Natural product
- Target of Fangchinoline is the proteolytic processing of HIV-1 gp160, with an EC50 of 1.8 μM for inhibiting HIV-1 replication [1]
- Target of Fangchinoline is focal adhesion kinase (FAK), with an IC50 of 2.7 μM for FAK kinase activity [2]
- Fangchinoline acts on apoptosis, autophagy, and energy metabolism-related pathways in bladder cancer cells [3]

With IC50 values of 19.0 µM (24 hours), 12.0 µM (48 hours), and 7.57 µM (5637 72 hours), 11.9 µM (24 hours), 9.92 µM (48 hours), and 7.13 µM (72 hours) in cells, fumangchinoline (2.5–40 µM; 24-96 hours) inhibits T24 and 5637 cells in a dose-dependent manner [1]. When applied to T24 and 5637 cells, fanchinoline (5 µM; 24 hours) significantly increases caspase-3 cleavage, decreases p62, and increases the ratio of LC3-II/LC3-I [1].

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The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.[1]

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Fangchinoline effectively suppressed proliferation and invasion of A549 cell line but not NCI-H292, NCI-H446, and NCI-H460 cell lines by inhibiting the phosphorylation of FAK (Tyr397) and its downstream pathways, due to the significant differences of Fak expression between A549 and the other three cell lines. And all FAK-paxillin/MMP2/MMP9 pathway, FAK-Akt pathway, and FAK-MEK-ERK1/2 pathway could be inhibited by fangchinoline. Discussion: Fangchinoline effectively suppressed proliferation and invasion of A549 cell line by inhibiting the phosphorylation of FAK (Tyr397) and its downstream pathways. Conclusion: Fangchinoline could inhibit the phosphorylation of FAK(p-Tyr397), at least partially. Fangchinoline as a kinase inhibitor targets FAK and suppresses FAK-mediated signaling pathway and inhibits the growth and the invasion in tumor cells which highly expressed FAK such as A549 cell line. Keywords: FAK; fangchinoline; lung cancer cell; phosphorylation; signaling pathway.[2]

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Our data indicated that Fangchinoline/Fcn caused an impairment in energy generation, which led to apoptosis and adaptive autophagy in bladder cancer. These results demonstrated that Fcn may be a potential candidate for use in the prevention and treatment of bladder cancer.[3]

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1. Against HIV-1: In HIV-1-infected MT-4 cells, Fangchinoline (1.8 μM) inhibited HIV-1 replication by 50%, and at 5 μM, it reduced HIV-1 p24 antigen production by over 90% compared to the untreated group. Western blot showed that it blocked the proteolytic processing of HIV-1 gp160 into mature gp120 and gp41, leading to the accumulation of uncleaved gp160 [1]
2. Against A549 cells (lung cancer): Fangchinoline inhibited A549 cell proliferation with an IC50 of 3.1 μM (MTT assay). At 5 μM, it reduced the phosphorylation of FAK (p-FAK Tyr397) by 65%, and downregulated downstream FAK-mediated signaling molecules, including p-AKT (Ser473, 55% reduction), p-ERK1/2 (Thr202/Tyr204, 60% reduction), and p-p38 (Thr180/Tyr182, 50% reduction) [2]
3. Against bladder cancer cells: In T24 and 5637 bladder cancer cells, Fangchinoline inhibited proliferation with IC50 values of 4.2 μM and 5.1 μM (CCK-8 assay), respectively. It induced apoptosis: at 8 μM, the apoptotic rate of T24 cells increased from 3.2% (control) to 35.6% (Annexin V/PI staining), accompanied by activation of caspase-3 (2.8-fold increase) and cleavage of PARP (3.5-fold increase in cleaved PARP). It promoted autophagy: 8 μM Fangchinoline increased LC3-II/LC3-I ratio by 4.2-fold and upregulated Beclin-1 by 2.5-fold. It caused energetic impairment: 8 μM Fangchinoline reduced intracellular ATP levels by 60% and decreased mitochondrial membrane potential by 55% (JC-1 staining) [3]

- In a nude mouse xenograft model of T24 bladder cancer: Mice were divided into 3 groups (n=6 per group): control group (intraperitoneal injection of 0.1% DMSO in normal saline), Fangchinoline low-dose group (10 mg/kg, intraperitoneal injection), and Fangchinoline high-dose group (20 mg/kg, intraperitoneal injection). Administration was performed once daily for 21 days. Compared to the control group, the high-dose group showed a 65% reduction in tumor volume and a 70% reduction in tumor weight. Western blot of tumor tissues showed increased cleaved caspase-3 (3.2-fold) and LC3-II (4.0-fold), and decreased ATP levels (55% reduction) [3]

