Rubipodanin A, the First Natural N-Desmonomethyl Rubiaceae-Type Cyclopeptide from Rubia podantha, Indicating an Important Role of the N 9-Methyl Group in the Conformation and Bioactivity

One new cyclic hexapeptide named rubipodanin A (1), which is the first identified natural N-desmonomethyl Rubiaceae-type cyclopeptide, together with six known Rubiaceae-type cyclopeptides (2–7) were obtained using the TLC cyclopeptide protosite detection method with ninhydrin from the roots and rhizomes of Rubia podantha. The cyclopeptide structures were elucidated by extensive spectroscopic analysis, including 1D-NMR, 2D-NMR, IR, UV and MS. The solution conformation and biological activities of 1 and RA-V (4) were evaluated, and the results demonstrated that the N 9-methyl group plays a vital role in the maintenance of the conformation and bioactivity.

For the unique bicyclic structure and significant antitumor activities in vitro and in vivo [1,2,12], RAs have recently attracted our interest. We have now isolated 28 RAs and synthesized several RA analogues, including 11 novel natural RAs consisting of two new skeleton RAs and one new O-seco-RA from R. yunnanensis and R. schumanniana, respectively [13][14][15][16][17]. We have also evaluated their cytotoxic activities and performed 2D-and 3D-QSAR studies on 54 RAs [18]. Our studies showed that RA-V exhibits anti-inflammatory activity by inhibiting NO production and NF-κB activation induced by TNF-α [13]. Furthermore, RA-V significantly suppresses angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells [19]. In addition, RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis and inhibits cell adhesion and invasion via the PI3K/AKT and NF-κB signaling pathways [20,21]. Importantly, the potential roles of RAs in cancer therapy have been highlighted [22].
Some investigations have shown that N-methylated amino acids of cyclopeptides play an important role in the maintenance of their conformation and bioactivity. Our previous NMR studies have indicated that both natural and synthetic RAs have two or four conformers [13][14][15][16][17][18], which prompted us to obtain the natural N-desmethyl RAs and investigate the functional role of amino acid N-methylation in conformation and bioactivity. In this study, we selected another Rubia plant, Rubia podantha Diels, which has been used as a substitute for the traditional Chinese medicine R. codifolia. The roots and rhizomes have been used to treat tuberculosis, menoxenia, rheumatism, contusion, hematemesis, anemia and lipoma for a long time in China. To the best of our knowledge, however, there is no literature on the chemical and antitumor constitutes of this plant. To expand the distribution of plant resources containing RAs and explore the functional role of N-methylated amino acids in natural RAs in the maintenance of the conformation and bioactivity, we structurally and pharmacologically investigated interesting RAs using the TLC cyclopeptide protosite detection method with ninhydrin [23] from R. podantha. Fortunately, a new cyclic hexapeptide, rubipodanin A (1), which is the first identified natural N-desmonomethyl RA, together with six known RAs (2-7) were obtained. Here, the isolation and structural elucidation of 1-7 as well as cytotoxic and NF-κB signaling pathway activities of 1 and 4 are described.

General experimental procedures
Optical rotations were obtained on a Jasco P-1020 polarimeter. IR spectra were measured by a Tensor 27 spectrometer using KBr pellets. UV spectra were performed using a Shimadzu UV-2401A spectrophotometer. 1D-NMR and 2D-NMR spectra were recorded on a Bruker AVANCE III-600 and Ascend ™ 800 spectrometer at 298K. Chemical shifts (δ) were expressed in parts per million (ppm) with reference to the solvent signals. Mass spectra were obtained on a Waters XEVO-TQD spectrometer or an Agilent G6230 TOF Mass spectrometer. Analytical or semi-preparative HPLC was performed on an Agilent 1100 with a Zorbax Eclipse-C18 (4.6 mm × 150 mm; 9.4 mm × 250 mm; 5 μm).

Plant material
The roots and rhizomes of R. podantha were collected in November of 2013 from Mount Chang Chong, Kunming, China. The locations/activities to collect R. podantha did not require specific permission because this studied plant is common in the field in Yunnan, China and is not cultivated in privately owned or protected areas. In addition, the filed studies did not involve endangered and protected species. The material was identified by Professor Ning-Hua Tan of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN0359279) was deposited in the Herbarium of Kunming Institute of Botany.

Cytotoxicity assay
Cytotoxicity was tested by the Sulforhodamine B (SRB) method (Sigma, USA). The assays were performed as described previously [24].

Cell transfection and luciferase assay
HEK293T cells were seeded in 24-well plates and transiently transfected with 5×κB-luciferase and pTK-Renilla reporters using Lipofectamine 2000 (Invitrogen, USA) for 18 h. The cells were then incubated with different concentrations of compounds for the indicated time, and subsequently stimulated with 10 ng/ml TNF-α for 2 h. The luciferase activity of the cell lysate was analyzed by the Dual Luciferase Reported Assay System (Promega, USA). Luciferase reporter assays were performed as described previously [25].

Western blot analysis
Cells were lysed using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, protease inhibitors and phosphatase inhibitors. After being disrupted on ice for 30 min, the cell lysates were centrifuged at 12,000 rpm for 15 min and boiled with SDS sample buffer at 95°C for 5 min. The samples were equally subjected to SDS-PAGE, electrophoresed, and transferred to PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk in TBST, the membranes were incubated with the indicated antibody for 2 h or overnight at 4°C, and the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The protein bands were detected with a SuperSingal West Pico Chemiluminescence ECL kit (Pierce, USA).

