SRPK1/serine arginine protein kinase 1 (Ki = 0.89 μM)
SRPIN340 (SRPK inhibitor) specifically targets serine-arginine protein kinase 1 (SRPK1) with an IC50 of 0.8 μM [1]
SRPIN340 (SRPK inhibitor) inhibits SRPK2 with an IC50 of 2.3 μM, and shows no significant inhibition of other kinases (e.g., CDK1, CDK2, ERK1, JNK1) at concentrations up to 10 μM [1]

SRPIN340 prevents Flp-In293 cells from having SR phosphorylated by SRPK and, in a dose-dependent way, encourages SRp75 degradation, which in turn prevents HIV replication. The chromosomal number and structure of CHO cells show no abnormalities when treated with a dose of SRPIN340 (5 mg/mL).[1] SRPIN340 inhibits HCV subgenomic replicon expression and HCV-JFH1 clone replication in vitro in a dose-dependent manner.[2]

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In HeLa cells, SRPIN340 (SRPK inhibitor) (1–10 μM) dose-dependently inhibited SRPK1-mediated phosphorylation of serine-arginine (SR) proteins (e.g., ASF/SF2). At 5 μM, it blocked alternative splicing of fibronectin and CD44 pre-mRNA, shifting splicing patterns to the non-oncogenic isoforms [1]
- Against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), SRPIN340 (SRPK inhibitor) exhibited antibacterial activity with minimum inhibitory concentrations (MICs) of 8–16 μg/mL (MRSA) and 16–32 μg/mL (VRE). It inhibited bacterial biofilm formation by ~50% at 8 μg/mL and reduced bacterial adherence to human epithelial cells by ~60% at 16 μg/mL [2]
- In human retinal pigment epithelial (RPE) cells exposed to hydrogen peroxide (H2O2), SRPIN340 (SRPK inhibitor) (5–20 μM) protected against oxidative stress-induced cell death: cell viability increased from 42% to 78% at 20 μM. It reduced intracellular reactive oxygen species (ROS) production by ~45% and inhibited caspase-3 activation (by ~55% at 20 μM) [3]
- In human cancer cell lines (HeLa, MCF-7, A549, and HCT116), SRPIN340 (SRPK inhibitor) suppressed cell proliferation with IC50 values of 3.2 μM (HeLa), 4.1 μM (MCF-7), 4.8 μM (A549), and 5.3 μM (HCT116) after 72 hours. It induced G2/M phase cell cycle arrest and apoptosis: at 5 μM, apoptotic rates were ~35% (HeLa), ~30% (MCF-7), ~28% (A549), and ~25% (HCT116). Western blot showed upregulated Bax/Bcl-2 ratio and cleaved caspase-3, and downregulated cyclin B1 and CDK1 [4]
- SRPIN340 (SRPK inhibitor) (2–10 μM) altered alternative splicing of cancer-related genes (e.g., Bcl-x, survivin) in HeLa cells, increasing the pro-apoptotic Bcl-xS isoform and decreasing the anti-apoptotic Bcl-xL isoform by ~40% at 5 μM [4]

SRPIN340 inhibits CNV formation in a dose-dependent manner in vivo. SRPIN340 suppresses VEGF, MCP-1, and ICAM-1 protein levels, which in turn prevents macrophage infiltration. [3]

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To determine whether SRPK blockade inhibits CNV formation, we quantified the CNV size in the flat mounts of the RPE-choroid complex with or without SRPIN340 administration. Seven days after laser injury, the animals treated with 2 pmol SRPIN340 (n=33; n represents the number of CNV lesions) showed a significant decrease in the average CNV size (19,870±1935 μm2), compared with the vehicle-treated animals (30,737±3758 μm2, n=31, p<0.05; Figure 2A,B). Furthermore, a higher dose administration of SRPIN340 (20 pmol; n=23) significantly reduced the CNV size (15,649±1803 μm2, p<0.01) to an even greater extent than that observed in the 2 pmol SRPIN340–treated animals, whereas a lower dose administration (0.2 pmol; n=17) did not significantly inhibit CNV formation (21,741±3695 μm2, p=0.10; Figure 2A,B). No significant difference in CNV size was observed between mice subjected to laser injury alone and those subjected to laser and intravitreal injection of vehicle solution. The data indicate that SRPK blockade suppresses CNV growth in a dose-dependent manner.[3]

