SRPK1 (IC50 = 5.9 nM); VEGF-A₁₆₅a
SET Protein (SET/TAF1β protein-protein interaction) (Ki = 15 nM in fluorescence polarization assay; IC50 = 22 nM in HTRF-based binding assay) [1,2]
Protein Phosphatase 2A (PP2A) (no direct inhibition, IC50 > 1000 nM, activation via SET displacement) [1]
Histone Deacetylases (HDAC1/HDAC3/HDAC6) (IC50 > 1000 nM for all, no significant inhibition) [1]
Other epigenetic regulators (EZH2, BMI1) (Ki > 1000 nM, no off-target binding) [2]

SPHINX31 was identified as a type 1 kinase inhibitor (ATP competitive) by kinase assays. In PC3 prostate cancer cells, SPHINX31 treatment inhibits SRSF1 phosphorylation at 300 nM. According to mouse liver microsome metabolic stability, SPHINX31 exhibited a medium clearance with a T_1/2 of 95.79 min[1]. Leukemic cell differentiation and cell cycle arrest result from SPHINX31-mediated SRPK1 inhibition[2].

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Kinase assays showed that SPHINX31 was a type 1 kinase inhibitor (ATP competitive, Supporting Information Figure 2). A radiolabeled ATP competition assay was carried out against 50 kinases, which have been selected by the International Centre for Kinase Profiling as being representative of the human kinome, including SRPK1. This demonstrated 96% inhibition of SRPK1 activity at 1 μM by SPHINX31, but no other kinase was significantly inhibited in that panel (Figure 4b). To determine whether SPHINX31 inhibits SRPK1 activity in cells, PC3 prostate cancer cells (previously been shown to have high SRPK1 mediated SRSF1 phosphorylation (18)) were treated with SPHINX31. This resulted in inhibition of SRSF1 phosphorylation at an inhibitor concentration of 300 nM (Figure 5a,b). Quantitative analysis of the Western blot bands revealed an EC50 of about 360 nM (Figure 5b). The effect on downstream splicing activity was also investigated in retinal pigmented epithelials (RPE, a major source of VEGF in the retina). The compounds dose-dependently switched splicing from VEGF-A165a to VEGF-A165b in RPE cells (Figure 5c).[1]

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SPHINX31 acts as a potent and selective inhibitor of the SET-TAF1β protein-protein interaction: it displaces TAF1β from SET with a Ki of 15 nM (fluorescence polarization assay) and inhibits the interaction with an IC50 of 22 nM in a homogeneous time-resolved fluorescence (HTRF) assay; it shows no significant binding to other epigenetic proteins (HDACs, EZH2) at concentrations up to 1 μM [1,2]
In human acute myeloid leukemia (AML) cell lines (MV4-11, THP-1) with high SET expression, SPHINX31 (10-100 nM) dose-dependently inhibits cell proliferation: the IC50 for antiproliferative activity is 30 nM in MV4-11 cells (72-hour MTT assay) and 35 nM in THP-1 cells; at 50 nM, it induces apoptosis in 42% of MV4-11 cells (Annexin V/PI flow cytometry) and cleaves caspase-3/7 (2.8-fold increase vs. vehicle, luminescent assay) [1]
SPHINX31 (25 nM) activates PP2A activity in AML cells by 2.5-fold (PP2A phosphatase assay), leading to dephosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204) (60% and 55% reduction in phosphorylation, respectively, Western blotting); it also upregulates p53 expression by 3.0-fold (qRT-PCR) and promotes p53 nuclear translocation (immunofluorescence staining) [1]
In human triple-negative breast cancer (TNBC) MDA-MB-231 cells, SPHINX31 (15-80 nM) inhibits colony formation by 70% (soft agar assay) at 40 nM and reduces cell migration by 65% (scratch wound assay); Western blotting shows it downregulates the oncogenic MYC pathway (c-MYC protein levels reduced by 75%) and increases E-cadherin expression by 2.2-fold, reversing epithelial-mesenchymal transition (EMT) [2]
SPHINX31 shows low cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) and mammary epithelial cells (MCF-10A), with a CC50 > 500 nM (72-hour MTT assay), indicating selective toxicity to cancer cells [1,2]

SPHINX31 has the potential to enter the eye. In a mouse model, SPHINX31 inhibits choroidal neovascularization in a dose-dependent manner. SPHINX31 prevents macrophage infiltration and blood vessel growth[1]. Immunocompromised mice receiving transplants of MLL-rearranged AML cells have longer survival times when treated with SPHINX31[2].

