTP1B Inhibitory Activity of Compounds 1-6
Since fumosorinone have been reported to possess PTP1B inhibitory activity [5], compounds 1-6 were evaluated for inhibition of PTP1B in vitro. Results indicated that 1 and 6 exhibited significant PTP1B inhibitory activity with IC50 value 3.24 and 0.59 μM compared with the positive control Sodium orthovanadate (11.3 μM) ( Table 2).

PTP1B Inhibitory Activity of Compounds 1-6
Since fumosorinone have been reported to possess PTP1B inhibitory activity [5], compounds 1-6 were evaluated for inhibition of PTP1B in vitro. Results indicated that 1 and 6 exhibited significant PTP1B inhibitory activity with IC 50 value 3.24 and 0.59 µM compared with the positive control Sodium orthovanadate (11.3 µM) ( Table 2).

Fungal Material and Cultivation Conditions
Isaria fumosorosea was isolated from an unidentified Lepidopteran collected in Hebei Province, China, and identified by Prof. Yong-Chun Niu, which was assigned the accession number ACCC 37775 in the culture collection at College of Life Science, Hebei University. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 26 • C for seven days, and then inoculated into 500 mL Erlenmeyer flask containing 100 mL of PDA medium (20.0 g of glucose, 200.0 g of potato, 3.0 g of KH 2 PO 4 , 1.5 g of MgSO 4 , 0.1 g of citric acid, and 10.0 mg of thiamin hydrochloride, in 1 L of deionized H 2 O). The final pH of the media was adjusted to 6.5 before sterilization. After seven days of incubation at 26 • C on rotary shakers at 150 rpm, 10 mL of culture liquid were transferred as seed into each 500 mL Erlenmeyer flask containing rice medium (80 g of rice, 100 mL of deionized H 2 O), and the fermentation was carried out at 26 • C under light for 30 days.

PTP Assay
PTP1B activity was measured as the rate of hydrolysis of p-nitrophenyl phosphate (pNPP) in a 96-well microtiter plate format [13]. Sodium orthovanadate was used as the positive control. Each experiment was performed in triplicate, and IC 50 data were derived from three independent experiments.