Discovery of Highly Potent Daphnane Diterpenoids Uncovers Importin-ß1 as a Druggable Vulnerability in Castration-Resistant Prostate Cancer.

Importins are overexpressed in many cancers and mediate the abnormal nuclear transport of oncogenic factors. The druggable potential of importins still remains unclear, largely because of the lack of potent inhibitors. Herein, the anti-castration-resistant prostate cancer (CRPC) screening of a Euphorbiaceae diterpenoid library followed by target fishing led to the identification of a highly potent importin-ß1 inhibitor, daphnane diterpenoid DD1. DD1 selectively inhibited the growth and survival of CRPC cells at subnanomolar concentrations and completely blocked tumor growth in preclinical models at an extremely low dosage. Mechanistic studies revealed that targeting of importin-ß1 by DD1 significantly reduced the nuclear accumulation of key CRPC drivers, shutting down their downstream oncogenic signaling. Disruption of the predicted binding sites of DD1 on importin-ß1 abolished this anti-CRPC effect. These findings suggest that importin-ß1 is an effective therapeutic target in CRPC and that DD1 as the most potent importin-ß1 inhibitor to date can be developed as therapeutics for treatment of this disease.

Tigliane and rhamnofolane glycosides from Euphorbia wallichii prevent oxidative stress-induced neuronal death in PC-12 cells.

Tigliane and rhamnofolane diterpenoids bearing glycosyl substituents are rarely found in nature. In the current study, seven new tigliane glycosides, euphorwallsides A - G (1-7), and five new rhamnofolane glycosides, euphorwallsides H - L (8-12), were isolated from the whole plants of Euphorbia wallichii. Their structures were elucidated by a combination of spectroscopic, computational, and chemical means. The aglycones of 1-5 represent a rare class of 13-deoxygenated tiglianes, while those of 8-12 represent the first examples of 4-deoxygenated rhamnofolanes. 2, 3, 5, 7, 8, and 12 showed significant neuroprotective effects on sodium nitroprusside (SNP)-induced neuronal death in pheochromocytoma cell line PC-12 at 10 µM, being more active than the clinical drug, edaravone. Mechanistic study revealed that the most active compound, 3, could inhibit reactive oxygen species (ROS) accumulation and restore the mitochondrial membrane potential via modulating the Nrf2 signaling pathway in PC-12 cells.

Structural Elucidation of Three 9,11-Seco Tetracyclic Triterpenoids Enables the Structural Revision of Euphorol J.

Compounds 1-3, the rare examples of 9,11-seco euphane or lanostane triterpenoids featuring an enol-hemiacetal functionality, were isolated from Euphorbia stracheyi. Their structures were elucidated by a combination of spectroscopic, computational, chemical, and single-crystal X-ray diffraction means, which enables the structure of previously published euphorol J to be revised as 1. 1-3 showed significant cytotoxicities on the breast cancer cell line MDA-MB-468 with IC50 values in the range of 2.9-3.9 µM.

Presegetane diterpenoids from Euphorbia sieboldiana as a new type of anti-liver fibrosis agents that inhibit TGF-ß/Smad signaling pathway.

Seven new diterpenoids, eupholenes A-G (1-7), including two presegetanes (1 and 2), four jatrophanes (3-6), and one paraliane (7), along with 19 known analogues (8-26) were obtained by anti-liver fibrosis bioassay-guided isolation of Euphorbia sieboldiana. Their structures were elucidated by extensive spectroscopic data analyses, chemical methods, ECD calculations, and single-crystal X-ray diffractions. Euphorbesulin A (10), a presegetane diterpenoid (5/9/5 ring system), was identified as a promising anti-liver fibrosis agent that could inhibit the expressions of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagen I in TGF-ß1-stimulated LX-2 cells at a micromolar level. Mechanistic study revealed that 10 suppressed liver fibrosis via inhibition of TGF-ß/Smad signaling pathway, and its potential target was TGF-ß type I receptor. These findings suggested that presegetane diterpenoid could serve as a new type of structural motif in future anti-liver fibrosis drug development.

Salviplenoid A from Salvia plebeia attenuates acute lung inflammation via modulating NF-κB and Nrf2 signaling pathways.