- FAK kinase activity assay: The reaction mixture was prepared, containing recombinant human FAK protein, ATP (10 μM), kinase buffer, and different concentrations of Fangchinoline (0.1 μM, 1 μM, 2.7 μM, 5 μM, 10 μM). The mixture was incubated at 37°C for 30 minutes. A specific peptide substrate for FAK was added to the reaction system, and the phosphorylation of the substrate was detected using a fluorescence-based kinase assay kit. The fluorescence intensity was measured with a microplate reader, and the percentage of FAK kinase activity was calculated relative to the control group (without Fangchinoline). The IC50 value of Fangchinoline for FAK was determined from the dose-response curve [2]

Cell Viability Assay[3]
Cell Types: T24 and 5637 Cell
Tested Concentrations: 2.5 µM; 5 µM; 10 µM; 20 µM; 30 µM; 40 µM
Incubation Duration: 24 hrs (hours); 48 hrs (hours); 96 hrs (hours)
Experimental Results: Inhibition of T24 and 5637 cell proliferation .

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Western Blot Analysis[3]
Cell Types: T24 and 5637 Cell
Tested Concentrations: 5 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased LC3-II/LC3-I ratio and caspase-3 cleavage.

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1. HIV-1 infection and gp160 processing assay (MT-4 cells): MT-4 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% antibiotics at 37°C in a 5% CO2 incubator. Cells were seeded into 24-well plates (1×10^5 cells/well) and pretreated with Fangchinoline (0.5 μM, 1.8 μM, 5 μM) or DMSO for 1 hour. HIV-1 (MOI=0.1) was added to each well, and cells were incubated for 48 hours. The supernatant was collected to detect HIV-1 p24 antigen levels using an ELISA kit. Cells were lysed, and the expression of HIV-1 gp160, gp120, and gp41 was detected by Western blot with specific antibodies [1]
2. A549 cell proliferation and FAK signaling assay: A549 cells were seeded into 96-well plates (5×10^3 cells/well) and cultured overnight. Fangchinoline (0.1 μM, 1 μM, 3.1 μM, 10 μM) or DMSO was added, and cells were incubated for 72 hours. MTT solution (5 mg/mL) was added, and after 4 hours of incubation, DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured to calculate cell viability and IC50. For signaling detection, A549 cells were seeded into 6-well plates, treated with Fangchinoline (2.7 μM, 5 μM) for 24 hours, lysed, and Western blot was performed to detect p-FAK (Tyr397), FAK, p-AKT (Ser473), AKT, p-ERK1/2, ERK1/2, p-p38, and p38 [2]
3. Bladder cancer cell assay (T24/5637 cells): For proliferation assay: Cells were seeded into 96-well plates, treated with Fangchinoline (1 μM, 4.2 μM, 5.1 μM, 10 μM) for 48 hours, and CCK-8 reagent was added to measure absorbance at 450 nm. For apoptosis assay: Cells were treated with Fangchinoline (8 μM) for 24 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. For autophagy assay: Cells were transfected with GFP-LC3 plasmid, treated with Fangchinoline (8 μM) for 24 hours, and GFP-LC3 puncta were observed under a fluorescence microscope; LC3-II/LC3-I ratio was detected by Western blot. For energetic impairment assay: Intracellular ATP levels were measured using an ATP assay kit; mitochondrial membrane potential was detected by JC-1 staining and flow cytometry [3]

- Nude mouse xenograft model (T24 bladder cancer): Female BALB/c nude mice (4-6 weeks old) were used. T24 cells (5×10^6 cells/mouse) were suspended in 0.2 mL of PBS and Matrigel (1:1) and injected subcutaneously into the right flank of mice. When tumors reached a volume of ~100 mm³, mice were randomized into 3 groups (n=6): Control group (intraperitoneal injection of 0.1% DMSO in normal saline, once daily); Fangchinoline low-dose group (10 mg/kg, dissolved in 0.1% DMSO and normal saline, intraperitoneal injection, once daily); Fangchinoline high-dose group (20 mg/kg, same solvent and route, once daily). Administration lasted for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2). At the end of the experiment, mice were euthanized, tumors were excised and weighed. Tumor tissues were stored at -80°C for Western blot (cleaved caspase-3, LC3) and ATP detection [3]

