Spliceosome
SF3b, a protein component of the spliceosome. [2]

FR901464 is a potent inhibitor of spliceosomes. It has shown remarkable anticancer activity against multiple human cancer cell lines. [2]
Researchers selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.[1]

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FR901464 exhibited potent cytotoxicity against multiple human cancer cell lines in vitro, with IC50 values ranging from 0.6 to 3.4 nM. [2]
In an in vitro splicing system using a synthetic pre-mRNA substrate, ATP, and nuclear extract from HeLa cells, FR901464 inhibited splicing with an IC50 of 0.05 μM. This was determined by quantifying the percent of pre-mRNA converted to mRNA using denaturing PAGE. [2]
In the same in vitro splicing system, FR901464 was shown to affect spliceosome assembly. Native gel analysis revealed that with increasing concentrations of FR901464, spliceosome assembly halted at a previously observed A-like complex, disrupting the normal progression from H/E → A → B → C complexes. [2]

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. [1]

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FR901464 exhibited prominent antitumor effects against human solid tumors implanted in mice, with a dosage range of 0.056 - 1 mg/kg. [2]

In Vitro Splicing Reactions[2]
Pre-mRNA substrate was derived from the adenovirus major late transcript. A 32P-UTP body-labeled G(5′)ppp(5′)G-capped substrate was generated by T7 runoff transcription followed by gel purification. Nuclear extract was prepared from HeLa cells grown in DMEM/F12 1:1 and 5% (v/v) newborn calf serum. For splicing reactions, 10 nM pre-mRNA substrate was incubated with 60 mM potassium glutamate, 2 mM magnesium acetate, 2 mM ATP, 5 mM creatine phosphate, 0.05 mg mL–1 tRNA, and 50% (v/v) HeLa nuclear extract at 30 °C.

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Denaturing Gel Analysis[2]
RNA was extracted from in vitro splicing reaction and separated on a 15% (v/v) denaturing polyacrylamide gel. 32P-labeled RNA species were visualized by phosphorimaging and quantified with ImageQuant software. Splicing efficiency is the amount of mRNA relative to total RNA and normalized to a DMSO control reaction. IC50 values for inhibitors are the concentration of inhibitor that causes 50% decrease of splicing efficiency, which were derived from averaged plots of splicing efficiency vs compound concentration.

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In Vitro Splicing Inhibition Assay: The biological properties of FR901464 were evaluated in an in vitro splicing system. Compounds were added to splicing reactions containing a synthetic pre-mRNA substrate, ATP, and nuclear extract isolated from HeLa cells. The splicing chemistry was examined by denaturing polyacrylamide gel electrophoresis (PAGE) to separate the substrate, intermediates (lariat intermediate, 5' exon intermediate), and the product mRNA. Splicing efficiency was quantified as the percentage of pre-mRNA converted to mRNA. The concentration required to reduce this splicing efficiency by half compared to the DMSO control was determined as the IC50. [2]
Spliceosome Assembly Assay: The effect of FR901464 on spliceosome assembly was also examined. Aliquots of the same in vitro splicing reactions were separated under native conditions and analyzed by gel electrophoresis. This allowed for the visualization of the ordered series of spliceosome intermediate complexes (H/E, A, B, and C). The impact of the compound on the normal progression of assembly was observed. [2]

Native Gel Analysis[2]
Splicing reactions were set up as described above and incubated at 30 °C for 4–30 min. Time point samples were kept on ice until all samples were ready for analysis. Amounts of 10 μL of splicing reactions were mixed with 10 μL of native gel loading buffer (20 mM Trizma base, 20 mM glycine, 25% (v/v) glycerol, 0.1% (w/v) cyan blue, 0.1% (w/v) bromophenol blue, 1 mg mL–1 of heparin sulfate) and incubated at room temperature for 5 min before loading onto a 2.1% (w/v) low-melting temperature agarose gel. Gels were run at 72 V for 3.5 h, dried onto Whatman paper, and exposed to phosphorimaging screens, which were digitized with a Typhoon Scanner.

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Cell Cycle Analysis by Flow Cytometry: M-8 cells were treated with FR901464 (10 ng/ml) or other drugs for various times. To measure DNA synthesis, cells were pulsed with BrdU for 30 minutes. Isolated nuclei were incubated with a fluorescein-conjugated anti-BrdU antibody and stained with propidium iodide. The cell cycle phase distribution was then analyzed using a FACScan flow cytometer. [1]
Cell Death Analysis (Apoptosis): M-8 cells were incubated with or without FR901464 (10 ng/ml) for various times. Cells were then lysed, and the enrichment of mono- and oligonucleosomes in the cytoplasm was quantified using a photometric enzyme immunoassay (Cell Death Detection ELISA). The results were measured as absorbance at 405 nm. [1]
Gene Expression Analysis by RT-PCR: M-8 cells were treated with or without FR901464 (10 ng/ml) for 16 hours. Total RNA was purified and subjected to RT-PCR amplification using specific primers for CAT, c-myc, E2F-1, p53, p21 cip-1, β-actin, and G3PDH. PCR products were resolved on a 2% agarose gel and stained with ethidium bromide for analysis. [1]
CAT Protein Quantification: M-8 cells were incubated with FR901464 (10 ng/ml) for various times. Cells were lysed by freeze-thaw cycles, and the CAT protein level in the lysates was determined using a colorimetric enzyme immunoassay (CAT ELISA). [1]

