Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Asperpyridone A: An Unusual Pyridone Alkaloid Exerts Hypoglycemic Activity through the Insulin Signaling Pathway Yuben Qiao,†,∥ Qianqian Xu,†,∥ Wenya Feng,† Li Tao,† Xiao-Nian Li,‡ Junjun Liu,† Hucheng Zhu,† Yuanyuan Lu,§ Jianping Wang,† Changxing Qi,*,† Yongbo Xue,*,†,⊥ and Yonghui Zhang*,†
Downloaded via UNIV OF MELBOURNE on September 6, 2019 at 22:08:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, People’s Republic of China ‡ State Key Laboratory of Phytochemistry and Plant Resourcses in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan Province, People’s Republic of China § Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, People’s Republic of China ⊥ School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China S Supporting Information *
ABSTRACT: A pyridone alkaloid, asperpyridone A (1), which possesses an unusual pyrano[3,2-c]pyridine scaffold, was isolated from solid cultures of the endophytic fungus Aspergillus sp. TJ23. Its structure, including its absolute configuration, was determined using a combination of nuclear magnetic resonance, highresolution electrospray ionization mass spectrometry, quantum chemical calculations (electronic circular dichroism), and X-ray crystallography. In vitro bioassays demonstrated that asperpyridone A (1) could function as a potential hypoglycemic agent, which exhibited pronounced glucose uptake effect in liver HepG2 cells, under both normal and insulin-resistant conditions, with higher efficacy than metformin. The underlying mechanism of asperpyridone A was elucidated by analyzing the genes expressed, the Gene Ontology (GO) function enrichment, the protein interaction network, and real-time quantitative reverse transcription polymerase chain reaction, which suggested that asperpyridone A exhibits hypoglycemic activity by activating the insulin signaling pathway. Moreover, on the basis of the hypoglycemic potency, fibroblast growth factor 21 (FGF21) was determined to be a potential target for asperpyridone A.
D
obese animal models and humans.7 Therefore, the development of a new therapeutic agent that activates FGF21 may improve treatment for type 2 diabetes and other disorders linked to obesity. Over the past 40 years, a significant number of the FDAapproved small-molecule drugs have been directly or indirectly derived from natural products (NPs), such that natural resources continue to play a significant role in NP-based drug discovery.8,9 Recently, our research group initiated a program to search for structurally diverse and bioactive products from plants of traditional Chinese medicines (TCMs) and their endophytic fungi.10−12 Previously, a chemical investigation of the endophytic fungus Aspergillus sp. TJ23 led to the isolation of a series of bioactive compounds by growing the fungus using a variety of conditions.13,14 Our current work on the secondary metabolites of this Aspergillus sp. TJ23 strain, grown on solid medium, resulted in the isolation and characterization of asperpyridone A
iabetes has become a serious threat to public health and is a growing burden on the global economy.1 Type 2 diabetes (T2D), characterized by insulin resistance and metabolic disorders, ranks as one of the world’s most prevalent diseases.2 Initially, T2D was predominantly reported in the Western countries; however, we now know that T2D occurs throughout the world, particularly in adults. In fact, China currently has the highest number of people with diabetes, which may be due to a higher insulin sensitivity and a much lower insulin response than those of people of European descent and African populations.3 In addition, on the basis of the latest research, the number of T2D patients is expected to increase by 230 million from 2015 to 2040, forecasted by the International Diabetes Federation.4 Characterized by impaired insulin secretion, T2D appears to result from an inadequate response to insulin,5 and an effective strategy for T2D treatment may improve the insulin sensitivity. Fibroblast growth factor 21 (FGF21) is an endocrine hormone that binds to a cell-surface receptor complex containing a classic fibroblast growth factor (FGF) receptor.6 Pharmacologically, FGF21 is a potent insulin sensitizer that improves metabolic dysfunction in a number of © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 28, 2019
A
DOI: 10.1021/acs.jnatprod.9b00188 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
(1), which contains an unusual pyrano[3,2-c]pyridine scaffold carbon skeleton and a 1,3-dimethyl-cyclohexane motif (Figure 1). A review of the literature reveals that pyrano[3,2-c]pyridine
NMR and HRESIMS data suggested that compound 1 was likely an unusual pyridone alkaloid composed of two subunits (units 1 and 2) (Figure 2).19 The heteronuclear multiple bond
Figure 1. Chemical structures of 1 and its analogues.