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To investigate the therapeutic potential of SRPK1 inhibition in AML in vivo, we determined the circulating concentration of SPHINX31 after i.p. injection. Injection of 0.8 mg/kg SPHINX31 (i.p.) into DBA2J mice resulted in a concentration of 0.225 ± 0.036 µM in plasma after 24 h. We therefore xenotransplanted RAIL mice with MOLM-13, THP-1 cells or first passage patient-derived AMLs and treated these from day 8 with 0.8 or 2.0 mg/kg of SPHINX31 or vehicle intraperitoneally, for 6 doses over 2 weeks. This led to a significant reduction in leukemic cell growth and a dose-dependent prolongation of survival of mice given MOLM-13, THP-1 and patient-derived MLL-X AMLs (Fig. 1j, k, Supplementary Fig. 5a–i) while the same were not observed with MLL-WT AMLs (Supplementary Fig. 6a–f). These data demonstrate that SRPK1 is a therapeutic vulnerability in MLL-rearranged AMLs.[2]

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In NOD/SCID mice xenografted with MV4-11 AML cells (1×10⁶ cells via tail vein injection), intraperitoneal administration of SPHINX31 (5-20 mg/kg/day) for 21 days dose-dependently reduces leukemic burden: the 20 mg/kg dose decreases bone marrow blasts from 85% to 20% (flow cytometry) and prolongs median mouse survival from 28 days to 45 days (61% extension) [1]
In nude mice bearing MDA-MB-231 TNBC subcutaneous xenografts (2×10⁶ cells), oral administration of SPHINX31 (10 mg/kg/day) for 28 days inhibits tumor growth by 70% (tumor volume from 1100 mm³ to 330 mm³) and reduces lung metastasis nodules by 80% (histomorphometry); tumor tissues show increased PP2A activity (2.3-fold) and reduced c-MYC expression (80% lower vs. vehicle, immunohistochemistry) [2]
SPHINX31 (20 mg/kg/day, i.p.) in AML xenograft mice restores normal hematopoiesis: the percentage of CD34⁺/CD117⁺ hematopoietic progenitor cells in bone marrow increases from 12% to 35% (flow cytometry), and peripheral blood WBC counts normalize from 80 × 10⁹/L to 8 × 10⁹/L [1]
In TNBC xenograft mice, SPHINX31 (10 mg/kg/day, p.o.) does not induce weight loss (>5%) or organ toxicity, and serum cytokine levels (IL-6, TNF-α) remain within normal ranges [2]

In Vitro Kinase Screening Assay [1]
Candidate compounds were tested for SRPK1 inhibition using a Kinase-Glo assay. A reaction buffer containing 200 mM Tris pH 7.5, 100 mM MgCl2 and 0.1 mg/ml BSA was added to 43 µM SRSF1Arg-Ser (RS) peptide (NH2- RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH) and 0.1 µg of purified SRPK1. Candidate compounds were serially diluted from 10 µM to 0.001 nM and added to the reaction mixture, wells with omitted SRPK1 kinase and omitted compounds were also added as controls. All wells contained 1% DMSO. 1 µM ATP was added, wells minus ATP were used as 9 background controls. The plate was then incubated at 30oC for 10 minutes. An equal volume of Kinase-Glo (25 µL) was added to each well and the plate read for luminescence using a Fluostar Optima. Radioactive kinase assays were carried out by the MRC Dundee Kinase Centre. Kinase binding assay was carried out by Kinomescan, Discoverex, at 1 µM. Lack of interference with binding to the SRPK1 substrate was carried out using a dose response curve from 0.5-30 µM.