Acute lung injury (ALI) involves series of inflammatory pathologies and cause high morbidity. Salviplenoid A (SA) was a new sesquiterpenoid from the traditional inflammatory herb Salvia plebeia. In our previous study, SA exhibited antiinflammatory activity in RAW264.7 cells. However, the extensive effects of SA in human cells and in vivo and the active mechanisms are unclear. Thus, in this study, we sought to access its effects in vitro and in vivo and to investigate its mechanisms. SA was proved to inhibit the induction of proinflammatory cytokines in human cell types, including pulmonary epithelial cells and endothetial cells. It also depressed monocyte adhesion. Moreover, SA potently attenuated the acute lung inflammation in the LPS-induced mouse model shown by down-regulation of proinflammatory mediators, inhibition of polymorphonuclear neutrophil infiltration, and alleviation of related symptoms like alveolar congestion and mucus secretion. Further evaluation confirmed that SA regulated NF-κB pathway by inhibiting the IκB-α phosphorylation. And it markedly mediated Nrf2/HO-1 pathway by activating the Nrf2/HO-1 expression and promoting Nrf2 nuclear translocation. Therefore, SA could attenuate acute lung inflammation via suppressing NF-κB and activating Nrf2, which provide a theoretical basis for the potential application of SA in clinic.

19-nor-, 20-nor-, and tetranor-Halimane-Type Furanoditerpenoids from Croton crassifolius.

The phytochemical investigation of the roots of Croton crassifolius led to the isolation of 16 new halimane furanoditerpenoids, crohalifuranes A-P (1-16), along with 15 known analogues, 17-31. The new structures including their absolute configurations were elucidated by NMR and MS data analysis, comparison of experimental and calculated electronic circular dichroism data, single-crystal X-ray diffraction data, and chemical methods. Crohalifuranes A (1) and B (2) are tetranor- and 19-nor-halimane diterpenoids featuring a rare decahydroacenaphthene core, respectively, which might be derived from the accompanying crassifoliusin A by loss of the furan ring or the C-19 substituent. Crohalifurane C (3) represents the first example of a 20-nor-halimane diterpenoid, and crohalifurane D (4) is characterized by an unusual 6,20-δ-lactone moiety. All compounds were examined for their inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells, and 2 and 23 exhibited moderate inhibition on NO production with IC50 values of 17.2 ± 1.3 and 23.7 ± 1.4 µM, respectively.

Euphoresulanes A-M, structurally diverse jatrophane diterpenoids from Euphorbia esula.

Thirteen new jatrophane diterpenoids, euphoresulanes A-M (1-13), and seven known analogues (14-20) were isolated from the whole plants of Euphorbia esula. Their structures were elucidated by extensive spectroscopic analysis, and the absolute configurations of 1, 6, and 10 were confirmed by single crystal X-ray diffraction. Compounds 1-20 were screened for the multidrug resistance (MDR) reversal activity on P-glycoprotein (Pgp)-dependent cancer cell line HepG2/ADR, and 1, 2, 4, 6, and 8 exhibited comparable activity to the positive drugs. Euphoresulane H (8), the most active MDR modulator, could enhance the efficacy of anticancer drug adriamycin to ca. 33 folds at 5 µM. The structure-activity relationship (SAR) study revealed that the acyloxy substitution at C-9 is essential to the activity and presence of H-2 was favorable.

Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata.

Twenty new ingol diterpenoids, euphornans A-T (1-20), representing a rare class of C-19-oxidated and H-2, H-3 ß-oriented ingols, were isolated from the seeds of Euphorbia marginata. Their structures including absolute configurations were elucidated by extensive spectroscopic analysis, ECD analysis, and single crystal X-ray diffraction. Compounds 1-20 were screened for the multidrug resistance (MDR) reversal activity on P-glycoprotein (Pgp)-dependent MDR cancer cell line HepG2/ADR, and 11, 14, and 18 were identified as potent MDR modulators that could enhance the efficacy of anticancer drug adriamycin to ca. 20 folds at 5 µM. The Pgp inhibition mechanism and brief structure-activity relationships (SARs) of these compounds were also discussed.

Two highly oxygenated nor-clerodane diterpenoids from Croton caudatus.

Two highly oxygenated nor-clerodane diterpenoids, crocleropenes A and B (1 and 2), together with four known compounds (3-6) were isolated from the leaves and twigs of Croton caudatus. Their structures were elucidated by combination of extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. 1 and 2 represent the first examples of nor-clerodane-3,5(10)-diene diterpenoids. Compounds 1 and 2 exhibited weak cytotoxicity in vitro against MCF-7 cancer cells with IC50 values of 35.8 and 40.2 µM, respectively. [Formula: see text].

Talaesthanes A-C, three new meroterpenoids from the endophytic fungus Talaromyces primulinus H21.

Three new meroterpenoids (1-3) and ten known ones (4-13) were obtained from the endophytic fungus Talaromyces primulinus H21 isolated from the plant of Euphorbia sikkimensis. Their structures including their absolute configurations were elucidated by extensive analysis of spectroscopic data such as HR-ESI-MS, 1D/2D NMR, and X-ray diffraction of single crystal together with comparison of experimental ECD with calculated ECD. All compounds were examined for their inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 cells, and compounds 3, 9, 12, and 13 exhibited certain inhibition on NO production, with IC50 values of 27.19, 41.55, 25.23, and 24.71 µM, respectively.

