Precursor messenger ribonucleic acid (pre-mRNA) spliceosome

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age. [1]

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Ca cells exhibited preferential sensitivity to E7107 relative to non-tumorigenic prostate epithelial cells RWPE1, with PC3 being more sensitive than LNCaP cells (Fig. 8a and Supplementary Fig. 13d). Experiments with Pladienolide B confirmed PC3 as the most sensitive line (Fig. 8b). Although a long-term E7107 treatment (6~7 days) induced massive cell death (Fig. 8a and Supplementary Fig. 13d), shorter treatments (<3 days) generally elicited limited apoptosis but instead arrested PCa cells at the G2/M phase of the cell cycle (Fig. 8c). Treatment of PCa cells with E7107 for 20~48 h also inhibited cell migration and invasion, as measured by both Boyden chamber (Fig. 8d and Supplementary Fig. 13e) and scratch-wound (Supplementary Fig. 13f) assays. Importantly, treatment of PCa cells with 5 nM E7107 for 6 h dramatically reshaped the splicing pattern of the selected genes (Supplementary Fig. 13g), suggesting an on-target effect of the drug. [2]

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E7107 molecularly reverses PCa cell aggressiveness [2]
To uncover the mechanisms of action of E7107 in PCa, we treated LNCaP and PC3 cells with the drug for 6 h followed by RNA-seq analysis (Fig. 8e). No gross defects were observed in cell growth (Supplementary Fig. 14a) but, as expected, E7107 dramatically inhibited the AS globally in both cell types (Fig. 8e) with SE being the major type affected (Fig. 8f). Sashimi plot visualization of the sequencing data and RT-PCR validated splicing analysis (Fig. 8g and Supplementary Fig. 14b). GO analysis of the top 1000 genes with significant SE events inhibited by E7107 in PC3 cells revealed many GO terms associated with cancer-promoting functions, e.g., cell cycle and proliferation, DNA repair, splicing, and cancer pathways (Supplementary Fig. 14c and Supplementary Data 6), suggesting that E7107 inhibits splicing of a subset of PCa-promoting genes. At the gene expression level, E7107 reshaped the transcriptomes and exhibited a slight suppressive effect, especially in LNCaP cells, on transcription (Fig. 8e and Supplementary Data 7). qRT-PCR analysis validated DEGs identified in RNA-seq (Supplementary Fig. 14d).

The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. [2]

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Spliceosome inhibition therapeutically targets CRPC in vivo [2]
We treated three distinct castration-resistant (AI) PCa xenograft models, i.e., the AR+/hi LNCaP-AI, AR−/lo LAPC9-AI and AR− PC3, with E7107 or vehicle (Fig. 9a). The LNCaP-AI and LAPC9-AI models were established by serially passaging the corresponding parent AD tumor cells in castrated immunodeficient mice. The LNCaP-AI was initially responsive to Enza but quickly became Enza-resistant, whereas LAPC9-AI was refractory to Enza de novo. Treatment of LAPC9-AI tumors with either one cycle (i.e., tail vein injection for 5 consecutive days) or two cycles (with 1 week of drug holiday between the 2 cycles) effectively inhibited tumor growth (Fig. 9b, c and Supplementary Fig. 15a, b, left). Similarly, treatment of mice bearing AR+/hi LNCaP-AI with two cycles of E7107 (Fig. 9d and Supplementary Fig. 15c, left) and PC3 xenografts with one cycle of E7107 (Fig. 9e and Supplementary Fig. 15d, left) also inhibited tumor growth. Although a certain degree of toxicity of E7107 was observed, treated mice returned to normal body weight within a week after cessation of treatment (Supplementary Fig. 15a-d, right). The endpoint tumors frequently displayed a more differentiated morphology manifested by enlarged and polynucleated cells (Supplementary Fig. 15e).