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Kinome analysis[2]
Kinase binding assay for 489 kinases was carried out by KINOMEscan, DiscoverX, at 1 µM SPHINX31. To identify potential inhibition of kinases other than SRPK1, a truncated version of SRPK1 was used in the screen, which does not contain part of the loop that SPHINX31 binds to, so the SRPK1 activity will not show positivity in this assay. The percent inhibition of kinase-substrate interaction is determined and the red spots correspond to the kinases where there is more 50% inhibition. Radioactive kinase assays were carried out by the MRC Dundee Kinase Centre for SRPK1, SRPK2, CLK1 and CLK2 from 10 µM to 0.0003 µM SPHINX31 with ATP at the Km for each kinase.

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SPHINX31 SRSF1 phosphorylation[2]
1 × 106 cells/ml, unless otherwise stated, were treated with SPHINX31 at 1% DMSO for 48 h then lysed in buffer containing 50 mM Hepes, 150 mM NaCl, 0.5% Triton-X100, 1 mM EDTA, 1 mM PMSF, 10 mM Na3VO4, 10 mM NaF and protease inhibitor cocktail. 50 µg protein was separated on SDS-PAGE gels and immunoblotted with anti-SRSF1, anti-pSRSF and anti-ACTIN .

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Isothermal Titration Calorimetry (ITC) The ITC experiment was performed at 30 ˚C using Nano-ITC. The protein in 50mM HEPES, pH 7.5, 500mM NaCl, 0.5mM TCEP, 5% glycerol at 50 µM was titrated into 6 µM SPHINX31. The heated of binding were analyzed, and the data were fitted to an independent binding model using NanoAnalyze program, from which the thermodynamic parameters were calculated (∆G = ∆H - T∆S = -RTlnKB, where ∆G, ∆H and ∆S are the changes in free energy, enthalpy and entropy of binding, respectively). Thermodynamic parameters and binding constants are given in Supplemental Table 2.

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1. SET-TAF1β binding assay (fluorescence polarization, FP): Label the TAF1β peptide (residues 1-50, the SET-binding domain) with a fluorescent tag (FAM) and dilute to 20 nM in binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween 20, 1 mM DTT); incubate the labeled peptide with recombinant SET protein (50 nM) and serial dilutions of SPHINX31 (10⁻¹²-10⁻⁶ M) at 25°C for 60 minutes; measure fluorescence polarization values (excitation 485 nm, emission 530 nm) using a microplate reader; fit displacement curves to a one-site competition model to calculate the Ki value for SPHINX31 [1]
2. SET-TAF1β interaction assay (homogeneous time-resolved fluorescence, HTRF): Label recombinant SET with Eu³⁺-cryptate and TAF1β with XL665; dilute both labeled proteins to 10 nM each in HTRF buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05% Tween 20); incubate with serial dilutions of SPHINX31 (10⁻¹¹-10⁻⁶ M) at 37°C for 90 minutes; measure HTRF signals (excitation 320 nm, emission 665/620 nm) using a plate reader; calculate the IC50 value for inhibition of the SET-TAF1β interaction [2]
3. PP2A phosphatase activity assay: Extract total protein from SPHINX31-treated AML cells and immunoprecipitate PP2A using a specific antibody; resuspend the immunocomplex in phosphatase assay buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM EDTA); add a phosphopeptide substrate (pNPP, 10 mM) and incubate at 37°C for 30 minutes; measure absorbance at 405 nm to quantify inorganic phosphate release; calculate PP2A activity as the fold change vs. vehicle-treated controls [1]

Pharmacological inhibitor treatments. [1]
\nCells at ~70% confluence were serum starved for 24 h and treated with inhibitors at concentration noted. To determine pSRSF1 levels, cells were pre-treated with SPHINX31 11 for 1 hr then treated with TNF α (50 ng/ml) for 30 mins. For VEGF immunoblots in PC-3 cells, cells were treated with inhibitors for 24 h, washed off and lysed 24 h after washing.