Statistical analysis
Student's t-test was used for statistical analysis. A p value < 0.05 was considered to be statistically significant.

Results and Discussion
The methanol extract of the air-dried powdered roots and rhizomes of R. podantha was subjected to fractionation using silica gel CC, which yielded the total cyclopeptide fraction guided by the TLC cyclopeptide protosite detection method with ninhydrin [23]. This fraction was repeatedly chromatographed over a series of silica gel, Sephadex LH-20, RP-18 CC, and ODS HPLC to obtain seven RAs (Fig 1),   10.59, 9.80, 8.90, 7.81). An extensive comparison of 1D-NMR and 2D-NMR spectra data of 1 with those of RA-V (4) in C 5 D 5 N indicated that both compounds were similar, except for the third amino acid residue (Tyr 3 ). Further analysis of the HMBC and 1 H-1 H COSY correlations confirmed the construction of the N-desmethyl Tyr 3 residue (Fig 2). In the 1 H-1 H COSY spectrum, cross-peaks of NH-3/H-3α/H-3β suggested that the amide N 9 -methyl was absent, which was also confirmed by the HMBC correlation from δ H 10.59 (NH-3) to δ c 173.0 (C-2-CO). In addition, the sequence of the amino acid residues in 1 was confirmed by The N-methylated amino acids of cyclopeptides play an important role for the conformation in solution, and RAs generally possess two or four conformers, including natural RAs, synthetic RAs and O-seco-RAs. In 1995, the DL Boger group synthesized several N-desmethyl derivatives of RA-VII and investigated their conformation property by 1 H-NMR spectra, and they reported that both N 9 -and N 15 -methyl groups are essential for maintenance of their conformation [29][30][31][32], which prompted us to investigate the difference between 1 and 4 in NMR spectra. Consistent with previous studies [31], the 1 H NMR spectra of 4 in C 5 D 5 N displayed the presence of two conformers in a ratio of 79:21, and 1 revealed a single solution conformation (Fig 3). The similar phenomenon occurred in 13 C NMR spectra ( Figure N in S1 File),

Biological Assay
Some studies have shown that natural bioactive cyclopeptides contain at least one N-methylated amino acid. For example, the N-methyl groups of cyclosporin A (CsA), a cyclic undecapeptide containing seven N-methylated amino acids, play an important role for maintenance of immunosuppressive activity [33]. Sansalvamide A, isolated from a marine fungus of the genus Fusarium, is a cyclic tetrapeptide, and the N-methyl derivatives of sansalvamide A exhibit better antitumor activity [34].
RAs are a type of potent antitumor agent and often contain three N-methylated tyrosines, which promotes researchers to explore the functional role of N-methyl groups on their biological activity. The DL Boger group evaluated the cytotoxicity of synthetic N-desmethyl RA derivatives and showed that the N 15 -methyl group is essential for their biological activities [32]. In the present study, we investigated the cytotoxicity against three tumor cell lines and inhibitory activity against the tumor-associated NF-κB signaling pathway of rubipodanin A (1) compared to RA-V (4) by using the SRB assay and the Dual Luciferse Reporter Assay System, respectively. As shown in Fig 4, 1 had weak cytotoxicity against HeLa, A549 and SGC-7901 with IC 50 values of 7.22 ± 0.76, 7.14 ± 0.81, 3.80 ± 0.17 μM, respectively, which was 420-650 times less than 4 ( Table 2). We also found that 4 exhibited a potent inhibitory activity against the NF-κB pathway activation induced by TNF-α, which was approximately 700 times greater than that of 1. In addition, we examined the effects of 1 and 4 on the expression of NF-κB-associated proteins using western blot analysis. As expected, 1 also had an inhibitory effect on TNF-α-induced IκBα phosphorylation, IκBα degradation and p65 phosphorylation only in the high concentration up to 20 μM, while 4 exhibited similar inhibitory effect at a concentration 200 nM. Taken together, these results suggested that the N 9 -methyl group is essential for maintenance of biological activities of natural RAs and that down-regulation of the NF-κB pathway might account for the mechanism of the antitumor activities of RAs. Of note, previous reports have shown that the N 9 -methyl group of synthetic RAs is not critical for biological activities [32], which was not consistent with our results. So more N-desmethyl derivatives of RAs from plants or by synthesis in future are needed to confirm this difference based on the structure-activity relationship analysis.

Conclusion
The phytochemical studies of Rubia podantha were performed using the TLC cyclopeptide protosite detection method with ninhydrin, which led to the identification of one new cyclic  hexapeptide (rubipodanin A, 1) and six known ones (2)(3)(4)(5)(6)(7). Importantly, 1 is the first identified natural N-desmonomethyl RA. Further studies revealed that 1 had only one solution conformation in NMR but two in RA-V (4), and it also had lower cytotoxicity and inhibitory activity against the NF-κB signaling pathway than 4, thus indicating that the N 9 -methyl group is essential for the conformation and biological activities of RAs. These findings not only expanded the distribution of plant resources containing RAs but also provided new insights into the role of the N-methyl group in maintaining conformational and biological properties of RAs.