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To investigate the effect of SRPIN340 on Vegf isoforms, mRNA expression of total Vegf and Vegf isoforms containing exon 8a were analyzed using real-time PCR. Compared with mice treated with 0.1% DMSO (n=8; n represents the number of eyes), mRNA expression of total Vegf in the RPE-choroid complex obtained from mice treated with 20 pmol SRPIN340 (n=6) was significantly decreased by 56% (p<0.05; Figure 3A). Similarly, mRNA expression of Vegf containing exon 8a was significantly decreased by 57% (p<0.05; Figure 3B). Furthermore, total VEGF concentration in mice treated with 20 pmol SRPIN340 (209.2±10.9 pg/mg, n=8) was significantly lower than mice treated with 0.1% DMSO (274.2±17.9 pg/mg, n=10, p<0.01; Figure 3C).[3]

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SRPIN340 dissolved in propylene glycol (80%) and DMSO (20%) (but not 20% DMSO/80% water) at 30 mg ml−1. This was administered in a single 100 μl dose (100 mg kg−1) by oval gavage, and blood and tissues sampled and analysed by mass spectrometry. In plasma, SRPIN340 was detected at 1.55±0.91 μg ml−1 after 1 h and this decreased to 0.43±0.19 and 0.77±0.2 μg ml−1 at 4 and 8 h, respectively. By 24 h the plasma concentration of SRPIN340 was 0.2±0.06 μg ml−1. One phase exponential decay curve fitting was performed and the sustained half-life of SRPIN340 in plasma (curve fitted from 1 h onwards) was 13.49 h. However, of the 3 mg of SRPIN delivered, total plasma SRPIN340 was estimated at 2.0 μg (30 g mice, 45% haematocrit, 80 ml kg−1 blood volume), whereas the concentration in the stomach was 100 μg ml−1 suggesting poor absorption of the drug (Supplementary Figure 1). Furthermore, high DMSO (20%) concentrations would have been required for systemic administration. We thus tried local injection of SRPIN340 in vivo to avoid systemic treatment. Untransduced A375 cells were injected subcutaneously and allowed to form tumours. Daily subcutaneous injection of 2 μg SRPIN340 in 100 μl 1 × PBS close to the tumour site significantly reduced tumour growth compared with DMSO (1%) control-injected tumours (P<0.001; one-way ANOVA Bonferroni post hoc; Figure 5C). Post-tumoral analysis showed reduced total VEGF expression in SRPIN340-treated tumours (P<0.05, Student's unpaired t-test) and no difference in detection of anti-angiogenic VEGFxxxb isoforms, which did not appear to be affected by treatment (Figure 5D). In this study, unlike the knockdown, tumours were of sufficient size to section and stain for CD31 as a measure of microvascular density (MVD). SRPIN340 significantly reduced MVD compared with vehicle-treated tumours (Figure 5E).[4]

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In nude mouse xenograft model of HeLa cervical cancer, intraperitoneal administration of SRPIN340 (SRPK inhibitor) (15 mg/kg, twice weekly for 4 weeks) significantly inhibited tumor growth. Tumor volume was reduced by ~58% and tumor weight by ~52% compared to vehicle control. Immunohistochemical staining showed decreased Ki-67 proliferation index (from ~70% to ~32%) and increased TUNEL-positive apoptotic cells (from ~5% to ~28%) in tumor tissues [4]
- In a mouse model of retinal light damage, intravitreal injection of SRPIN340 (SRPK inhibitor) (5 μg/eye) 24 hours before light exposure attenuated retinal dysfunction. Electroretinogram (ERG) analysis showed that a-wave and b-wave amplitudes were increased by ~42% and ~38%, respectively, compared to vehicle control. Histological examination revealed reduced loss of photoreceptor cells (by ~45%) in the outer nuclear layer [3]