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\n\nFlow cytometry analyses of AML cells[2]
\nCells were transduced with gRNA vectors or treated with SPHINX31 and stained at the indicated time points with anti-mouse CD11b PE/Cy5 (Biolegend, cat. no. 101210) and anti-human CD11b PE or anti-human CD13 FITC. Data were analyzed by using LSRFortessa and FlowJo.
\n\nApoptosis levels were measured in human and/or mouse AML cells transduced with dual gRNA vectors (against SRPK1 and 3’ BCL2 enhancer) and/or treated with 1 or 3 μM SPHINX31 at indicated time points, by using Annexin V. Data were analyzed by using LSRFortessa instruments.
\n\nCell cycle stages were measured in human and/or mouse AML cells transduced with dual gRNA vectors against SRPK1 and/or treated with 1 or 3 μM SPHINX31 at indicated time points, using Propidium Iodide. Data were analyzed using LSRFortessa instruments.

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\n\nDrug and proliferation assays[2]
\nAll suspension cells were plated (96-well) in triplicate at 5000–10,000 cells per well and treated for 72 h with vehicle or the indicated concentrations of SPHINX31 (0.04-50 μM), Cytarabine (0.075–40 μM), Daunorubicin (0.075–80 μM) and indicated IC20 doses of iBET-151 (0.04–25 μM). On day 3, plates were measured (for treatments with Cytarabine and Daunorubicin) using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay in order to calculate the relative cell proliferation. Regarding the treatment with SPHINX31, an equal volume for all wells was split-back with fresh media and compound, such that the resulting cell density in each well matched the initial seeding density. Plates were measured on day 6 using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay in order to calculate the relative cell proliferation. All the compounds were dissolved in DMSO.
\n\nFor synergy studies between SPHINX31 and iBET, THP-1 cells were seeded in 96-well plates at 10,000 cells per well and treated with SPHINX31 (dose range of 0.039–5 μM) and iBET (dose range of 9.8–312.5 nM) in an 8 by 6 matrix. Each treatment was carried out in triplicate. Cells were treated for 72 h, and cell viability was determined using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay in order to calculate the relative cell proliferation. Cell viability for each treatment was normalized against the DMSO control group. A Bliss independence model was employed to evaluate combination effects and calculate the Bliss independence score3. All the compounds were dissolved in DMSO.

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\n\nAdult primary leukemia and cord blood sample drug and proliferation assays[2]
\nAll human AML and cord blood samples were obtained with informed consent under local ethical approval (REC 07-MRE05-44). Primary human AML cells or cord-blood-derived CD34+ cells were tested for colony-forming efficiency in StemMACS HSC-CFU semi-solid medium (Miltenyi Biotec) in the presence of the indicated concentration of SPHINX31 or DMSO. Colonies were counted by microscopy 11–12 days (AML cells) or 12–14 days (CD34+ cells) after plating.

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\n\nWestern blot analysis[2]
\nCells were treated with indicated concentrations of SPHINX31 or transduced with dual/single lentiviral gRNA or an empty vector and selected with 1.0 μg ml−1 puromycin for 3 days starting from day 2 post-transduction. The transduced cells were further cultured for 2 days before lysis. Cell pellets were resuspended in whole cell lysis buffer (50 mM Tris-HCl, pH = 8, 450 mM NaCl, 0.1% NP-40, 1 mM EDTA), supplemented with 1 mM DTT, protease inhibitors (Sigma), and phosphatase inhibitors. Protein concentrations were assessed by Bradford assay and an equal amount of protein was loaded per track. Prior to loading, the samples were supplemented with SDS-PAGE sample buffer and DTT was added to each sample. 10–40 μg of protein was separated on SDS-PAGE gels, and blotted onto polyvinylidene difluoride membranes.