Figure 2. Key 2D NMR correlations of 1.
containing organic compounds possess a variety of biological activities, attracting broad attention from pharmacologists and chemists.15,16 For instance, leporin B was reported to activate Hexokinase II (HKII), the key enzyme in the muscle responsible for insulin-stimulated glucose phosphorylation.17 The biosynthesis of leporin C, an analogue of leporin B, was reported in a recent article.18 In the present study, in vitro bioactive assays have demonstrated that compound 1 can act as a potential hypoglycemic agent. The isolation, structural elucidation, and potent hypoglycemic activity of compound 1 and its potential targets are discussed. Asperpyridone A (1) was obtained in the form of colorless massive crystals. The high-resolution electrospray ionization mass spectrometry (HRESIMS) coupled to the 13C nuclear magnetic resonance (NMR) data of 1 inferred the molecular formula C16H23NO3, corresponding to six degrees of unsaturation. The absorption band at 1763 cm−1 in the infrared (IR) spectrum of 1 showed the presence of a carbonyl group. The 1H NMR, 13C NMR, and distortionless enhancement by polarization transfer (DEPT) spectra data of 1 (Table 1) indicated that it may have a tricyclic ring system. On the basis of the heteronuclear single quantum coherence (HSQC) spectrum analysis, all proton resonances could be assigned to their respective carbons. Detailed analyses of the 2D
correlation (HMBC) correlations from H3-15 to C-10 and C9, from H3-16 to C-12 and C-7, and from H3-14 to C-8 and C-13 coupled to the key 1H−1H COSY correlations of H-7/H-8/H29/H-10/H2-11/H-12, H3-14/H-13/H-8, H3-15/H-10, and H316/H-12 sequences revealed the presence of cycloxane ring A in unit 1. Subsequently, the substructure of unit 2 (ring B) was determined by analyzing the 1H−1H COSY spectrum of H-5/H6 and determining the key HMBC correlations from H-5 to C-3 and C-4 and from H-6 to C-2 and C-3. Additionally, the dramatically downfield carbon chemical shift of a methoxyl group (δC 64.6) was directly correlated with N-1 after the detailed analysis by NMR. The connectivity of units 1 and 2 was determined to occur by the direct carbon−carbon bond between at C-7 and C-3 based on the key HMBC correlations from H316, H-8, and H-7 to C-3 (Figure 2). Subsequently, the existence of an O-linkage between C-13 and C-4 and the presence of ring C in 1 were established to satisfy the requirement pertaining to degrees of unsaturation and were further supported by a key HMBC correlation from H-13 (δH 3.63) to C-4 (δC 163.5). The resulting planar structure of 1 is shown in Figure 1. The relative configuration of 1 was established via the detailed analysis of its nuclear Overhauser effect spectroscopy (NOESY) spectrum (Figure 2 and Figure S6). The NOE correlations of H3-14/H-8, H-8/H-10, and H-10/H-12 indicated that these protons are cofacial and tentatively assigned a β-orientation, whereas H3-15, H3-16, H-13, and H-7 have the opposite αorientation. The verification of the planar structure and the absolute configurations of 1 was determined by a combination of electronic circular dichroism (ECD) calculations based on timedependent density functional theory (TDDFT) method at the B3LYP/6-311++G** level with the polarizable continuum model (PCM) in MeOH, and a single-crystal X-ray diffraction experiment using Cu Kα radiation was applied (Figure 3 CCDC 1564050).20−22 The resulting structure of 1 was unequivocally established as shown in Figure 3 and Figure S11. Referring to