Discovery of the First Raptor (Regulatory-Associated Protein of mTOR) Inhibitor as a New Type of Antiadipogenic Agent.

Raptor, a regulatory-associated protein of mTOR, has been genetically proved to be an important regulator in lipogenesis. However, its druggable potential is rarely investigated, largely due to the lack of an inhibitor. In this study, the antiadipogenic screening of a daphnane diterpenoid library followed by target fishing led to the identification of a Raptor inhibitor, 1c (5/7/6 carbon ring with orthoester and chlorine functionalities). Pharmacodynamic studies verified that 1c is a potent and tolerable antiadipogenic agent in vitro and in vivo. Mechanistic studies revealed that the targeting of Raptor by 1c could block the formation of mTORC1 and then downregulate the downstream S6K1- and 4E-BP1-mediated C/EBPs/PPARγ signaling, eventually retarding adipocyte cell differentiation at the early stage. These findings suggest that Raptor can be explored as a novel therapeutic target for obesity and its related complications, and 1c as the first Raptor inhibitor may provide a new therapeutic option for these conditions.

Diversity of sesquiterpenoids from Stellera chamaejasme with neuroprotective effects.

Chromatographic fractionation of the 95% EtOH extract of the roots of Stellera chamaejasme yielded 20 sesquiterpenoids of four different types, guaiane-, carotane-, sesquicarane-, and alpiniane-types. Among them, sesquistrachanoids A-F were previously undescribed ones, whose structures including absolute configurations were elucidated by spectroscopic methods, the Mo2(OAc)4-induced ECD experiment, and analysis of experimental and calculated 1D NMR and ECD data. Sesquistrachanoid A is a 2,3-seco-guaiane-type sesquiterpenoid with a α-pyrone core and sesquistrachanoid B is the first example of 8,9-seco-guaiane-type sesquiterpenoid featured with an 1,8-δ-lactone core. Sesquistrachanoid C is a guaiane sesquiterpenoid characterized by a peroxide bridge between C-8 and C-10. All sesquiterpenoids were evaluated for their neuroprotective effects on cell damage induced by sodium nitroprusside in PC-12 cells. The bioassay results showed that six compounds at 10 µM could restore the cell viability, being comparable to that of the positive control edaravone. The mechanistic study on the most pronounced activity compound, stelleraguaianone B, demonstrated that it played a neuroprotective role by promoting the mRNA expression of antioxidant enzymes to reduce oxidative stress.

Discovery of a highly potent and orally available importin-ß1 inhibitor that overcomes enzalutamide-resistance in advanced prostate cancer.

Nuclear transporter importin-ß1 is emerging as an attractive target by virtue of its prevalence in many cancers. However, the lack of druggable inhibitors restricts its therapeutic proof of concept. In the present work, we optimized a natural importin-ß1 inhibitor DD1 to afford an improved analog DD1-Br with better tolerability (>25 folds) and oral bioavailability. DD1-Br inhibited the survival of castration-resistant prostate cancer (CRPC) cells with sub-nanomolar potency and completely prevented tumor growth in resistant CRPC models both in monotherapy (0.5 mg/kg) and in enzalutamide-combination therapy. Mechanistic study revealed that by targeting importin-ß1, DD1-Br markedly inhibited the nuclear accumulation of multiple CRPC drivers, particularly AR-V7, a main contributor to enzalutamide resistance, leading to the integral suppression of downstream oncogenic signaling. This study provides a promising lead for CRPC and demonstrates the potential of overcoming drug resistance in advanced CRPC via targeting importin-ß1.

Jolkinolide B sensitizes bladder cancer to mTOR inhibitors via dual inhibition of Akt signaling and autophagy.

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB + mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer.

Euphylonoids A and B, Two Highly Modified Jatrophane Diterpenoids with Potent Lipid-Lowering Activity from Euphorbia hylonoma.

Euphylonoids A (1) and B (2), two highly modified jatrophane diterpenoids, were isolated from Euphorbia hylonoma. 1 represents a new 9(10→18)-abeo-8,12-cyclojatrophane skeleton containing a cage-like 3,8-dioxatricyclo[5.1.2.04,9]decane core, while 2 is a 14(13→20)-abeo-8,12-cyclojatrophane featuring an unusual 17-oxatetracyclo[12.2.1.01,5.09,13]heptadecane framework. Their structural elucidation was completed by spectroscopic, chemical, computational, and single-crystal X-ray diffraction means. 2 significantly inhibited early adipogenesis in 3T3-L1 adipocytes via activating AMP-activated protein kinase signaling.

