Open Access This article is
- freely available
Molecules 2017, 22(12), 2058; doi:10.3390/molecules22122058
PTP1B Inhibitors from the Entomogenous Fungi Isaria fumosorosea
College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China
College of Pharmaceutical Science, Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Hebei University, Baoding 071002, China
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Correspondence: Tel: +86-898-6698-9095 (Y.-X.Z.); +86-159-3128-7995 (D.-Q.L.)
Received: 8 November 2017 / Accepted: 23 November 2017 / Published: 24 November 2017
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.
Keywords:Isaria fumosorosea; PTP1B; pyrrolidinedione
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 . More recent evidence has suggested protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling pathway . 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 . 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 . 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 . 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 , which was found to improve insulin resistance in type II diabetes . 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.
2. Results and Discussion
2.1. Identification of Compounds 1–6
Compound 1 was obtained as red oil. It was assigned a molecular formula of C29H37NO4 (12 degrees of unsaturation) by HRESIMS (m/z 464.2792 [M + H]+; calcd for C29H38NO4, 464.2795). The 13C-NMR data (Table 1) of 1 along with analysis of the DEPT spectra displayed signals of 5 methyl, 3 methylene, 13 methine, and 8 quaternary carbons (Supplementary Figure S2). The NMR data of 1 were similar to those of militarinone C , 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 1H-1H 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 1H-1H 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 1H-1H COSY signals. The connectivity from C-15 to C-22 was also supported by the 1H-1H 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 H3-24 (δH 2.06, s) to C-7, C-8, and C-9 and from H3-23 (δH 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 1H-1H 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 13C-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.
2.2. PTP1B Inhibitory Activity of Compounds 1–6
Since fumosorinone have been reported to possess PTP1B inhibitory activity , 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).
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 341 spectropolarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were measured on an UV-210 spectrometer (Analytikjena, Jena, Germany). IR Spectra were acquired on a PerkinElmer 577 instrument (PerkinElmer, Waltham, MA, USA). NMR Spectra were recorded on a Bruker AM-600 spectrometer (Bruker, Fllanden, Switzerland). HR-MS Spectra were recorded on a Bruker apex-ultra 7.0T spectrometer (Bruker, Billerica, MA, USA). Column chromatography (CC) were conducted over silica gel (SiO2; 200–300 mesh; Yantai Zhi Fu chemical Co., Yantai, China), and Sephadex LH-20 gel (25–100 μm, GE Healthcare Co., Ltd., Uppsala, Sweden). TLC were conducted with silica gel GF254 plates (Yantai Zhi Fu chemical Co., Ltd., Yantai, China).
3.2. 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 KH2PO4, 1.5 g of MgSO4, 0.1 g of citric acid, and 10.0 mg of thiamin hydrochloride, in 1 L of deionized H2O). 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 H2O), and the fermentation was carried out at 26 °C under light for 30 days.
3.3. Extraction and Isolation
The fermented material was extracted three times with AcOEt (15 L for each time). Evaporation of the solvent in vacuo gave a yellow oily residue (200 g), which was subjected to CC [SiO2; petroleum ether (PE)/AcOE 100:0, 95:5, 90:10, 80:20, 60:40, 50:50 (v/v)] to afford six fractions, Fr. 1–6. Fr. 4 (35 g) was further purified by repeated CC (Sephadex LH-20; Methanol) and prep. TLC [PE/acetone 5:1 (v/v)] to afford compound 1 (10 mg), compound 2 (15 mg), compound 5 (8 mg), compound 6 (13 mg). Fr. 4 (12 g) was further purified to afford compound 3 (21 mg) and compound 4 (17 mg).
3.4. Spectroscopic Data
Fumosorinone A (1), C29H37NO4, obtained as red oil; −207 (c 0.1, MeOH); UV(MeOH) λmax (log ε): 203 (4.46), 225 (4.40), 307 (4.29) nm; IR (KBr) λmax 3395, 2959, 1655, 1515, 1444, 1239 cm−1; 1H- and 13C-NMR data, see Table 1. Positive HR-ESI-MS [M + H]+ m/z 464.2792 (calcd for C29H38NO4 464.2795).