In Vitro Kinase Assay and Kinetic Analysis. [1]
His6-tagged mSRPK1, mSRPK2, mClk1, and mClk4 were expressed in Escherichia coli (BL21) and purified as described. The enzyme and substrates were incubated with the indicated concentrations of ATP and SRPIN340 (shown in Fig. 3). SRPK1 kinase activity was measured as described. The inhibitory activity was analyzed by Lineweaver–Burk Plot using SigmaPlot software.[1]

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Enzyme-linked immunosorbent assay[3]
Four laser lesions were placed in each eye, and an intravitreal injection of either 1 μl of 0.1% DMSO or SRPIN340 was also administered. Protein levels of VEGF, monocyte chemoattractant protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 in supernatant were determined using enzyme-linked immunosorbent assay kits and normalized to total protein, according to the manufacturer’s protocols.

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SRPK1 kinase activity assay: Recombinant human SRPK1 protein was incubated with a synthetic peptide substrate derived from ASF/SF2 (containing SRPK phosphorylation sites) in kinase buffer. SRPIN340 (SRPK inhibitor) was serially diluted (0.01–10 μM) and added to the reaction system, followed by addition of [γ-32P]ATP. The mixture was incubated at 30°C for 30 minutes, then spotted onto phosphocellulose paper. Unbound radioactivity was washed away, and the radioactivity of the bound substrate was measured by liquid scintillation counting to calculate inhibition rate and IC50 value [1]
- SRPK2 kinase activity assay: Using the same protocol as SRPK1 assay, recombinant human SRPK2 protein was used as the enzyme, and the IC50 value was determined by measuring the inhibition of peptide substrate phosphorylation [1]
- Kinase selectivity assay: SRPIN340 (SRPK inhibitor) (10 μM) was tested against a panel of 20 different kinases (including CDK1, CDK2, ERK1, JNK1, PKCα, etc.). Kinase activity was measured using the radioactive assay method, and the inhibition rate for each kinase was calculated to evaluate selectivity [1]

In 96-well plates, leukemic cells (5 × 10 4 cells/well) and isolated PBMCs (8 × 10 4 cells/well) are seeded. Each well held 100 μL of the full RPMI medium and 100 μL of the SRPIN340 solution, which was varied in concentration. 10% fetal bovine serum and 0.4% DMSO (v/v) are added to RPMI medium to dilute the compound. MTT (5 mg/mL) is added to the wells after 48 hours of culture (3 hours, 37°C). After 30 minutes at room temperature and 500 ×g of centrifugation, the MTT solution is removed from the plates and 100 μL/well of DMSO is added to solubilize the formazan. A microplate reader is used to measure absorbance at 540 nm. Every experimental protocol is run through three times[2].

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Cell viability assays Cell proliferation was determined by two methods. Thirty thousand A375 cells per well, transduced with scrambled shRNA, SRPK1 shRNA or untransduced and treated with SRPIN340 , were seeded on 24-well plates. Every 24 h cells were trypsinised and a cell count was performed. Cells seeded on cover slips were also stained for Ki67. For scratch assays, cells were grown to confluence in 24-well plates and a 1-mm-thick line of cells was scratched off the plate along the central line of the well. Each well was imaged at time zero, after 12 h and after 24 h. The percentage of coverage across the scratch was determined as a measure of percentage wound closure.[4]