the natural origin of leporins B and C,15,18 a plausible biosynthesis of 1 was proposed (Scheme S1). A polyketide chain (a) derived from one unit of acetyl-CoA and four units of malonyl-CoA was catalyzed by polyketide synthase (PKS) and two units of S-adenosyl methionine (SAM). A series of reactions including dehydration, oxidation, ring cleavage, and reduction occurred to yield d. Subsequently, the key cyclization via a classical Diels−Alder reaction afforded the tricyclic system of e, which was further methylated to yield 1. Compound 1 did not have cytotoxic activities against cell lines HepG2, AsPC-1, BXPC-3, Panc02, and CT-26. Published reports have established that leporin B exerts antidiabetic activity.17 Because the structure of compound 1 is similar to that
Table 1. 1H and 13C NMR Data for Compound 1 in CDCl3 (δ in ppm and J in Hz) 1 position
δC
type
δH (J in Hz)
2 3 4 5 6 7 8 9a 9b 10 11a 11b 12 13 14 15 16 1−OMe
159.9 113.8 163.5 99.7 132.0 44.2 49.2 37.2 37.2 32.8 45.8 45.8 40.6 77.9 18.8 22.4 22.8 64.6,
C C C CH CH CH CH CH2 CH2 CH CH2 CH2 CH CH CH3 CH3 CH3 OMe
5.78, d (7.7) 7.26, d (7.7) 2.27, t (10.1) 1.45, m 0.81, q (12.0) 1.79, m 1.64, m 1.07, m 1.76, m 1.63, m 3.63, dq (10.0, 6.3) 1.31, d (6.3) 0.94, d (6.5) 1.13, d (6.7) 3.99, s B
DOI: 10.1021/acs.jnatprod.9b00188 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
significantly increased by compound 1 at 10 μM (Figure 6A). Phosphoenolpyruvate carboxykinase (PEPCK, PCK2) is well known for its role in promoting gluconeogenesis and insulin resistance, whereas compound 1 increased glucose consumption in HepG2 cells. However, FGF21 can act as an insulin sensitizer to overcome the peripheral insulin resistance. Hence, we further explored the activation effect of compound 1 on FGF21 using qRT-PCR. As expected, FGF21 was significantly decreased in HepG2 cells under insulin-resistant conditions. In the normal control group, compound 1 administration at 5 μM was able to effectively return FGF21 expression levels (Figure 6B). Furthermore, compound 1 dose-dependently increased FGF21 at dosage concentrations ranging from 5 to 25 μM (Figure 6B). The symptoms of type 2 diabetes, in particular, insulinresistant type 2 diabetes, rely heavily on glucose metabolism. The present study demonstrates that compound 1 exhibits a pronounced glucose uptake effect on liver HepG2 cells under both normal and insulin-resistant conditions, with higher efficacy than metformin and lower cytotoxicity. We further identified the gene expression profile of treated HepG2 cells to reveal the potential targets of compound 1. As expected, compound 1 was found to modulate cellular glucose homeostasis by regulating the PPAR signaling pathway, insulin signaling pathway, and insulin resistance signaling pathway. Moreover, the protein interaction analysis uncovered FGF21 as the most likely target. In addition to the RNA-seq results, we repeatedly determined FGF21 levels using qRT-PCR. The results confirmed FGF21 as a potential target for compound 1 based on hypoglycemic potency. The remarkable hypoglycemic effect of compound 1 against insulin resistance demonstrates its potential use for developing effective small-molecule agents for diabetes treatments. Natural sources are known to be a good place to search for novel bioactive compounds for the treatment of diseases. More importantly, the work herein underscores the advantages of using existing NP resources to identify novel antidiabetic compounds.
Figure 3. X-ray crystallographic structure of 1.