A pair of novel bisindole alkaloid enantiomers from marine fungus Fusarium sp. XBB-9.

A preliminary research on the marine fungus Fusarium sp. XBB-9 resulted in a pair of novel bisindole alkaloid enantiomers, (+)- and (-)-fusaspoid A (1a/1b) and 12 diverse compounds. One strain many compound (OSMAC) method was used to enhance as many biosynthetic pathways as possible. The structures of the compounds were elucidated by spectroscopic analysis, and the absolute configuration of 1a was determined by X-ray single-crystal diffraction analysis. Compounds 1a and 1b were tested for cytotoxic activity against HCT-15, RKO cell lines, but were inactive. Compounds 1a and 1b were the first example of bisindole alkaloids isolated from fungus Fusarium sp. XBB-9.

Euphanoids A and B, two new lathyrane diterpenoids with nitric oxide (NO) inhibitory activity from Euphorbia kansuensis.

Two new lathyrane diterpenoids, euphanoids A and B (1 and 2), along with five known compounds (3-7) were isolated from the roots of Euphorbia kansuensis. Their structures were elucidated by spectroscopic analysis, and the absolute configuration of 1 was determined by single crystal X-ray diffraction. All of the isolates were screened for the inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 cells, and compounds 1 and 2 showed pronounced inhibition on NO production with IC50 values of 4.7 and 9.5 µM, respectively, being more active than the positive control, quercetin (IC50 = 10.8 µM).

Discovery of 8,9-seco-ent-Kaurane Diterpenoids as Potential Leads for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a lethal malignancy without safe and effective therapeutic drugs. In this study, the anti-TNBC bioassay-guided isolation of the medicinal plant Croton kongensis followed by the structural modification led to the construction of a small ent-kaurane diterpenoid library (1-25). With subsequent biological screening, 20 highly potent compounds (IC50s < 3 µM) were identified. Among them, 8,9-seco-ent-kaurane 6 displayed comparable activity (IC50s ∼ 80 nM) to doxorubicin but with better selectivity. The analysis of structure-activity relationships suggested that the cleavage of the C8-C9 bond and the presence of α,ß-unsaturated ketone moiety were essential for the activity. The mechanistic study revealed that 6 induced apoptosis, autophagy, and metastasis suppression in TNBC cells via inhibition of Akt. In vivo, 6 significantly suppressed the TNBC tumor growth without causing side effects. All these results suggested that 6 may serve as a promising lead for the development of novel anti-TNBC agents in the future.

Jolkinolide B targets thioredoxin and glutathione systems to induce ROS-mediated paraptosis and apoptosis in bladder cancer cells.

Bladder cancer is a clinically heterogeneous disease with a poor prognosis. In the current study, anti-proliferation assay of a Euphorbiaceae diterpenoid library led to the identification of an anti-bladder cancer agent Jolkinolide B (JB). JB showed significant cytotoxicity against a panel of bladder cancer cell lines and suppressed the growth of cisplatin (CDDP)-resistant bladder cancer xenografts in single or combination treatments. Mechanistic study revealed that, besides inducing mitogen-activated protein kinase (MAPK)-related apoptosis, JB could trigger the paraptosis via activation of reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and extracellular signal-regulated kinase (ERK) pathway. The excessive production of ROS could be induced by JB via inhibition of thioredoxin reductase 1 (TrxR1) and depletion of glutathione (GSH). Collectively, JB that targets thioredoxin and GSH systems to induce two distinct cell death modes may serve as a promising candidate in future anti-bladder cancer drug development.

Diterpenoids from Euphorbia royleana reverse P-glycoprotein-mediated multidrug resistance in cancer cells.

Eight previously undescribed diterpenoids, euphoroyleans A-H, including two cembranes, three ingenanes, two ent-atisanes, and one ent-kaurane, along with 22 known analogues were isolated from the whole plants of Euphorbia royleana. The structures of euphoroyleans A-H, including the absolute configurations, were elucidated by extensive spectroscopic analyses, chemical transformation, and single crystal X-ray diffractions. All the isolates were screened for their chemoreversal abilities on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) cancer cell line HepG2/DOX, and eight compounds exhibited significant activities. Among them, ingol-3,7,12-triacetate-8-benzoate, the most active MDR modulator with no obvious cytotoxicity, could enhance the efficacy of anticancer drug DOX to ca. 105 folds at 10 µM, being stronger than the positive drug verapamil. Mechanistic study revealed that ingol-3,7,12-triacetate-8-benzoate could inhibit the transport activity of P-gp rather than its expression, and the possible recognition mechanism between compounds and P-gp was predicted by molecular docking.