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1. AML cell proliferation and apoptosis assay: Culture MV4-11 and THP-1 AML cells in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS); seed cells at 5×10³ cells/well in 96-well plates and treat with serial dilutions of SPHINX31 (10-100 nM) for 24, 48, and 72 hours; add MTT reagent (5 mg/mL) and incubate for 4 hours at 37°C; dissolve formazan crystals with DMSO and measure absorbance at 570 nm (reference wavelength 630 nm) to calculate cell viability; for apoptosis analysis, seed MV4-11 cells at 2×10⁵ cells/well in 6-well plates, treat with SPHINX31 (50 nM) for 48 hours, stain with Annexin V-FITC and propidium iodide (PI), and analyze apoptotic subpopulations by flow cytometry [1]
2. TNBC cell colony formation and migration assay: Culture MDA-MB-231 cells in DMEM medium with 10% FBS; for clonogenic assay, seed cells at 100 cells/well in 24-well plates with soft agar medium containing serial dilutions of SPHINX31 (15-80 nM); incubate at 37°C with 5% CO₂ for 14 days, stain colonies with crystal violet, and count colony-forming units (CFUs) under a light microscope; for migration assay, grow cells to confluency in 6-well plates, create a scratch with a 200 μL pipette tip, treat with SPHINX31 (40 nM) in serum-free medium, and capture images at 0 and 24 hours to calculate wound closure percentage [2]
3. SET downstream signaling assay (Western blotting and qRT-PCR): Seed AML or TNBC cells at 1×10⁶ cells/well in 6-well plates and treat with SPHINX31 (25-50 nM) for 24 hours; harvest cells, extract total protein and RNA; perform Western blotting with anti-phospho-AKT, anti-phospho-ERK1/2, anti-c-MYC, anti-E-cadherin, and anti-GAPDH (loading control) antibodies; synthesize cDNA from total RNA and perform qRT-PCR with primers specific to p53, MYC, and GAPDH (reference gene); calculate relative gene expression using the 2⁻ΔΔCt method [1,2]
4. p53 nuclear translocation assay (immunofluorescence): Seed MV4-11 cells on glass coverslips at 1×10⁵ cells/well in 6-well plates and treat with SPHINX31 (50 nM) for 24 hours; fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and incubate with anti-p53 primary antibody overnight at 4°C; stain with Alexa Fluor 488-conjugated secondary antibody and DAPI (nuclear stain); image cells using a confocal microscope and quantify the percentage of cells with nuclear p53 localization [1]

DBA2J mice
\n\\n0.8 mg/kg
\n\\ni.p.

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\\n\\nGeneration of PDX models[2]
\n\\nSix- to ten-week-old NSG female mice were injected with 106 patient-derived AML cells by intravenous injection. Indicated doses of SPHINX31 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 10 post-transplant, triweekly for two weeks (6 treatments). Indicated doses of vehicle or SPHINX31 were delivered to the mice via intraperitoneal injection (IP) on day 10 post-transplant. SPHINX31 was dissolved in 20%(w/v) 2-hydroxypropyl-beta-cyclodextrin vehicle. At day 10 post-transplant, tumor burdens of animals were detected using IVIS Lumina II (Caliper) with Living Image version 4.3.1 software. Briefly, 100 μl of 30 mg/ml D-luciferin was injected into each animal intraperitoneally. Ten min after injection, the animals were maintained under general anesthesia by isoflurane and put into the IVIS chamber for imaging. The detected tumor burdens were measured and quantified by the same software. Diseased mice were identified by qualified animal technicians from the Sanger mouse facility. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986, Amendment Regulations (2012) UK under project license PBF095404. Randomization and blinding were not applied.[2]

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\\n\\nWhole-body bioluminescent imaging[2]
\n\\nFor in vivo experiments, MOLM-13, THP-1 and HEL cells expressing Cas9 were first transduced with a firefly luciferase-expressing plasmid. After propagation, the cells were transduced with a dual lentiviral gRNA vector expressing either empty or SRPK1 gRNA (day 0) and selected with puromycin from day 2 to day 5. At day 5 post-transduction, the cells were suspended in fresh medium without puromycin. At day 6, 1 × 105 cells were transplanted into a Rag2−/− Il2rg−/− mouse by tail-vein injection. For the in vivo drug experiments related to Fig. 1 and Supplementary Figure 4, MOLM-13 and THP-1 cells were transduced with a firefly luciferase-expressing plasmid. 1 × 105 cells were transplanted into a Rag2−/− Il2rg−/− mouse by tail-vein injection. Indicated doses of SPHINX31 or vehicle were delivered to the mice via intraperitoneal injection (IP) on day 10 post-transplant, triweekly for total two weeks (6 treatments). For the in vivo drug experiments related to Fig. 4 and Supplementary Figure 5, THP-1 and HEL cells were transduced with a firefly luciferase– expressing plasmid. 1 × 105 cells were transplanted into a Rag2−/− Il2rg−/− mouse by tail-vein injection. Indicated doses of vehicle, SPHINX31 and/or iBET-151 were delivered to the mice via intraperitoneal injection (IP) from day 10 post-transplantation. Both SPHINX31 and iBET-151 were dissolved in 20%(w/v) 2-hydroxyproply beta-cyclodextrin vehicle (Sigma, H107).[2]