3.5. PTP Assay
PTP1B activity was measured as the rate of hydrolysis of p-nitrophenyl phosphate (pNPP) in a 96-well microtiter plate format . Sodium orthovanadate was used as the positive control. Each experiment was performed in triplicate, and IC50 data were derived from three independent experiments.
A new tetramic acid possessing a rare pyrrolidinedione skeleton named fumosorinone A (1), along with four known 10-membered macrolides 2–5 and a known cyclopeptide 6 identified as cepharosporolides C (2), cepharosporolides E (3), cepharosporolides F (4), 2-carboxymethyl-4-(3′-hydroxybutyl)furan (5), and beauvericin (6), have been isolated from the an EtOAc extract of entomogenous fungus Isaria fumosorosea. Fumosorinone A (1) and beauvericin (6) showed significant PTP1B inhibitory activity with IC50 value of 3.24 μM and 0.59 μM, suggesting novel kinds of protein tyrosine phosphatase inhibitors.
The 1D- and 2D-NMR spectra are available as Supplementary Materials.
This work was funded by National Natural Science Foundation of China (31371957 and 31672070), Changjiang Scholars and Innovative Research Team in University (IRT_15R16), the High-Level Talents Programs of Hebei Province (GCC2014034), and the national Key Research and Development Program of China (2017YFD0201400 and 2017YFD0201401).
Jun Zhang designed and performed the experiments; Lin-Lin Meng, Jing-Jing Wei, and Peng Fan contributed to data collection and analysis. Sha-Sha Liu and Yu-Yuan Wei contributed to literature search and figure design. You-Xing Zhao and Du-Qiang Luo wrote the paper. All the authors reviewed and approved the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
- Hunter, T. Signaling—2000 and beyond. Cell 2000, 100, 113–127. [Google Scholar] [CrossRef]
- Huijsduijnen, R.H.V.; Sauer, W.H.B.; Bombrun, A.; Swinnen, D. Prospects for inhibitors of protein tyrosine phosphatase 1B as antidiabetic drugs. J. Med. Chem. 2004, 47, 4142–4146. [Google Scholar] [CrossRef] [PubMed]
- Barr, A.J. Protein tyrosine phosphatases as drug targets: Strategies and challenges of inhibitor development. Future Med. Chem. 2010, 2, 1563–1576. [Google Scholar] [CrossRef] [PubMed]
- Liu, G. Protein tyrosine phosphatase 1B inhibition: Opportunities and challenges. Curr. Med. Chem. 2003, 10, 1407–1421. [Google Scholar] [CrossRef] [PubMed]
- Liu, L.X.; Zhang, J.; Chen, C.; Teng, J.T.; Wang, C.S.; Luo, D.Q. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus isaria fumosorosea. Fungal Genet. Biol. 2015, 81, 191–200. [Google Scholar] [CrossRef] [PubMed]
- Liu, Z.Q.; Liu, T.; Chen, C.; Li, M.Y.; Wang, Z.Y.; Chen, R.S.; Wei, G.X.; Wang, X.Y.; Luo, D.Q. Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice. Toxicol. Appl. Pharmacol. 2015, 285, 61–70. [Google Scholar] [CrossRef] [PubMed]
- Schmidt, K.; Riese, U.; Li, Z.Z.; Hamburger, M. Novel tetramic acids and pyridine alkaloids, militarinones B, C, and D, from the insect pathogenic fungus paecilomyces militaris. J. Nat. Prod. 2003, 66, 378–383. [Google Scholar] [CrossRef] [PubMed]