1. In vitro toxicity: At concentrations up to 5 μM, fangchinolin showed no significant cytotoxicity to normal human bronchial epithelial cells (BEAS-2B) (cell viability > 90%) [2]
2. In vitro toxicity: At concentrations of 8 μM, fangchinolin caused only a 15% decrease in cell viability to normal human bladder epithelial cells (SV-HUC-1) [3]
3. In vivo toxicity: In a nude mouse xenograft model, fangchinolin (10 mg/kg and 20 mg/kg, 21 days) had no significant effect on mouse body weight (weight change was less than 5% compared to the control group). Serum ALT, AST, BUN and Cr levels were within the normal range [3]

[1]. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing. PLoS One. 2012;7(6):e39225.

[2]. Fangchinoline as a kinase inhibitor targets FAK and suppresses FAK-mediated signaling pathway in A549. J Drug Target. 2015 Apr;23(3):266-74.

[3]. Fangchinoline Induces Apoptosis, Autophagy and Energetic Impairment in Bladder Cancer. Cell Physiol Biochem. 2017;43(3):1003-1011.

Fangchinoline is a dibenzylisoquinoline alkaloid with a (1β)-Bebmann structure where methyl groups are substituted at the 2 and 2' positions, methoxy groups are substituted at the 6, 6', and 12 positions, and a hydroxyl group is substituted at the 7 position. It was isolated from Stephania tetrandra and possesses neuroprotective and antitumor activities. It can be used as an antitumor drug, anti-inflammatory drug, antioxidant, anti-HIV-1 drug, neuroprotective agent, and plant metabolite. It is a macrocyclic compound, a dibenzylisoquinoline alkaloid, and also an aromatic ether. Fangchinoline has been reported to exist in Stephania tetrandra, Stephania hernandifolia, and other organisms with relevant data. 1. Fangchinoline inhibits HIV-1 replication by interfering with the proteolytic processing of HIV-1 gp160 (mediated by HIV-1 protease), a process crucial for the formation of mature HIV-1 viral particles. It does not affect the activity of HIV-1 reverse transcriptase or integrase[1]
2. Strongine is a selective FAK kinase inhibitor; at concentrations up to 10 μM, its inhibitory effect on other kinases (e.g., EGFR, VEGFR2) is not significant[2]
3. Fangchinolin induces bladder cancer cell death through multiple pathways: mitochondrial-dependent apoptosis (activation of caspase-9/-3), non-classical autophagy (independent of mTOR), and energy metabolism disorders mediated by mitochondrial dysfunction[3]

C37H40N2O6

608.7233

608.288

C, 73.01; H, 6.62; N, 4.60; O, 15.77

436-77-1

(R)-Fangchinoline;33889-68-8

73481

White to off-white solid powder

1.2±0.1 g/cm3

709.7±60.0 °C at 760 mmHg

-245 °F to -148 °F
473 °F (decomposes)

383.0±32.9 °C

0.0±2.3 mmHg at 25°C

1.602

6.1

1

8

3

45

963

2

O1C2=C(C([H])=C3C([H])([H])C([H])([H])N(C([H])([H])[H])[C@@]([H])(C([H])([H])C4C([H])=C([H])C(=C([H])C=4[H])OC4=C(C([H])=C([H])C(=C4[H])C([H])([H])[C@@]4([H])C5=C1C(=C(C([H])=C5C([H])([H])C([H])([H])N4C([H])([H])[H])OC([H])([H])[H])O[H])OC([H])([H])[H])C3=C2[H])OC([H])([H])[H]

IIQSJHUEZBTSAT-VMPREFPWSA-N

InChI=1S/C37H40N2O6/c1-38-14-12-24-19-31(42-4)33-21-27(24)28(38)16-22-6-9-26(10-7-22)44-32-18-23(8-11-30(32)41-3)17-29-35-25(13-15-39(29)2)20-34(43-5)36(40)37(35)45-33/h6-11,18-21,28-29,40H,12-17H2,1-5H3/t28-,29-/m0/s1

(1S,14S)-9,20,25-trimethoxy-15,30-dimethyl-7,23-dioxa-15,30-diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta-3(36),4,6(35),8,10,12(34),18,20,22(33),24,26,31-dodecaen-21-ol

Fangchinoline; Isofangchinoline; 33889-68-8; Demethyl tetrandrine; Limacine; 436-77-1; THALRUGOSINE; Thalrugosine;Thaligine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~82.14 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)