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. To address the involvement of transcriptional activation ability of the three FR compounds in the antitumor effect, we selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.[1]

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Drug Formulation:** For in vivo studies, FR901464 was dissolved and diluted in a 10% polyoxyethylated (60 mol) hydrogenated castor oil in saline (HCO60 solution). All drugs were administered to mice at a volume of 10 ml/kg body weight. [1]
* **Murine Ascitic P388 Leukemia Model:** P388 cells (1 × 10⁶) were inoculated intraperitoneally (ip) into BDF₁ mice on Day 0. FR901464 was administered ip once daily for 4 days (Days 1-4). Antitumor efficacy was assessed by the percentage of median survival time of the treated group (T) to that of the control group (C), calculated as T/C (%). [1]
* **Murine Solid Tumor Models (Colon 38 and Meth A):** For Colon 38, tumor fragments (2×2×2 mm) were implanted subcutaneously (sc) in BDF₁ mice on Day 0. FR901464 was administered intravenously (iv) once a day on Days 1, 4, 7, and 10. For Meth A, cells (1 × 10⁵) were inoculated intradermally (id) in BALB/c mice on Day 0. FR901464 was administered iv once a day on Days 8, 11, and 14. Tumor sizes were measured with calipers, and tumor weight was calculated. Efficacy was expressed as growth inhibition (1 - T/C) %. [1]
* **Human A549 Lung Adenocarcinoma Xenograft Model (Subrenal Capsule Assay):** A 1 mm³ tumor fragment was implanted under the kidney capsule of BDF₁ mice on Day 0. The immunosuppressive agent FK-506 (32 mg/kg) was injected subcutaneously on Days 1, 2, 5, 7, 9, and 12 to prevent xenograft rejection. FR901464 was administered ip on Days 1, 5, and 9 (every 4 days). On Day 14, mice were sacrificed, and the final tumor size was measured. Efficacy was calculated as growth inhibition (1 - T/C) %. [1]

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Drug Formulation: For in vivo studies, FR901464 was dissolved and diluted in a 10% polyoxyethylated (60 mol) hydrogenated castor oil in saline (HCO60 solution). All drugs were administered to mice at a volume of 10 ml/kg body weight. [1]
Murine Ascitic P388 Leukemia Model: P388 cells (1 × 10⁶) were inoculated intraperitoneally (ip) into BDF₁ mice on Day 0. FR901464 was administered ip once daily for 4 days (Days 1-4). Antitumor efficacy was assessed by the percentage of median survival time of the treated group (T) to that of the control group (C), calculated as T/C (%). [1]
Murine Solid Tumor Models (Colon 38 and Meth A): For Colon 38, tumor fragments (2×2×2 mm) were implanted subcutaneously (sc) in BDF₁ mice on Day 0. FR901464 was administered intravenously (iv) once a day on Days 1, 4, 7, and 10. For Meth A, cells (1 × 10⁵) were inoculated intradermally (id) in BALB/c mice on Day 0. FR901464 was administered iv once a day on Days 8, 11, and 14. Tumor sizes were measured with calipers, and tumor weight was calculated. Efficacy was expressed as growth inhibition (1 - T/C) %. [1]
Human A549 Lung Adenocarcinoma Xenograft Model (Subrenal Capsule Assay): A 1 mm³ tumor fragment was implanted under the kidney capsule of BDF₁ mice on Day 0. The immunosuppressive agent FK-506 (32 mg/kg) was injected subcutaneously on Days 1, 2, 5, 7, 9, and 12 to prevent xenograft rejection. FR901464 was administered ip on Days 1, 5, and 9 (every 4 days). On Day 14, mice were sacrificed, and the final tumor size was measured. Efficacy was calculated as growth inhibition (1 - T/C) %. [1]

In the murine P388 leukemia model, a dose of 3.2 mg/kg of FR901464 was toxic, as indicated by a T/C value of <86% (specifically, 22% T/C). [1]
In the human A549 xenograft SRC assay, a dose of 5.6 mg/kg of FR901464 was toxic, resulting in a survival rate of less than 65% on the evaluation day. [1]
In the murine Colon 38 solid tumor model, a dose of 1.0 mg/kg of FR901464 was toxic, resulting in a survival rate of less than 65% on the evaluation day. [1]

[1]. New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action. J Antibiot (Tokyo). 1996 Dec;49(12):1204-11.

[2]. Enantioselective total syntheses of FR901464 and spliceostatin A and evaluation of splicing activity of key derivatives. J Org Chem. 2014 Jun 20;79(12):5697-709.