of the former compound, it was predicted that it may also exhibit antidiabetic activity. The liver is the largest glucose metabolizing organ in the body and is responsible for insulin resistance. The present study tested the influence of 1 on the glucose utilization in HepG2 liver cells. 50 μM of compound 1 was found to promote the glucose consumption in HepG2 cells (Figure 4A), indicating potential hypoglycemic activity. We further constructed an insulin-resistant cell model in which 2-NBDG uptake was decreased to 33.4%. We found that compound 1 could improve the glucose uptake by insulin-resistant cells (Figure 4B). RNA-seq was used to investigate the gene expression profile of HepG2 cells treated with 10 μM compound 1. As shown in Figure 5A, compound 1 increased the expression of 81 genes and decreased the expression of 24 genes in HepG2 cells (≥2-fold change, q value ≤0.001). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the differential expressed genes were significantly enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, insulin signaling pathway, and insulin resistance pathway (Figure 5B). Protein interaction analysis uncovered regulatory networks of the differential expressed genes, indicating four potential target genes (PCK2, IRS1, LIPE, and FGF21) for compound 1 (Figure 5C). Subsequently, the transcription levels of the four genes were determined using realtime quantitative reverse transcription polymerase chain reaction (qRT-PCR) to further elucidate the targets of compound 1. PCK2 and FGF21 transcription levels were
■
EXPERIMENTAL SECTION
General Experimental Procedures. The melting point was measured using a microscopic melting point apparatus (Beijing Tech.X5). The optical rotation value was obtained using a PerkinElmer 341
Figure 4. Hypoglycemic activity of compound 1. (A) Glucose concentrations of HepG2 cells treated with compound 1 (50 μM) and control (DMSO). (B) 2-NBDG uptake of HepG2 insulin-resistant cells administrated by compound 1. Metformin was used as the positive control. ***P < 0.001. C
DOI: 10.1021/acs.jnatprod.9b00188 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Figure 5. RNA-seq uncovered the mechanism and potential target of compound 1. (A) Differentially expressed genes (DEGs) of HepG2 cells treated by compound 1. Lighter red in the heat-map indicates high expression, lighter green indicates low expression, and black denotes medial expression. (B) Functional enrichment analysis of the KEGG pathway. (C) Interaction network of DEGs reveals potential targets for compound 1. Asperpyridone A (1): colorless crystals; mp 212−214 °C; [a]24D −352.9 (c 0.41, MeOH); UV (MeOH) λmax (log ε): 218 (4.47) and 290 (3.81) nm; IR (KBr) νmax: 2940, 1763, 1607, 1488, 1284, and 1041 cm−1; CD (MeOH) λmax (Δε) 205 (+2.27), 218 (−0.48), 231 (+2.68), 252 (+2.39), and 290 (−3.43); ECD (MeOH) λmax (Δε) 213 (−7.41), 244 (+1.55), and 283 (−1.78) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 278.1750 [M + H]+ (calcd for 278.1756) and 300.1531 [M + Na]+ (calcd for 300.1576). X-ray Crystallography. Asperpyridone A (1) was recrystallized from CH2Cl2−MeOH to obtain colorless crystals. Crystallographic data (excluding structure factor tables) for 1 (CCDC 1564050)22 have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be acquired free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK (fax: + 44-1223-336-033; or E-mail:
[email protected]). Crystallographic data for 1: C16H23NO3, M = 277.35, a = 8.40150(10) Å, b = 8.82050(10) Å, c = 19.3619(3) Å, α = 90°, β = 90°, γ = 90°, V = 1434.82(3) Å3, T = 100(2) K, space group P212121, Z = 4, μ(CuKα) = 0.708 mm−1, 7785 reflections measured, 2402 independent reflections (Rint = 0.0270). The final R1 values were 0.0270 (I > 2σ(I)). The final wR(F2) values were 0.0707 (I > 2σ(I)). The final R1 values were 0.0271 (all data). The final wR(F2) values were 0.0708 (all data). The goodness of fit on F2 was 1.091. Flack parameter = 0.12(4). ECD Calculations. The theoretical calculation of compound 1 and conformations generated by BALLOON21 were subjected to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program.20 The conformation optimization, ECD spectra calculation of each conformation, and the final ECD combination were performed according to the previously reported procedures.23 Cell Culture and 2-NBDG Uptake. HepG2 cells were purchased from the China Center for Type Culture Collection (CCTCC). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and 1% penicillin/streptomycin (100 units/mL) (Invitrogen, USA) at 37 °C in a humidified atmosphere containing 5% CO2.24 The glucose concentration in HepG2 cell (control group treated with 0.1% DMSO, sample group treated with 50 μM of compound 1) supernatants was determined by the glucose oxidase−peroxidase method with a glucose test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).25 HepG2 cells were treated with 0.1 units/mL insulin in DMEM without FBS for 24 h to construct the insulin-resistant cell model. HepG2 cells were pretreated with compound 1 at 10, 25, and 50 μM concentrations for 24 h and subsequently incubated with 2-NBDG for 30 min, after which the cells were analyzed for glucose uptake using flow cytometery.26 Metformin (50, 500, 750, and 1000 μM) served as the positive control.