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E7107 inhibits the progression in transgenic Hi-Myc PCa model [2]
As Myc overexpression and function represent a critical early oncogenic driver of PCa, many SRGs are co-amplified with the MYC gene and Myc-driven lymphomas are susceptible to spliceosome interference, we treated the Myc-driven murine PCa (Hi-Myc tumors) with E7107 (Fig. 10a) and observed significant inhibition by E7107 of Hi-Myc tumors (Fig. 10b). Histological examination of whole-mount images revealed large areas of Ads in vehicle-treated prostates that were frequently fused (Fig. 10c; top, solid circles). In contrast, most E7107-treated Hi-Myc prostates contained reduced tumor areas and prominent benign and hyperplasic glands (Fig. 10c, bottom, dashed circles and Fig. 10d, e). Immunohistochemistry (IHC) analysis revealed that the benign/hyperplastic glands in E7107-treated animals expressed AR and MYC to similar levels in the vehicle-treated tumors (Fig. 10d, e). Compared with the vehicle-treated Hi-Myc tumors, the benign/hyperplastic glands in E7107-treated prostates showed heterogeneous and generally reduced Ki-67+ cells (Fig. 10f).

The SF3b subcomplex was first titrated against a fixed concentration (25 nM) of fluorescent label (NTA-647) to determine the optimal concentrations of protein and label for the MST experiment. After 15 min of incubation at room temperature, samples were loaded into Monolith NT.115 capillaries, and MST analysis was performed using a Monolith NT.115Pico. The KD for NTA-647 was determined to be 8 nM. For subsequent compound-binding experiments, the SF3b subcomplex bound to NTA-647 was fixed at 10 nM, and the compound was titrated from 50 µM to 3 pM. After 15 min of incubation, samples were loaded into capillaries, and analysis was performed as described previously. A KD of 3.6 nM was determined for the interaction with E7107. The same analysis was applied to the PHF5AY36C mutant complex, and no binding was detected for E7107. [1]

Wound-healing, migration, and invasion assays [2]
For wound-healing assays, PCa cells in six-well culture dishes were allowed to grow to 80~90% confluence, and a sterilized tip was utilized to introduce a scratch “wound” with the same width on the bottom of the dishes. We generally made two scratch wounds per well as technical replicates, and wound closure under multiple microscopic views per well was recorded. Images were captured at 0 and 20–48 h after the wounding depending on cell types. Data shown were representative of three independent repeats. Moreover, cell migration and invasion assays were performed using Boyden chambers according to manufacturer’s instructions. Briefly, PCa cells were loaded into the chambers and cultured in media with or without varying concentrations of E7107 for 1–2 days, and results were visualized by PROTOCOL™ Hema 3 staining kit. Images of the membranes were captured by Olympus IX71. Data were quantified based on the cell number counting of at least five 10× images.

E7107 drug treatment [2]
For all in-vitro experiments, E7107 was dissolved in dimethyl sulfoxide. For in-vivo administration, E7107 was dissolved in vehicle (10% ethanol and 5% Tween-80 in sterile PBS) and administered via intravenous tail vein injection at 5 mg/kg/day (d). Xenograft-bearing mice were treated for 5 consecutive days (i.e., one cycle) or 10 days (7 days of ‘drug holiday’ between the 2 cycles). Previously, a dose of 4 mg/kg/d was used for treating blood diseases63. Given that PCa is a solid tumor, a dose of 5 mg/kg/d was recommended by the company (H3 Biomedicine) based on pilot studies. For drug-efficacy studies in xenograft models, randomization was done when tumor volume reached 150~200 mm3 using a calculation of 1/2 (length × width2). Animal body weight and tumor growth were measured twice weekly during the experiments. At the end of experiments, tumors were collected and tumor incidence, weight, and gross images were recorded. No blinding was done in the in vivo drug studies or in data analysis. For RNA-seq analysis in the CRPC xenograft models, tumor cells were first implanted in castrated male mice and then subjected to randomization when tumor size reached 150~200 mm3, followed by one cycle of E7107 treatment. Mice were killed for tumor collection exactly 4 h after receiving the last injection of five consecutive injections of E7107. For E7107 treatment of FVB Hi-Myc tumors, male mice at 42 weeks (when Ads fully developed) were randomized into two groups (n = 16 and 17, respectively). Animals were tail vein injected with one cycle of E7107 (at 5 mg/kg/d) or vehicle (as in the xenograft treatment) for 5 consecutive days followed by 1 week of drug holiday and another cycle of 5-day treatment at 44 weeks (Fig. 10a). All animals were terminated at week 46 and the genitourinary track and prostate were isolated and weighed. Whole-mount prostate was subjected to HE and IHC (MYC, AR, and Ki67) staining and Aperio Scanscope analysis. [2]

[1]. The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action. Genes Dev. 2018 Feb 1;32(3-4):309-320.
[2]. Intron retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer. Nat Commun. 2020 Apr 29;11:2089.