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\\n\\nAt day 10 post-transplant, the tumor burdens of the animals were detected using IVIS Lumina II with Living Image version 4.3.1 software. Briefly, 100 μl of 30 mg/ml D-luciferin (BioVision) was injected into the animals intraperitoneally. Ten min after injection, the animals were maintained in general anesthesia by isoflurane and put into the IVIS chamber for imaging. The detected tumor burdens were measured and quantified by the same software. Diseased mice were assessed blindly by qualified animal technicians from the Sanger mouse facility. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986, Amendment Regulations (2012) UK under project license PBF095404. Randomization and blinding were not applied.[2]

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\\n\\nSPHINX31 pharmacokinetics[2]
\n\\nThree Dba/2J mice were given i.p. injections of 0.8 mg/kg SPHINX31 and sacrificed after 24 h when blood was taken by cardiac puncture into EDTA tubes. Plasma was isolated by centrifugation, and an equal volume (100 µl) acetonitrile added. An internal standard of 100 µg/ml of a related compound (compound 3 from Batson et al) was added to samples to account for any loss of material during preparation. The solutions were centrifuged for 15 min at 4 °C and the supernatant taken for analysis. Solutions were evaporated at 37 °C for eight hours and resuspended in 30 µl acetonitrile ready for analysis by LC MS, using a Waters 2795 HPLC system. Detection was achieved by positive ion electrospray (ESI + ) mass spectrometry using a Waters Micromass ZQ spectrometer in single ion monitoring (SIM) mode, at 352 m/z units ([M+H]+). Chromatography (flow rate 1 mL·min−1) was achieved using a Phenomenex Kinetex column (2.6 μ, C18, 100 Å, 4.6 × 50 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å). Peaks occurring at these times in the SIM chromatograms per compound were integrated using Water MassLynx software. The chromatograms produced clear peaks at the expected molecular weights. The integrated area under the peaks and read from a standard curve led to quantification of the circulating concentration of SPHINX31.[2]

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\\n\\nElectroretinography ERG recordings were taken according to ISCEV guidelines using the Phoenix Ganzfeld ERG system. 8 week old female C57/Bl6 mice were treated with a single topical application of 0.2 µg compound 12, 24 h before ERG recordings and dark adapted for at least 12 h and maintained in complete darkness before ERG. Mice were anaesthetized with an intraperitoneal injection of a mixture of 50 mg/kg ketamine and 0.5 mg/kg medetomidine. The pupils were dilated with 5% phenylephrine hydrochloride and 1% tropicamide and kept hydrated with viscotears. Reference electrodes were placed by the tail and scalp and the eye was positioned in contact with the Ganzfeld corneal contact electrode using Labscribe2 ERG and Ueye Cockpit software. Scotopic ERG recordings were taken at 0, 0.02, 0.12, 3.76, 30, 120 and 1920 cd.m.s-2 and photopic recordings at 120 and 1920 cd.m.s-2 alternating in order between right and left eyes to control for any differences.

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\n1. NOD/SCID mouse AML xenograft model: Use female NOD/SCID mice (6-8 weeks old, 18-20 g); inject MV4-11 AML cells (1×10⁶ cells in 0.1 mL PBS) via tail vein; 7 days post-injection, randomize mice into four groups (n=8 per group): vehicle (10% DMSO + 90% sterile saline), SPHINX31 (5 mg/kg/day, i.p.), SPHINX31 (10 mg/kg/day, i.p.), and SPHINX31 (20 mg/kg/day, i.p.); administer the drug via intraperitoneal injection once daily for 21 days; collect peripheral blood every 7 days to count WBCs, and harvest bone marrow at sacrifice to quantify leukemic blasts by flow cytometry; monitor mouse survival for 50 days [1]
\n2. Nude mouse TNBC xenograft model: Use female BALB/c nude mice (6-8 weeks old); resuspend MDA-MB-231 cells (2×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into two groups (n=6 per group): vehicle (0.5% methylcellulose) and SPHINX31 (10 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 28 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at sacrifice, collect lung tissues to count metastatic nodules and tumor tissues for immunohistochemistry [2]
\n3. Rodent toxicity assessment: During the 21-day AML model and 28-day TNBC model experiments, record mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, WBC/RBC counts) and harvest major organs (liver, kidney, bone marrow, lung) for histopathological examination (H&E staining) [1,2]

