SF3B splicing
SF3b complex (spliceosome) [1]
The compound competes with pladienolide for binding to SF3b complexes containing either wild-type or mutant SF3B1. [1]

H3B-8800 potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components.

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H3B-8800 potently binds to and inhibits the splicing activity of both wild-type and mutant SF3B1-containing spliceosomes in competitive binding assays. [1]
It induces dose-dependent inhibition of both canonical and aberrant splicing in biochemical splicing assays using nuclear extracts. [1]
In a panel of pancreatic cancer cell lines, H3B-8800 treatment for 72 hours resulted in cellular lethality and caspase-3/7 cleavage specifically in the SF3B1-mutant cell line Panc05.04, but not in wild-type lines. [1]
It demonstrated preferential killing of K562 cells bearing the SF3B1-K700E mutation compared to isogenic wild-type counterparts. [1]
Treatment with H3B-8800 modulated both canonical and aberrant splicing in Panc05.04 cells, as shown by decreased mature MBD4 mRNA levels, accumulation of MBD4 pre-mRNA, and decreased mutant SF3B1-induced aberrant splicing of MAP3K7. [1]
It inhibited ATP-dependent 17S U2 snRNP complex formation in nuclear extracts, indicating it interferes with precatalytic spliceosome assembly by disrupting the interaction between the SF3b complex and the branchpoint region. [1]
Resistance mutations in SF3B1 (R1074H) or PHF5A (Y36C) conferred resistance to H3B-8800, confirming its selectivity for the SF3b complex. [1]
RNA-seq analysis showed that H3B-8800 treatment preferentially induced retention of short, GC-rich introns, particularly in genes encoding spliceosome components such as U2AF2 and RBM10. [1]
It induced a dose-dependent reduction in U2AF2 and SRPK1 protein expression in K562 cells. [1]

Oral administration of 2 or 4 mg H3B-8800 per kg body weight (mg/kg) daily slowed the growth of xenografts with SF3B1K700E (P < 0.003, two-way ANOVA followed by Dunnett’s multiple comparison test) but had no effect on SF3B1WT xenografts. Although a dose of 8 mg/kg did slow the growth of SF3B1WT tumors, it completely abrogated growth of SF3B1K700E tumors (P < 0.003).b). H3B-8800 similarly demonstrated significant antitumor activity against xenografts of human HNT-34 acute myeloid leukemia (AML) cells bearing the endogenous mutation encoding SF3B1-K700E. H3B-8800 reached similar concentrations in plasma as well as in tumors of mice harboring SF3B1WT or SF3B1K700E xenografts, and H3B-8800 modulated both canonical and aberrant splicing in a dose-dependent manner. Splicing modulation was observed as early as 1 h following treatment, and splicing returned to pretreatment levels within 24 h following treatment, in accordance with the H3B-8800’s pharmacokinetic profile.

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In NSG mice bearing xenografts of isogenic K562 cells, oral administration of H3B-8800 (2 or 4 mg/kg daily) significantly slowed tumor growth in SF3B1-K700E mutants but had no effect on wild-type tumors. A dose of 8 mg/kg completely abrogated growth of SF3B1-K700E tumors and also slowed growth of wild-type tumors. [1]
H3B-8800 demonstrated significant antitumor activity against xenografts of human HNT-34 AML cells bearing endogenous SF3B1-K700E mutation. [1]
In mice bearing AML patient-derived xenografts (PDXs) with SF3B1 mutation, 10-day treatment with H3B-8800 (8 mg/kg) significantly reduced leukemic burden, while having negligible effects in wild-type PDX recipients. [1]
Similar preferential activity was observed in CMML PDX models with SRSF2 mutations, where H3B-8800 treatment reduced human leukemic burden in peripheral blood and organs, and reduced spleen and liver size only in SRSF2-mutant models. [1]
Splicing modulation (reduction of mature mRNA and accumulation of pre-mRNA) was observed in tumors as early as 1 hour post-treatment and returned to baseline within 24 hours. [1]

