Cryptopleurine Analogs with Modification of E Ring Exhibit Different Mechanism to Rac-Cryptopleurine and Tylophorine

Tylophorine analogs exhibit a broad range of pharmacological activities, including anti-cancer, anti-inflammatory, anti-autoimmune, and anti-virus effects. Structure-activity relationship study of different structure tylophorine analogs can provide further understanding of their biological activity. Modifications on the E ring of the quinolizidine moiety of cryptopleurine analogs changed the potency and the selective inhibitory effect on NF-κB, AP-1, and CRE signaling pathways. Functional cryptopleurine analogs showed potent inhibition of NF-κB signaling pathway in both HepG2 and HEK-293 cell lines. The E ring structure analogs also differed in suppression of protein translation, and expression of cyclin D1. Our results showed that DCB-3503 or Rac-cryptopleurine could be a scaffold for modification to yield compounds with different mechanisms of action.


Introduction
Tylophorine alkaloids are natural products originally identified in the Asclepiadaceae and Moracea family. Their claimed medical uses include the treatment of cancer, lupus, and inflammation [1,2,3,4,5,6]. NCI's COMPARE program indicated that their activity was distinct from other known anticancer compounds, suggesting that this group of analogs have a novel mode of action that is different from current chemotherapeutic compounds [1]. Tylophorine alkaloids, such as DCB-3500, DCB-3503, or Raccryptopleurine (chemical structures shown in Fig. 1), inhibit synthesis of protein, DNA, and RNA. DCB-3503 preferentially downregulated express of proteins with short a half-life, e.g. cyclin D1 [7,8]. They exhibit inhibitory effect on NF-kB signaling pathway, but are less potent against activator protein-1 (AP-1), and cyclic AMP response elements (CREs) signaling pathways [1,7,9]. Tylophorine analog DCB-3503 but not DCB-3500 is active against HepG2 and PANC-1 xenografts in nude mice [1,2], suggesting that the R14 hydroxyl group is important for in vivo activity.
Due to the diverse and potent pharmacological activities of tylophorine analogs, many groups including ours synthesize and modify different tylophorine alkaloids analogs and study their structure-activity relationship (SAR). This group of compounds shares a common pentacyclic structure with the phenanthrene ring conjugated with the indolizidine (five-member E ring) or quinolizidine (six-member E ring) moiety. Previous SAR study has found that cryptopleurine analogs with quinolizidine moiety (Fig. 1) were more active than analogs with indolizidine moiety against several cancer cell lines and NF-kB signaling pathway in vitro [9]. In the present study, we further evaluated SAR of a series of newly synthesized cryptopleurine analogs with modifications especially on the quinolizidine moiety.
The cytotoxicity of these compounds against human hepatoma cell lines (HepG2 and Huh-7) is shown in Table 1 (The dose-cell viability curves used to obtain the IC 50 value is shown in Figure  S1a and S1b). These compounds showed different selectivity against HepG2 and Huh-7 cell lines, suggesting that the mechanisms of action of different analogs could be cell-type specific. Structurally, YXM-109, 110, 139, and 140 differ only in the chirality and position of hydroxyl (-OH) group. The addition of a (R)-hydroxyl group on the R13 position (YXM-110) decreased the cytotoxicity to about seven fold in HepG2 cells and three fold in Huh-7 cells compared to Rac-cryptopleurine. The addition of an (S)-hydroxyl group on the R13 position (YXM-109) further decreased the cytotoxicity in both cells lines. YXM-140 with an (R)-hydroxyl group on the R12 position showed similar cytotoxic potencies compared to YXM-109. However, the addition of an (S)-hydroxyl group on the R12 position (YXM-139) resulted in loss of potency against the tested cancer cell lines. Modifications on the E-ring quinolizidine moiety, including substitution of carbon by nitrogen or oxygen (YXM-66, 82, 93, and 101), replacing the sixmembered E-ring with a seven-membered E-ring (YXM-83) or with a five-membered N-cyclopropylpyrrolidinyl E-ring (YXM-93), caused the loss of cytotoxicity towards HepG2 and Huh-7 cell lines. Together with our previous report, these newly synthesized compounds showed similar spectrum of potency against A549, DU145, KB, KBvin, SKBR3 cell lines to HepG2 and Huh 7 cell lines as we reported in the present study [12]. The more potent cryptopleurine analogs (Rac-cryptopleurine, YXM-109, YXM-110, and YXM-140) exhibited moderate selectivity (nM IC 50 ) against KB, KBvin, and Huh 7 cell line. These results provided important information that the structure requirements for the most potent cytotoxic analogs for different cell lines appear to be the same.