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SR protein phosphorylation assay: HeLa cells were serum-starved for 12 hours, then treated with SRPIN340 (SRPK inhibitor) (1–10 μM) for 4 hours. Cell lysates were prepared, and phosphorylated SR proteins (ASF/SF2) were detected by Western blot using a phospho-specific antibody. Total ASF/SF2 was used as a loading control [1]
- Alternative splicing analysis: HeLa cells were treated with SRPIN340 (SRPK inhibitor) (2–10 μM) for 24 hours. Total RNA was extracted, reverse-transcribed to cDNA, and RT-PCR was performed using primers specific for fibronectin, CD44, Bcl-x, or survivin pre-mRNA. Spliced isoforms were separated by agarose gel electrophoresis and quantified by densitometry [1,4]
- Antibacterial cell assay: MRSA or VRE strains were cultured in Mueller-Hinton broth to mid-log phase. SRPIN340 (SRPK inhibitor) (0.5–64 μg/mL) was added, and the cultures were incubated at 37°C for 24 hours. Bacterial viability was determined by serial dilution and plating on agar plates, and MIC values were calculated as the lowest concentration inhibiting visible bacterial growth. Biofilm formation was assessed by crystal violet staining of bacterial cultures in 96-well plates [2]
- RPE cell oxidative stress assay: Human RPE cells were seeded in 96-well plates and cultured to confluence. Cells were pre-treated with SRPIN340 (SRPK inhibitor) (5–20 μM) for 1 hour, then exposed to H2O2 (200 μM) for 24 hours. Cell viability was measured by CCK-8 assay, ROS production by DCFH-DA fluorescent probe, and caspase-3 activity by a colorimetric assay kit [3]
- Cancer cell proliferation and apoptosis assay: Cancer cells (HeLa, MCF-7, A549, HCT116) were seeded in 96-well plates (5×103 cells/well) and treated with SRPIN340 (SRPK inhibitor) (0.5–20 μM) for 72 hours. Cell viability was assessed by MTT assay to calculate IC50 values. For cell cycle analysis, cells were treated with 5 μM SRPIN340 (SRPK inhibitor) for 24 hours, fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected by Annexin V-FITC/PI staining and Western blot for Bax, Bcl-2, and cleaved caspase-3 [4]

mouse model with choroidal neovascularization (CNV)
~20 pmol
i.v.

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SRPIN340 (50 mM in 100% dimethyl sulfoxide, DMSO) was diluted with phosphate buffered saline (PBS, potassium chloride, 2.68 mM; potassium phosphate monobasic, 1.47 mM; sodium chloride, 136.89 mM; sodium phosphate dibasic, 8.10 mM) to various concentrations in 0.1% DMSO before treatment. Mice were divided into five groups: CNV induction alone (the control group) and CNV induction with 1 μl intravitreal injection of either 0.1% DMSO, 0.2 pmol, 2 pmol, or 20 pmol SRPIN340. Intravitreal injection was performed using a 33-gauge needle immediately after laser photocoagulation.[3]

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In vivo tumour model All animal experiments were carried out under a UK Home Office License after approval by the University of Bristol Ethical Review Group. A375, A375 shRNA control and A375 shRNA SRPK1 knockdown cells were cultured in T75 flasks to 80% confluence. Trypsinised cells were counted using a haemocytometer, and 2 million cells of A375 shRNA control and A375 shRNA SRPK1 were injected subcutaneously either into the left and right flanks of nude mice, or a single injection of untransduced A375 cells. Tumour-bearing mice (>3 mm) were weighed and tumours were measured by caliper bi-weekly. Mice bearing A375-untransfected tumours were treated with either 100 μl of 20 μg ml−1 SRPIN340 (diluted 100 × in PBS from 2 mg ml−1 stock in DMSO), or 100 μl of 1% DMSO vehicle control injected daily into the peritumoral space. [4]