Figure 6. qRT-PCR identified FGF21 as a possible target of compound 1 on hypoglycemic activity. (A) Verification of possible targets PCK2, IRS1, LIPE, and FGF21 in RNA-seq data. (B) FGF21 transcription levels in insulin resistance HepG2 cells treated with compound 1 at different doses. Control groups: normal HepG2 cells treated with 1% DMSO (solvents for compound 1). Model groups: HepG2 cells conducted with insulin resistance model. *P < 0.05, **P < 0.05.
polarimeter. HRESIMS was obtained from a Thermo Fisher LC-LTQOrbitrap XL spectrometer. UV and IR spectroscopic data were measured using Varian Cary 50 and Bruker Vertex 70 instruments, respectively. Experimental ECD spectra were recorded on a JASCO810 spectropolarimeter. NMR spectroscopic data were recorded on a Bruker AM-400 spectrometer, referring to CDCl3 (δH 7.26 and δC 77.0). Compound purity was obtained using a Dionex high-performance liquid chromatography (HPLC) system with a reversed-phased C18 column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). Column chromatography was performed using silica gel (80−100, 100−200, and 200−300 mesh, Qingdao Marine Chemical, China), octadecylsilyl (ODS) (50 μm, Merck, Germany), and Sephadex LH-20 (Merck, Germany). Fungus Cultivation, Extraction, and Isolation. The fungus was grown on rice (rice−water, 1:0.9 m/v) cultural medium (20 kg) for 21 days, after which metabolites were extracted six times using 95% alcohol (30 L), and the solvent was evaporated under reduced pressure to obtain the extract part. The resulting extract was suspended in H2O and partitioned successively with EtOAc to give the EtOAc part (75.0 g). The EtOAc part was chromatographed using silica gel (100−200 mesh) and eluted with petroleum ether−EtOAc (80:1 → 1:1 v/v) to give eight fractions (F1−F8) based on the TLC analysis. F5 (2.4 g) was fractioned using Sephadex LH-20 (CH2Cl2−MeOH) to afford seven fractions (SF1−SF7). SF3 (2.2 g, wet weight) was further chromatographed using an ODS C18 column (MeOH−water, 30% → 100%) to yield nine subfractions. Subfraction 7 (64.0 mg) was purified by semipreparative HPLC (MeCN−H2O, 54:46 v/v) to yield 1 (5.6 mg; 2 mL/min, tR 46.5 min). D
DOI: 10.1021/acs.jnatprod.9b00188 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
RNA-seq and Identification of Differentially Expressed Genes. Total RNAs were extracted with Trizol (Invitrogen, USA) from HepG2 cells treated with 10 μM of compound 1 or DMSO (control) for 24 h. Three biological replicates for the control group and the sample group were sequenced using the BGISEQ-500 platform for each group.27 The differentially expressed genes (DEGs) were screened out with a q value (adjusted p value) ≤ 0.001 and |fold change| ≥ 2.28 Pathway Functional Enrichment and Protein−Protein Interaction Analysis. The KEGG pathway functional enrichment analysis was utilized to reveal the pathways influenced by compound 1. Cytoscape was used to construct a protein−protein interaction network to investigate the distribution of DEGs in the regulation network of compound 1 on HepG2 cells.28 Quantitative Real-Time PCR Tests. Total RNA was reversetranscribed into cDNA using a transcription kit (Promega, Madison, WI). qRT-PCR was performed using SYBR Green qPCR mix (ABI, USA). The resulting cDNA was amplified according to the manufacturer’s instructions. Values are expressed as arbitrary units relative to β-actin.29 The corresponding primer sequences are listed in Table S1. Statistical Analysis. Experimental results are expressed as means ± SD of triplicate measurements. Statistical analyses employed one-way analysis of variance, followed by Dunnett’s t test.30 The difference was considered to be statistically significant when the p value was