NF-kB plays important role in controlling inflammation, cancer cell survival and death, and formation of chemoresistance [10,11]. Regulation of transcription factors including AP-1 and CRE governs key steps in controlling cell proliferation, inflammation, and apoptosis [13,14]. Those three signal transduction pathways also interplayed among themselves. Inhibition of NF-kB signaling pathway is involved in the inhibition of cancer cell growth and suppression of inflammatory diseases in DCB-3503-treated mice model [2,5,13,15]. DCB-3503, Rac-cryptopleurine, and their functional analogs preferentially inhibited NF-kB to AP-1, and CRE signaling pathways in HepG2 cell line [1,7,9]; therefore, we determined the activities of these eleven new cryptopleurine analogs to the above three signaling pathways in HepG2 and HEK-293 cell lines (The dose-luciferase activity curves used to obtain the IC 50 value is shown in Figure S2a-S3c for HepG2 cells and S3a-S3c for HEK-293 cells). Results in Table 2 obtained from HepG2 cells showed that DCB-3503 and Rac-cryptopleurine preferentially inhibited NF-kB signaling pathway. YXM-140 had similar pattern of selectivity. YXM-109 and 110 had almost equal potency against NF-kB and AP-1 signaling pathways. YXM-139 with a R12-(S)-OH substitution showed at least 1000-fold less potent than its R13 -OH isomers (YXM-109, -110) and R12 enantioisomer (YXM-140). YXM-142 exhibited about 3-fold more potent against AP-1 than against NF-kB signaling pathway. YXM-93 and -139 inhibited CRE pathway preferentially in HepG2 cells. While YXM-66, 82, 83, and 101 exhibited almost equal activities  This result suggests that inhibition of NF-kB rather than AP-1 and CRE signaling pathway could be one of the key factors related to the potency of cryptopleurine analogs. Activation of NF-kB will induce the expression of Cox 2 [16] and iNOS [17], we then analyzed the effect of the treatment of cryptopleurine analogs on these two NF-kB pathway downstream targets by Western blot. Cryptopleurine analogs down-regulated the expression of both Cox 2 and iNOS in HepG2 cells at their IC 50 concentration against NF-kB pathway (Fig. 2). This confirmed the inhibition of NF-kB pathway by cryptopleurine analogs. These results showed that modifications on the E-ring of cryptopleurine analogs are directly related to their selectivity against NF-kB, AP-1, and CRE signaling pathways in HepG2 cell line. Structural analogs can not only lead to altered potency [9], but also change mechanisms of action.
DCB-3503 suppressed the expression of cellular proteins with a short half-life, for instance cyclin D1 and p53 [8]. Therefore, we examined the effect of the treatment of functional crytopleurine analogs on the expression of cyclin D1. Figure 3 showed that the treatment of Rac-cryptopleurine, and DCB-3503 with about three times IC 50 concentration decreased more than 70% of cyclin D1 expression. The treatment of YXM-109, -110, and -140 also showed more than 50% inhibitory effect on cyclin D1 expression at their IC 50 concentration in HepG2 cells (Fig. 3). However, the treatment of YXM-139 did not change cyclin D1 expression level at its IC 50 concentration (Fig. 3).
We previously demonstrated that DCB-3503 inhibited protein synthesis at the elongation step of translation, which could be the basis of inhibiting cell growth and TNF/NF-kB pathway [8]. The effect of some cryptopleurine analogs on synthesis of cellular proteins was examined. Figure 4 showed that DCB-3503, Raccryptopleurine, YXM-109, 110, and 140 inhibited incorporation of [ 35 S]-methionine/cysteine into newly synthesized proteins after treatment for 4 hours at their IC 50 concentration. However, the  The effect of cryptopleurine analogs on protein translation was examined by capped luciferase mRNA with T3 promoter and poly (A) tail in Retic lysate in vitro translation system. DCB-3503 and Rac-cryptopleurine inhibited about 50% of translation of luciferase mRNA at 250 nM and 500 nM, respectively (Fig. 5). YXM-109 and 110 has significantly inhibitory effect on luciferase mRNA translation at 250 nM (p,0.05); however, YXM-139 and 140 did not inhibit translation under the same condition at 500 nM and 250 nM, respectively (Fig. 5). This suggested that modifications of the E ring not only altered cytotoxicity, but also changed their activity against protein translation.