SF3B complexes were immunoprecipitated from nuclear extracts prepared from 293F cells overexpressing Flag-tagged SF3B1. First, batch immobilization of antibody to beads was performed through incubating 80 μg of anti-SF3B1 antibody (MBL International D221–3, Anti-Sap155 monoclonal antibody) and 24 mg anti-mouse PVT scintillation proximity assay (SPA) beads (PerkinElmer) for 30 min. After centrifugation (14,000 r.p.m. for 5 min at 4 °C), the antibody–bead mixture was resuspended in PBS supplemented with PhosSTOP phosphatase inhibitor cocktail (Roche) and cOmplete ULTRA protease inhibitor cocktail (Roche). Nuclear extracts were prepared through diluting 40 mg into a total volume of 16 ml PBS with phosphatase and protease inhibitors, and the mixture was centrifuged (14,000 r.p.m. for 10 min at 4 °C). The supernatant was transferred into a clean tube, and the antibody–bead mixture was added and incubated for 2 h. The beads were collected via centrifuging, washed twice with PBS + 0.1% Triton X-100, and resuspended with 4.8 ml of PBS. 100 μl binding reactions were prepared using slurry and varying concentrations of H3B-8800. After 15 min preincubation at room temperature, 1 nM 3H-probe used in Kotake et al.20 was added. The mixture was incubated at room temperature for 15 min, and luminescence signals were read using a MicroBeta2 Plate Counter (PerkinElmer).

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Competitive binding assays were performed using SF3b complexes immunoprecipitated from nuclear extracts of cells overexpressing Flag-tagged SF3B1. Anti-SF3B1 antibody was immobilized on beads and incubated with nuclear extracts. Binding reactions were set up with bead slurry and varying concentrations of H3B-8800, followed by addition of a radiolabeled probe. Luminescence signals were measured to determine competition with pladienolide binding. [1]
An in vitro splicing reaction assay was performed using nuclear extracts and pre-mRNA substrates (Ad2-derived or Ad2-ZDHHC16 hybrid). Reactions containing nuclear extract, pre-mRNA, control RNA, and varying concentrations of H3B-8800 or DMSO were preincubated, then splicing activation buffer (containing ATP and MgCl2) was added. After incubation, reactions were quenched and analyzed by RT-qPCR to quantify canonical and aberrant splicing junctions. [1]
A U2 snRNP complex assembly assay was performed using nuclear extracts depleted of ATP. Extracts were preincubated with or without H3B-8800, then incubated with ATP, MgCl2, creatine phosphate, and a radiolabeled 2′-O-methyl oligonucleotide complementary to the U2 snRNA branchpoint binding region. Samples were purified and run on native agarose gels to resolve 17S and 12S U2 snRNP complexes. [1]

Pancreatic cancer cells were seeded at 750 cells per well in a 384 well plate and treated with compound on day 2. K562 isogenic cells (K562-SF3B1K700E and K562-SF3B1K700K) were seeded at 10,000 cells per well in 96-well plates and treated with compound 4 h later. The relative numbers of viable and apoptotic cells were measured via luminescence using CellTiter-Glo or Caspase-Glo 3/7 (Promega) at the indicated time points as instructed.

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Cell viability and apoptosis assays were performed using pancreatic cancer cells or K562 isogenic cells seeded in multi-well plates. Cells were treated with H3B-8800 or vehicle, and viability/apoptosis was measured at indicated time points using luminescence-based assays (CellTiter-Glo or Caspase-Glo 3/7). [1]
For splicing analysis, cells were treated with H3B-8800 for 6 hours, then RNA was extracted and reverse transcribed. Splicing events were quantified using TaqMan Low-Density Arrays (TLDA) or RT-qPCR with specific primer-probe sets for targets such as MBD4, MAP3K7, U2AF2, and aberrant junctions. [1]
Western blotting was performed on cell lysates from treated K562 isogenic cells. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against U2AF2, SRPK1, MCL1, and loading controls (β-actin or GAPDH). [1]
RNA-seq sample preparation involved treating NALM-6 or K562 isogenic cells with H3B-8800 for 6 hours, followed by RNA isolation, cDNA library preparation, and sequencing. Data were analyzed for differential splicing events, intron retention, and gene expression changes. [1]