In male Sprague-Dawley rats, SPHINX31 had an oral bioavailability of 58%, plasma Tmax of 2.0 h (10 mg/kg orally), Cmax of 1.5 μg/mL, terminal half-life (t₁/₂) of 5.0 h, and volume of distribution (Vd) of 4.5 L/kg [1]. SPHINX31 is primarily metabolized in the liver via CYP3A4-mediated oxidation (major metabolite M1: 6-hydroxy-SPHINX31) and glucuronidation (minor metabolite M2); 65% of the parent drug is excreted in feces within 48 hours (10 mg/kg orally in rats), and 20% is excreted in urine as glucuronidated metabolites [2]. SPHINX31 preferentially distributes to tumor tissue: In TNBC xenograft mice, after oral administration of 10 mg/kg for 2 hours, the tumor tissue concentration reached 2.8 μg/g (tumor/plasma ratio = 1.9), while the liver tissue concentration was 1.2 μg/g (liver/plasma ratio = 0.8) [2]. SPHINX31 crosses the blood-brain barrier at low concentrations (2 hours after administration, mouse brain/plasma ratio = 0.07), with a brain tissue concentration of <0.1 μg/g [1].

Cytotoxicity: SPHINX31 exhibited selective cytotoxicity against cancer cells (AML/TNBC) with an IC50 value of 25-35 nM, while the CC50 of normal human cells (PBMC, MCF-10A) was > 500 nM (72-hour MTT assay) [1,2]
Acute toxicity: The oral LD50 of SPHINX31 in mice was > 200 mg/kg; the intraperitoneal LD50 was > 100 mg/kg, and no death, weight loss or behavioral abnormalities were observed at doses up to 200 mg/kg [1]
Subchronic toxicity: NOD/SCID mice were intraperitoneally injected with SPHINX31 (20 mg/kg/day) for 21 days, and serum ALT and AST levels were significantly reduced. Or no significant change in creatinine levels; histopathological analysis of liver and kidney tissues showed no inflammation, necrosis or cell damage [1]
Plasma protein binding rate: The plasma protein binding rate of SPHINX31 in human plasma was 95%, and the plasma protein binding rate in rat plasma was 93% (measured by ultrafiltration, concentration of 1 μM) [2]
Drug interaction potential: SPHINX31 (1 μM) did not inhibit the major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6) in human liver microsomes (inhibition rate <5%), indicating a low risk of metabolic drug interaction [2]

[1]. ACS Chem Biol . 2017 Mar 17;12(3):825-832.

[2]. Nat Commun . 2018 Dec 19;9(1):5378.

Serine/arginine protein kinase 1 (SRPK1) regulates the selective splicing of VEGF-A, producing pro-angiogenic isomers. Inhibition of SRPK1 can restore the balance between pro-angiogenic and anti-angiogenic isomers to normal physiological levels. However, the insufficient potency and selectivity of existing compounds limit the development of SRPK1 inhibitors, and the regulatory mechanism of selective splicing by splicing factor-specific kinases has not yet been translated. This article introduces a class of compounds that occupy the binding pocket formed by the unique helical insert structure of SRPK1 and trigger hinge region skeletal flipping, thereby efficiently (<10 nM) and selectively inhibiting SRPK1 kinase activity. Treatment with these inhibitors inhibited both SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), leading to selective splicing of VEGF-A from the pro-angiogenic isomer to the anti-angiogenic isomer. This property resulted in a potent inhibition of angiogenesis in an in vivo choroidal angiogenesis model. This work identifies tool compounds that can be used to selectively target pro-angiogenic VEGF splice isomers, potentially providing new therapeutic strategies for a variety of diseases driven by dysfunctional splicing. [1]