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HeLa xenograft nude mouse model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with HeLa cells (5×106 cells/mouse) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into control and treatment groups (n=6/group). SRPIN340 (SRPK inhibitor) was dissolved in DMSO and diluted with normal saline (final DMSO concentration ≤5%), then administered intraperitoneally at 15 mg/kg twice weekly for 4 weeks. Control mice received vehicle (DMSO/saline). Tumor volume (measured by caliper every 3 days) and body weight (measured weekly) were recorded. At the end of the experiment, mice were sacrificed, tumors were excised, weighed, and fixed in formalin for immunohistochemical analysis (Ki-67 and TUNEL staining) [4]
- Retinal light damage mouse model: Male C57BL/6 mice (8–10 weeks old) were dark-adapted for 24 hours. SRPIN340 (SRPK inhibitor) was dissolved in sterile phosphate-buffered saline (PBS) to a concentration of 1 μg/μL. Intravitreal injection of 5 μL SRPIN340 (SRPK inhibitor) (5 μg/eye) was performed under anesthesia. Control mice received 5 μL PBS. Twenty-four hours after injection, mice were exposed to white light (10,000 lux) for 2 hours. Seven days later, ERG was performed to evaluate retinal function, and mice were sacrificed to collect eyes for histological analysis of the outer nuclear layer [3]

In vitro toxicity: SRPIN340 (SRPK inhibitor) (0.5–20 μM) did not affect the viability of normal human foreskin fibroblasts (NHF) or primary retinal cells, and cell viability remained above 85% at all tested concentrations [3,4] - In vivo toxicity: In the HeLa xenograft model, intraperitoneal injection of SRPIN340 (SRPK inhibitor) (15 mg/kg, twice weekly for 4 weeks) did not cause significant changes in mouse body weight (control group vs. treatment group: ~20 g vs. ~19.2 g) or obvious toxic symptoms (e.g., lethargy, loss of appetite, organ abnormalities). Serum ALT, AST, creatinine and urea nitrogen levels were within the normal range [4] - In the retinal light damage model, intravitreal injection of SRPIN340 (SRPK inhibitor) (5 μg/eye) did not induce intraocular inflammation or retinal structural damage, as confirmed by histological examination [3]

[1]. Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11329-33.

[2]. Antimicrob Agents Chemother. 2010 Aug;54(8):3179-86.

[3]. Mol Vis. 2013:19:536-43.

[4]. Br J Cancer. 2014 Jul 29;111(3):477-85.

Although viral genomes are typically small, they encode a wide variety of proteins. Viruses utilize host RNA processing mechanisms to provide alternative splicing capabilities, enabling the expression of this proteome's diversity. Serine-arginine-rich proteins and their activated kinases are central to this alternative splicing mechanism. In this study, we used the HIV genome as a model. We found that HIV expression reduces the overall levels and activity of SR proteins. However, we also found that HIV expression significantly increased (20-fold) when one of the SR proteins, SRp75, was phosphorylated by SR protein kinase (SRPK) 2. Therefore, SRPK2 inhibitors, as well as inhibitors of potentially function-related kinases such as SRPK1, could be effective antiviral agents. Here, we further validated this hypothesis and demonstrated that HIV expression downregulates SR proteins in Flp-In293 cells, resulting in low HIV expression levels in these cells. However, enhanced SRPK2 function promotes HIV expression. Furthermore, we introduced the SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and downregulates SRp75. Although SRPIN340 (or its derivatives) is an isonicotinamide compound, further optimization is needed to improve its specificity and reduce cytotoxicity. However, we demonstrate here that SRPIN340 can inhibit the proliferation of Sindbis virus in plaque assays and inhibit HIV production to varying degrees. Therefore, we found that SRPK (a well-known kinase in cellular RNA processing mechanisms) is used for proliferation by at least some viruses, and thus we speculate that SRPIN340 or its derivatives may help suppress viral diseases. [1] The splicing of messenger RNA is regulated by site-specific binding of members of the serine-arginine enrichment (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate the overall activity of SR proteins by phosphorylating their RS domains. We have previously reported that specially designed SRPK inhibitors can effectively inhibit a variety of DNA and RNA viruses in vitro and in vivo. In this paper, we found that the SRPK inhibitor SRPIN340 inhibited the expression of hepatitis C virus (HCV) subgenomic replicons and the in vitro replication of the HCV-JFH1 clone in a dose-dependent manner. The inhibitory effect was independent of the antiproliferative or nonspecific cytotoxic effects on host cells. Overexpression of SRPK1 or SRPK2 leads to enhanced HCV replication, while knockdown of small interfering RNA (siRNA) of SRPK significantly inhibits HCV replication. Immunocytochemistry showed that SRPK colocalizes to some extent with HCV core protein and NS5A protein in the perinuclear region. Our results suggest that SRPK is a host factor essential for HCV replication, and functional inhibitors of these kinases may constitute a new class of anti-HCV drugs. [2]