E7107 has been used in clinical trials for cancer treatment research. The pradinolone derivative E7107 is a synthetic carbamate derivative of pradinolone D with potential antitumor activity. E7107 is derived from the 12-membered macrolide pradinolone D, one of several macrolide compounds derived from Streptomyces Mer-11107. This compound appears to bind to the 130 kDa subunit 3 of splice factor 3b (SF3b) (splicing body-associated protein 130; SAP130), thereby inhibiting the splicing of precursor messenger RNA and arresting cell cycle progression. Splice factor SF3b is a multi-protein complex essential for the precise excision of introns from precursor messenger RNA; subunit SAP130 binds to U2 snRNP and is recruited to the splice precursor complex. The role of mRNA alternative splicing (AS) dysregulation in the development and progression of solid tumors remains to be elucidated. This paper provides the first comprehensive description of the AS atlas in the evolution of human prostate cancer (PCa). We found that the severity of splicing dysregulation is associated with disease progression and established intron retention as a marker of PCa stem cell characteristics and invasiveness. A systematic analysis of 274 splice regulatory genes (SRGs) revealed ubiquitous genomic copy number variations (CNVs), resulting in misexpression of approximately 68% of SRGs during PCa development and progression. Therefore, many SRGs have prognostic value. Surprisingly, the splicing program controlled by androgen receptors is quite different from its transcriptional regulatory mechanism. The spliceosome regulator E7107 reverses cancer invasiveness and inhibits castration-resistant prostate cancer (CRPC) in xenograft and autologous prostate cancer models. In conclusion, our study suggests that the abnormal AS atlas caused by SRG dysregulation is a marker of prostate cancer invasiveness, and that the spliceosome is a therapeutic target for CRPC. [2]

C40H66N2O9

718.97

718.477

C, 66.82; H, 9.25; N, 3.90; O, 20.03

630100-90-2

630100-90-2;

16202132

Typically exists as solid at room temperature

4.925

4

10

11

51

1220

10

CC[C@@H]([C@@H](C)[C@@H]1[C@H](O1)C[C@](C)(/C=C/C=C(\C)/[C@@H]2[C@H](/C=C/[C@@H]([C@](CC[C@H](CC(=O)O2)O)(C)O)OC(=O)N3CCN(CC3)C4CCCCCC4)C)O)O

MNOMBFWMICHMKG-MGYWSNOQSA-N

InChI=1S/C40H66N2O9/c1-7-32(44)29(4)37-33(49-37)26-39(5,47)19-12-13-27(2)36-28(3)16-17-34(40(6,48)20-18-31(43)25-35(45)51-36)50-38(46)42-23-21-41(22-24-42)30-14-10-8-9-11-15-30/h12-13,16-17,19,28-34,36-37,43-44,47-48H,7-11,14-15,18,20-26H2,1-6H3/b17-16+,19-12+,27-13+/t28-,29+,31+,32-,33+,34-,36+,37+,39-,40+/m0/s1

[(2S,3S,4E,6S,7R,10R)-7,10-Dihydroxy-2-[(2E,4E,6R)-6-hydroxy-7-[(2R,3R)-3-[(2R,3S)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate

E-7107; E7107; E7107; 630100-90-2; UNII-R60DZX1E2N; R60DZX1E2N; E 7107; [(2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-2-[(2E,4E,6R)-6-hydroxy-7-[(2R,3R)-3-[(2R,3S)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; 1-Piperazinecarboxylic acid, 4-cycloheptyl-, (2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-2-((1E,3E,5R)-5-hydroxy-6-((2R,3R)-3-((1R,2S)-2-hydroxy-1-methylbutyl)oxiranyl)-1,5-dimethyl-1,3-hexadienyl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl ester; (3R,6R,7S,8E,10S,11S,12E,14E,16R,18R,19R,20R,21S)-7-[(4-cycloheptylpiperazin-1-yl)carbonyl]oxy-3,6,16,21-tetrahydroxy-6,10,12,16,20-pentamethyl-18,19-epoyxtrichosa-8,12,14-trien-11-olide; E 7107

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)