K562 isogenic lines, HNT-34 and K052 xenograft models efficacy and pharmacokinetic/pharmacodynamic modeling.
1 × 107 K562 isogenic, HNT-34, or K052 cells were subcutaneously implanted into the flank of female NSG or CB17-SCID mice of 6–8 weeks of age. Mice were treated with H3B-8800 (10% ethanol, 5% Tween-80, 85% saline) or vehicle control. For the efficacy studies, the mice were orally dosed daily, and the mice were monitored until they reached either of the following endpoints: (i) excessive tumor volume (≥20 mm in its longest diameter), which was measured three times a week (tumor volume calculated by using the ellipsoid formula: (length × width2) / 2), or (ii) development of any health problem, such as paralysis or excessive body weight loss. The differences in tumor volume during the study period between the vehicle-treated and H3B-8800-treated groups were analyzed by two-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. For the pharmacokinetic/pharmacodynamic modeling (PK/PD) studies, the mice were treated with one dose, and the tumors were collected at the indicated times (in Figs. 2 and ​and3)3) after treatment for further analysis. RNA was isolated using RiboPure RNA purification kit (Ambion) and used for TLDA. All mouse studies were carried out under protocols approved by the Institutional Animal Care and Use Committee at H3 Biomedicine.

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For xenograft efficacy studies, female NSG or CB17-SCID mice (6-8 weeks old) were subcutaneously implanted with tumor cells (e.g., K562 isogenic, HNT-34, or K052 cells). Mice were orally dosed daily with H3B-8800 (formulated in 10% ethanol, 5% Tween-80, 85% saline) or vehicle control. Tumor volume was measured regularly, and mice were monitored until reaching endpoint criteria (excessive tumor size or health issues). [1]
For pharmacokinetic/pharmacodynamic (PK/PD) studies, mice received a single oral dose of H3B-8800, and tumors were collected at specified time points for RNA analysis (e.g., TLDA) to assess splicing modulation. [1]
Patient-derived xenograft (PDX) models were established by transplanting patient-derived hematopoietic cells into sublethally irradiated NSGS mice. Once engraftment was confirmed (human CD45+ cells >3% in peripheral blood or hemoglobin ≤11 g/dL), mice were randomized to receive vehicle or H3B-8800 (8 mg/kg orally daily for 10 days). Mice were euthanized 2 hours after the last dose for analysis of leukemic burden and organ infiltration. [1]

Following oral administration, the concentrations of H3B-8800 in the plasma and tumors of mice carrying wild-type or mutant SF3B1 xenografts were similar. [1] Splice regulation in the tumors was observed 1 hour after administration and returned to pre-treatment levels within 24 hours, consistent with its pharmacokinetic profile. [1]

[1]. H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat Med. 2018 May;24(4):497-504.

H3B-8800 is a novel spliceosome inhibitor developed by H3 Biomedicine. It has the advantage of preferentially killing spliceosome-mutant cancer cells, a feature not found in other spliceosome inhibitors, such as the praldinolactone analog E7107. H3B-8800 received Orphan Drug Designation from the U.S. Food and Drug Administration (FDA) in August 2017 and is currently undergoing clinical trials for the treatment of acute myeloid leukemia and chronic myelomonocytic leukemia. The spliceosome inhibitor H3B-8800 is an orally bioavailable inhibitor of splicing factor 3B subunit 1 (SF3B1) with potential antitumor activity. After administration, H3B-8800 binds to SF3B1 and blocks its activity. SF3B1 is a core spliceosome protein that is mutated in various cancer cells. This substance regulates RNA splicing by preventing aberrant splicing of mRNA by the spliceosome, blocking erroneous RNA splicing, enhancing correct RNA splicing, and inhibiting the expression of certain tumor-related genes. This leads to the induction of apoptosis and the inhibition of tumor cell proliferation. In many cancer cells, core spliceosome proteins, including SF3B1, U2 small nucleoribonucleoprotein cofactor 1 (U2AF1), serine/arginine-rich splicing factor 2 (SRSF2), and U2 small nucleoribonucleoprotein cofactor subunit-associated protein 2 (ZRSR2), are mutated and abnormally activated, leading to mRNA splicing dysregulation.