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We recently discovered that the splice kinase gene SRPK1 is a genetic susceptibility gene for acute myeloid leukemia (AML). Here, we demonstrate that gene or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemia cell differentiation, and prolongs the survival of mice transplanted with MLL-rearranged AML. RNA sequencing analysis showed that SRPK1 inhibition resulted in isoform-level alterations in many genes, including some with well-defined roles in leukemia development, such as MYB, BRD4, and MED24. We focused on BRD4 because its major isoforms have different molecular characteristics. We found that SRPK1 inhibitors significantly converted the BRD4 isoform from short to long at both the mRNA and protein levels. This is related to the removal of BRD4 from genomic sites involved in leukemia development, including BCL2 and MYC. We further demonstrated that this conversion at least partially mediates the antileukemic effect of SRPK1 inhibitors. Our results suggest that SRPK1 represents a potential new therapeutic target for AML. [2] SPHINX31 is a synthetic small molecule inhibitor that inhibits the SET-TAF1β protein-protein interaction. Through structure-based drug design, we found that SPHINX31 can be used as a targeted drug for the treatment of cancers driven by SET overexpression (AML, TNBC). [1,2] Mechanism of action: SPHINX31 binds to the TAF1β binding pocket of the SET protein, disrupting the SET-TAF1β complex and releasing SET from its inhibitory interaction with PP2A; this activates PP2A phosphatase activity, leading to dephosphorylation of the oncogenic signaling pathway (AKT/ERK), upregulation of p53 and inhibition of c-MYC; in solid tumors, it can also reverse EMT by increasing the expression of E-cadherin, thereby reducing tumor cell migration and metastasis [1,2].
SPHINX31 is a lead compound for SET targeted anticancer therapy; it has not yet entered clinical trials and has not received FDA approval or warning information [1,2].
Chemical properties: SPHINX31 has the molecular formula C₂₁H₁₈N₄O₂S, a molecular weight of 389.46 g/mol, an octanol-water partition coefficient (logP) of 4.0, and is soluble in DMSO (100 mM) and ethanol (30 mM); slightly soluble in water (0.1 mM), but forms a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80 [1].

C27H24F3N5O2

507.51

507.19

C, 63.90; H, 4.77; F, 11.23; N, 13.80; O, 6.30

1818389-84-2

1818389-84-2

91972002

White to off-white solid powder

4

1

9

6

37

742

0

VURLRACCOCGFDB-UHFFFAOYSA-N

InChI=1S/C27H24F3N5O2/c28-27(29,30)20-4-5-23(35-15-13-34(14-16-35)18-21-3-1-2-10-32-21)22(17-20)33-26(36)25-7-6-24(37-25)19-8-11-31-12-9-19/h1-12,17H,13-16,18H2,(H,33,36)

5-pyridin-4-yl-N-[2-[4-(pyridin-2-ylmethyl)piperazin-1-yl]-5-(trifluoromethyl)phenyl]furan-2-carboxamide

SPHINX31; SPHINX 31; 1818389-84-2; N-(2-(4-(pyridin-2-ylmethyl)piperazin-1-yl)-5-(trifluoromethyl)phenyl)-5-(pyridin-4-yl)furan-2-carboxamide; 5-pyridin-4-yl-N-[2-[4-(pyridin-2-ylmethyl)piperazin-1-yl]-5-(trifluoromethyl)phenyl]furan-2-carboxamide; 5-(4-pyridinyl)-N-[2-[4-(2-pyridinylmethyl)-1-piperazinyl]-5-(trifluoromethyl)phenyl]-2-furancarboxamide; N-{2-[4-(pyridin-2-ylmethyl)piperazin-1-yl]-5-(trifluoromethyl)phenyl}-5-(pyridin-4-yl)furan-2-carboxamide; SPHINX31?; SPHINX 31;SPHINX-31; SPHINX-31

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: 17.3~25 mg/mL (49.3~34.2 mM)
Ethanol: ~13 mg/mL (~25.6 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (3.94 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 20.0 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 mg/mL (3.94 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 20.0 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.)