SRPK1/serine arginine protein kinase 1 (Ki = 0.89 μM)

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]

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]

---

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]

---

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]

---

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]

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]

---

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.

In 96-well plates, leukemic cells (5 × 10⁴ cells/well) and isolated PBMCs (8 × 10⁴ 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].

---

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]

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

---

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]

---

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]

[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 the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.[1]

---

Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.[2]

---

Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model.[3]
Methods: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR.[3]
Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms.[3]
Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.[3]

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.

---

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

---

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