FR901464 is a spirocyclic oxide with potent anticancer activity, capable of reducing the mRNA levels of oncogenes and tumor suppressor genes. It was isolated from Pseudomonas strain 2663. It can be used as an antibacterial agent, antitumor agent, and bacterial metabolite. It is an acetate, cyclic hemiketal, pyran compound, monocarboxylic acid amide, and spirocyclic oxide.
(2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpentan-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate has been reported in Burkholderia thailandensis, and relevant data are available.

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FR901464 (1) and splice repressin A (2) are potent inhibitors of the spliceosome. These compounds have shown significant anticancer activity against a variety of human cancer cell lines. This paper describes the efficient enantioselective synthesis of FR901464, splice repressor A, and six corresponding diastereomers, and evaluates their splicing activities. The synthesis of splice repressor A and FR901464 followed the longest linear sequences, involving 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A ring, we employed CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The Michael addition reaction exhibited significant diastereoselectivity, which we validated for different substrates and reaction conditions. Side chain B was prepared from an optically active alcohol, followed by acetylation and Lindlar-catalyzed hydrogenation. Another highly functionalized tetrahydropyran C ring was derived from readily available (R)-isopropylglyceraldehyde, and its synthetic route involved 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were later coupled together via amidation and cross-metathesis reactions in a convergent synthesis. Six key diastereomers were then synthesized to investigate the importance of specific stereochemical features of FR901464 and splicing repressor A to their in vitro splicing activity. [2]

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FR901464 is a potent anticancer compound originally isolated in 1996 from a fermentation broth of the bacterium Pseudomonas sp. No. 2663 by Fujisawa Pharmaceutical Co. [2]
FR901464 enhances the activity of a promoter of the SV40 DNA tumor virus at a concentration of 10 nM in M-8 cells. [2]
The primary mechanism of action for FR901464 involves inhibiting in vitro splicing and promoting pre-mRNA accumulation by binding to SF3b, a protein component of the spliceosome. This represents a novel mechanism for generating an antitumor response. [2]
Along with the structurally distinct pladienolide B, FR901464 is one of the only known molecular scaffolds capable of modulating splicing to produce an antitumor effect. [2]
Spliceostatin A, a methyl ketal derivative of FR901464 at the C1 position, shows better chemical stability and similar antitumor activity. In this study, the synthetic FR901464 was obtained by hydrolysis of the ketal moiety in synthetic spliceostatin A using PPTS in wet THF at 0°C. [2]
This paper describes an efficient, enantioselective total synthesis of FR901464 in 20 total steps with a longest linear sequence of 10 steps. The synthesized FR901464 was fully characterized and its spectral data were in full agreement with those reported for the natural product. [2]
Six diastereomers of FR901464 and spliceostatin A were also synthesized and tested. All diastereomers showed a significantly reduced potency (over 100-fold less potent than spliceostatin A) in inhibiting in vitro splicing, with IC50 values ranging from 1 to 35 μM. This highlights the critical importance of the correct stereochemistry for biological activity. [2]

C27H41NO8

507.624

507.283

C, 63.89; H, 8.14; N, 2.76; O, 25.21

146478-72-0

10553647

White to off-white solid powder

1.21g/cm3

702.7ºC at 760mmHg

378.8ºC

7.94E-23mmHg at 25°C

1.553

3.152

3

8

9

36

900

9

O1C([H])([H])C21C([H])([H])[C@@](C([H])([H])[H])(O[H])O[C@]([H])(/C(/[H])=C(\[H])/C(/C([H])([H])[H])=C(\[H])/C([H])([H])[C@@]1([H])[C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([C@@]([H])(C([H])([H])[H])O1)N([H])C(/C(/[H])=C(\[H])/[C@]([H])(C([H])([H])[H])OC(C([H])([H])[H])=O)=O)[C@@]2([H])O[H]

PJKVJJDQXZARCA-QHYZBLTGSA-N

InChI=1S/C27H41NO8/c1-16(8-11-23-25(31)27(15-33-27)14-26(6,32)36-23)7-10-22-17(2)13-21(19(4)35-22)28-24(30)12-9-18(3)34-20(5)29/h7-9,11-12,17-19,21-23,25,31-32H,10,13-15H2,1-6H3,(H,28,30)/b11-8+,12-9-,16-7+/t17-,18-,19+,21+,22-,23+,25+,26-,27+/m0/s1

[(Z,2S)-5-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]amino]-5-oxopent-3-en-2-yl] acetate

FR 901464; FR901464; FR901464; 146478-72-0; (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate; CHEBI:65915; [(Z,2S)-5-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-Dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]amino]-5-oxopent-3-en-2-yl] acetate; FR-901464; [(E,2S)-4-[[(2R,3R,5S,6S)-6-[(2E,4E)-5-[(3R,4R,5R,7S)-4,7-dihydroxy-7-methyl-1,6-dioxaspiro[2.5]octan-5-yl]-3-methylpenta-2,4-dienyl]-2,5-dimethyloxan-3-yl]carbamoyl]but-3-en-2-yl] acetate; CHEMBL494107; FR-901464

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~197.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.92 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 25.0 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: ≥ 2.5 mg/mL (4.92 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 25.0 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.)