Conclusion
Tylophorine analogs with introduction of different moieties (e.g. N or O) in the E-ring have less potency against the growth of HepG2 and Huh-7 cell lines, and some of those are no longer functional analogs. Introduction of the hydroxyl group into the Ering altered the cytotoxicity of cryptopleurine ananlogs, and the chirality of the hydroxyl group is critical determination factor in the cytotoxicity. Cryptopleurine analogs with 6-member E-ring are more potent than 7-member E-ring analogs (Fig. 6 for results of HepG2 cells and Fig. S4 for results of HEK-293 cells). Together with results from our previous SAR studies [9], we propose the following order of potency in terms of NF-kB inhibition and cytotoxicity: six member E-ring with R14a-(R)-hydrogen.five member E-ring with R13a-(R)-hydrogen.five member E-ring with R13a-(S)-hydrogen..six member E-ring with R14a-(R)hydrogen. The results we obtained suggest that the E ring size may be a critical determination factor for the interaction of cryptopleurine analogs to their molecular targets, and molecular target of cryptopleurine analogs may not be identical. Changing structure of compound may not only be reflected in potency, but also determined the mode of action. Since the biochemical determinants of the primary target(s) of these compounds may vary, the selectivity against different signaling pathways could be cell type specific. This was demonstrated by the comparative study of HepG2 and HEK-293 cell lines. Their potency against HepG2 cell growth correlates well with the inhibitory activity against  protein synthesis and TNF/NF-kB pathway. Tylophorine or Raccryptopleurine could be a good scaffold for synthesis of biological active compound with diverse action. We are in the process of evaluating the in vivo antitumor activity of selected cryptopleurine analogs with R15 hydroxyl group.

Cell Lines and Growth Conditions
Cell lines were obtained from the American Type Culture Collection (ATCC). HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. HepG2 stable cell lines harboring NF-kB, AP-1, CRE response elements in pGL4 vector (Promega) were maintained in the presence of 0.8 mg/ml G418. Huh-7 and HEK-293 cells were maintained in DMEM containing 4.5 g/l glucose supplemented with 10% FBS. All cell lines were maintained in a humidified incubator with an atmosphere of 95% air and 5% CO 2 at 37uC.

Cytotoxicity Assay
Ten thousands cells/well were plated in 24-well plates. After overnight incubation, cells were treated with drugs for 72 hours. Cells were fixed and stained with 0.5% methylene blue in 50% ethanol for 2 hours at room temperature, followed by washing with tap water to remove excess color. Plates were dried and then resuspended in 1% sarkosyl and incubate for 3 hours at room temperature. Cell growth was quantitated based on the amount of methylene blue adsorbed into cellular proteins measured by spectrophotometer (Molecular Devices) at 595 nm. IC 50 was defined as the concentration of drug that inhibited cell growth by 50% after continuous drug exposure for 72 hours [1].

Signaling Pathway Reporter Assay
HepG2 and HEK-293 cell lines stably harboring NF-kB, AP-1, and CRE response elements in pGL4.0 luciferase vector (Promega) were used for the signaling pathway reporter assay [1]. Cells were treated with 50 ng/ml TNF-a to stimulate NF-kB signaling pathway, 10 ng/ml TPA to stimulate AP-1, and 1 mM forskolin to stimulate CRE signaling pathway for 1 hour priory to addition of compounds for another 4 hours. Medium was removed at the end of the treatment, and cell extracts were prepared and luciferase activity was measured by Luciferase assay kit (Promega) according to the manufacturer's instructions. IC 50 was defined as the concentration of drug that inhibited stimulator-triggered luciferase reporter activation by 50% after continuous drug exposure for 4 hours.

[ 35 S]-amino Acid Mixture Incorporation Assay
The incorporation assay was done followed by the protocol described previously [8]. In brief, HepG2 cells treated with drugs were labeled with 50 mCi/ml [ 35 S]-methionine/cysteine (Perki-nElmer) for 30 minutes before harvest. Incorporation of [ 35 S]methionine/cysteine was determined by scintillation counter.

In vitro Transcription
The detailed protocol for in vitro transcription was reported previously [8]. Luciferase encoding plasmid T3 luciferase was linearized by BamHI, and was used as the template for in vitro transcription. Capped luciferase mRNA was generated by mMESSAGE mMACHINE high yield capped RNA transcription kit containing T3 RNA polymerase (Ambion). The in vitro transcribed mRNAs were purified by MEGAclear kit (Ambion); and the integrity of mRNA was examined by Bioanalyzer (Agilent Technologies, Santa Clara, CA). The purified mRNAs were used for in vitro translation experiments.

In vitro Translation
In vitro translation was performed by Retic Lysate IVT TM (Ambion) as described previously [8]. The in vitro translation mixtures containing 50 ng/ml T3 luciferase mRNA was incubated for 90 minutes at 30uC. Translation products of T3 luciferase were measured by luciferase assay.

Statistical Analysis
Data were analyzed by ANOVA and the Bonferroni multiple comparision test by GraphPad Prism 5 software. The difference was considered to be statistically significant when p,0.05.