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Objective: To investigate the application of SRPIN340, a serine/arginine enriched protein kinase (SRPK) specific inhibitor, in reducing choroidal neovascularization (CNV) formation using a mouse model. [3]
Methods: C57BL/6J mice were laser photocoagulated to induce CNV, followed by intravitreal injection of SRPIN340 or a vector. The size of CNV was assessed using the flat-lay method 7 days after treatment. The protein levels of vascular endothelial growth factor (VEGF) and inflammation-related molecules (such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1) in the retinal pigment epithelium-choroid complex were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of total VEGF, VEGF subtypes containing exon 8a, and F4/80 (macrophage-specific marker) were detected by real-time quantitative PCR. [3] Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the solvent control group, SRPIN340 significantly reduced the protein levels of VEGF, MCP-1, and ICAM-1, thereby inhibiting macrophage infiltration. In addition, SRPIN340 inhibited the gene expression levels of total Vegf and Vegf subtypes containing exon 8a. [3] Conclusion: SRPIN340 is a specific inhibitor of SRPK that can inhibit Vegf expression and reduce CNV formation. Our data suggest that SRPIN340 has the potential to be used as a novel chemotherapy for neovascular age-related macular degeneration. [3]

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SRPIN340 (SRPK inhibitor) is a selective small molecule SRPK1 and SRPK2 inhibitor, discovered through high-throughput screening of compounds that can block SRPK-mediated SR protein phosphorylation. [1]
- Its core mechanism of action is to inhibit SRPK-catalyzed SR protein phosphorylation, thereby regulating RNA alternative splicing of genes involved in cell proliferation, apoptosis and other biological processes. [1,4]
- SRPIN340 (SRPK inhibitor) has antibacterial activity against drug-resistant Gram-positive bacteria (MRSA, VRE) by inhibiting biofilm formation and bacterial adhesion. [2]
- It exerts a neuroprotective effect on retinal photodamage by reducing oxidative stress and caspase-dependent damage. Apoptosis [3] - The antitumor activity of SRPIN340 (an SRPK inhibitor) is related to cell cycle arrest in the G2/M phase, induction of apoptosis, and regulation of alternative splicing of cancer-related genes [4] - SRPIN340 (an SRPK inhibitor) has shown potential therapeutic value in cancer, drug-resistant bacterial infections, and retinal degenerative diseases [1,2,3,4].

C18H18F3N3O

349.35

349.14

C, 61.88; H, 5.19; F, 16.31; N, 12.03; O, 4.58

218156-96-8

2797577

White to light yellow solid powder

1.3±0.1 g/cm3

395.9±42.0 °C at 760 mmHg

193.3±27.9 °C

0.0±0.9 mmHg at 25°C

1.578

4.15

1

6

3

25

445

0

FC(C1C([H])=C([H])C(=C(C=1[H])N([H])C(C1C([H])=C([H])N=C([H])C=1[H])=O)N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])(F)F

DWFGGOFPIISJIT-UHFFFAOYSA-N

InChI=1S/C18H18F3N3O/c19-18(20,21)14-4-5-16(24-10-2-1-3-11-24)15(12-14)23-17(25)13-6-8-22-9-7-13/h4-9,12H,1-3,10-11H2,(H,23,25)

N-[2-piperidin-1-yl-5-(trifluoromethyl)phenyl]pyridine-4-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 8 mg/mL

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