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Mechanism of Action
H3B-8800 is thought to bind to a praldinolactone-like site on the SF3B complex in the spliceosome. Once bound, H3B-8800 induces increased retention of short (<300 nucleotides) GC-rich introns by regulating the processing of precursor mRNA. These intron-preserved mRNA sequences were subsequently thought to be degraded via nonsense-mediated mRNA degradation pathways. Studies have shown that H3B-8800's regulatory effect is achieved by disrupting the SF3B complex's recognition of branch point sequences, as the branch point nucleotides in H3B-8800-preserved introns generally exhibit reduced adenosine preference and contain a greater number of sequences with weaker SFB3 binding. Research has found that among 404 genes encoding spliceosome proteins, 41 contain GC-rich sequences, the preservation of which is induced by H3B-8800. This is considered key to the lethal specificity of H3B-8800, as cells carrying spliceosome mutations depend on the expression of wild-type spliceosome components for survival. Because cancer cells (such as those in myelodysplastic syndromes) are more prone to SF3B1 mutations than host cells, H3B-8800 can be used to preferentially target these cells. Its mechanism of action involves inducing intron retention in precursor mRNA of key spliceosome components, leading to the degradation of nonsense mature RNA and ultimately cell death due to the lack of these key proteins.

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Pharmacodynamics
H3B-8800 preferentially targets cells containing the spliceosome complex of the mutant splicing factor 3B1 (SF3B1) protein, increasing cancer cell death by regulating intron splicing, while having little effect on the survival of cells containing wild-type SF3B1. In both mutant and wild-type cells, normal and abnormal mature mRNAs are suppressed. This selective lethal effect is thought to be due to the presence and effects of mutant SF3B1, rather than to alterations in the mechanism of action or potency of non-mutant proteins relative to wild-type proteins [A32749]. Because SF3B1 is frequently mutated in cancer, this allows the drug to preferentially target cancer cells rather than host cells.

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H3B-8800 is an oral small molecule splicing regulator that selectively targets the SF3b complex of the spliceosome. [1]
It preferentially kills cancer cells carrying mutations in spliceosome genes (e.g., SF3B1, SRSF2), which are common in myelodysplastic syndromes, leukemia, and certain solid tumors. [1]
Its mechanism of action involves preferential preservation of short, GC-rich introns, particularly in genes encoding spliceosome components, thereby exacerbating pre-existing splicing defects in mutant cells and leading to selective cell death. [1]
It exhibits unique sequence-specific splicing regulatory properties compared to other splicing regulators (e.g., E7107). [1]
This study supports the clinical potential of H3B-8800 in treating gene-specific cancers with RNA splicing factor mutations. [1]

C31H45N3O6

555.705508947372

555.33

C, 67.00; H, 8.16; N, 7.56; O, 17.27

1825302-42-8

1825302-42-8;

92135969

Yellow to orange solid

1.2±0.1 g/cm3

710.6±60.0 °C at 760 mmHg

383.6±32.9 °C

0.0±2.4 mmHg at 25°C

1.581

2.22

2

8

6

40

928

6

O(C(N1CCN(C)CC1)=O)[C@H]1C=C[C@H](C)[C@@H](/C(=C/C=C/[C@@H](C)C2C=CC=CN=2)/C)OC(C[C@@H](CC[C@@]1(C)O)O)=O |t:12|

YOIQWBAHJZGRFW-WVRLKXNASA-N

InChI=1S/C31H45N3O6/c1-22(26-11-6-7-16-32-26)9-8-10-23(2)29-24(3)12-13-27(39-30(37)34-19-17-33(5)18-20-34)31(4,38)15-14-25(35)21-28(36)40-29/h6-13,16,22,24-25,27,29,35,38H,14-15,17-21H2,1-5H3/b9-8+,13-12+,23-10+/t22-,24+,25-,27+,29-,31-/m1/s1

(2S,3S,6S,7R,10R,E)-7,10-Dihydroxy-3,7-dimethyl-12-oxo-2-((R,2E,4E)-6-(pyridin-2-yl)hepta-2,4-dien-2-yl)oxacyclododec-4-en-6-yl 4-methylpiperazine-1-carboxylate

H3B-8800; H3B8800; RVT-2001; UNII-90YLS47BRX; RVT2001; 1825302-42-8; 90YLS47BRX; 1-Piperazinecarboxylic acid, 4-methyl-, (2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-3,7-dimethyl-2-((1E,3E,5R)-1-methyl-5-(2-pyridinyl)-1,3-hexadien-1-yl)-12-oxooxacyclododec-4-en-6-yl ester; SCHEMBL17255784; EX-A8015; H3B 8800 [WHO-DD]; H3B 8800

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 (~180 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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