- Stahl, M.; Schopfer, U.; Frenking, G.; Hoffmann, R.W. Assignment of relative configuration to acyclic compounds based on 13C NMR shifts. A density functional and molecular mechanics study. J. Org. Chem. 1996, 61, 8083–8088. [Google Scholar] [CrossRef] [PubMed]
- Clark, A.J.; Ellard, J.M. Synthesis of the C9-C25 fragment of L-755, 807. Evidence for the relative configuration of the side-chain. Tetrahedron Lett. 1998, 39, 6033–6036. [Google Scholar] [CrossRef]
- Ackland, M.J.; Hanson, J.R.; Hitchcock, P.B. Structure of the Cephalosporolides B-F, A Group of C10 Lactones from cephalosporium aphidicola. J. Chem. Soc. Perkin Trans. 1 1985, 843–847. [Google Scholar] [CrossRef]
- Rukachaisirikul, V.; Pramjit, S.; Pakawatchai, C.; Isaka, M.; Supothina, S. 10-membered macrolides from the insect pathogenic fungus cordyceps militaris BBC 2816. J. Nat. Prod. 2004, 67, 1953–1955. [Google Scholar] [CrossRef] [PubMed]
- Zhan, J.X.; Burns, A.M.; Liu, M.X.; Faeth, S.H.; Gunatilaka, A.A. Search for cell motility and angiogenesis inhibitors with potential anticancer activity: Beauvericin and other constituents of two endophytic strains of fusarium oxysporum. J. Nat. Prod. 2007, 70, 227–232. [Google Scholar] [CrossRef] [PubMed]
- Chen, Y.P.; Yang, C.G.; Wei, P.Y.; Li, L.; Luo, D.Q.; Zheng, Z.H.; Lu, X.H. Penostatin derivatives, a novel kind of protein phosphatase 1B inhibitors isolated from solid cultures of the entomogenous fungus Isaria tenuipes. Molecules 2014, 19, 1663–1671. [Google Scholar] [CrossRef] [PubMed]
- Sample Availability: Samples of the compounds are available from the authors.
Figure 1. Structures of compounds 1–6.
Figure 2. 1H-1H COSY (bold) and key HMBC (arrow) correlations of fumosorinone A (1).
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data of compound 1 (in CD3OD).
|No.||δC (Dept)||δH (Mult, J in Hz)||No.||δC (Dept)||δH (Mult, J in Hz)|
|2||174.8, C||17a||44.6, CH2||1.16 a, m|
|3||99.7, C||17b||1.34 a, m|
|4||194.1, C||18||32.3, CH||1.34 a, m|
|5||61.6, CH||4.05, t (4.8)||19a||29.8, CH2||1.16 a, m|
|6a||36.5, CH2||2.89, dd (14.1, 6.1)||19b||1.34 a, m|
|6b||3.03, dd (14.1, 4.1)||20||10.3, CH3||0.88, t (7.4)|
|7||185.0, C||21||18.2, CH3||0.88, d (6.9)|
|8||128.5, C||22||20.4, CH3||0.98, d (6.6)|
|9||142.7, CH||7.67, d (9.5)||23||11.4, CH3||1.84, s|
|10||126.4, CH||6.70, dd (15.2, 9.5)||24||11.2, CH3||2.06, s|
|11||142.8, CH||6.70, dd (15.2, 9.5)||1′||126.4, C|
|12||126.2, CH||6.42, dd (15.2, 9.5)||2′||130.4, CH||7.03, d (8.2)|
|13||142.8, CH||6.54, d (15.2)||3′||114.7, CH||6.71, d (8.2)|
|14||132.7, C||4′||155.9, C|
|15||143.3, CH||5.45, d (9.8)||5′||114.7, CH||6.71, d (8.2)|
|16||30.5, CH||2.68, m||6′||130.4, CH||7.03, d (8.2)|
a Overlapped signals.
Table 2. PTP1B inhibitory activities of 1–6.
|1||3.24 ± 0.37|
|6||0.59 ± 0.15|
|Sodium orthovanadate||11.3 ± 0.87|
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).