Novel analgesic compounds inducing alternative splicing of VEGF-A through SRPK1 inhibition

A. A. Tranholm1, Z. Blackley1,2, J. Batson3,2, H. D. Toop4,2, S. Oltean5, J. C. Morris4,2, D. O. Bates2,3, L. F. Donaldson1,2. 1School of Life Sciences, University of Nottingham, Nottingham, UNITED KINGDOM, 2Exonate Ltd, Cambridge, UNITED KINGDOM, 3TVBL, Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, UNITED KINGDOM, 4School of Chemistry, University of New South Wales, Sydney, AUSTRALIA, 5Bristol Renal, School of Clinical Sciences, University of Bristol, Bristol, UNITED KINGDOM.

Introduction

Alternative mRNA splicing can produce proteins with highly varying functions affecting a range of different physiological conditions including neuropathic pain (1). Novel serine-arginine protein kinase (SRPK1) inhibitors have been developed, preventing the SRPK1 mediated SRSF1 phosphorylation involved in downstream splicing of Vascular Endothelial Growth Factor-A (VEGF-A) (2). VEGF-A can be spliced at a proximal or a distal splice site at the end of exon 7 to produce the pro-nociceptive and -angiogenic splice-variants VEGFxxxa or the anti-nociceptive and -angiogenic variants VEGFxxxb respectively, (where xxx denotes the number of amino acids) (1, 2). We are developing SRPK1 inhibitors as novel analgesic compounds targeting VEGF-A splicing.

Methods

Cytotoxicity assays were performed on 50B11 cells, (immortalized rat embryonic dorsal root ganglion sensory neuronal cell line) (3). Forskolin-differentiated cells were treated with SRPK1 inhibitors (up to 10μM) and toxicity was evaluated using different assays measuring metabolic activity (WST-1), cell viability (Neutral Red) and lactate dehydrogenase release (LDH). EC50 was calculated for each compound. A stable cell line of transfected PC3 (prostate cancer) cells, pRG8ab, was created to express fluorescent outputs corresponding to VEGF-A splice-variants, and used to evaluate the efficacy of SRPK1 inhibitors. Cells were treated with SRPK1 inhibitors for 24 hours, fixed in 4% PFA and nuclei stained with DAPI. Fluorescence for DAPI, eGFP (VEGF-A165b) and dsRED (VEGF-A165a) was assessed using confocal microscopy and fluorescence plate readers. eGFP (VEGF-A165b) and dsRED (VEGF-A165a) were normalised to DAPI to correct for cell number variation. Potency (IC50) for induction of VEGF-A165b was compared to cytotoxicity EC50 to identify suitable compounds for future studies.

Results

Compounds showed variable levels of cytotoxicity, however even the most toxic compounds had IC50 at least 100 times lower than their EC50 for toxicity. SRPK1 inhibitor treatment of pRG8ab cells increased the eGFP:dsRED ratio (VEGF-A165b:VEGF-A165a) in a dose-dependent manner. Compound EXN109 at 2uM increased the eGFP:dsRED ratio (n=3) 2-fold over DMSO vehicle control when assessed in a high throughput screening format, which was confirmed with confocal microscopy and image analysis. EXN109 also increased expression of VEGF165b by more than 2-fold in pRG8ab cells when assessed using western blotting.

Conclusions

Our novel compounds induce alternative splicing in favour of VEGF-A165b at non-toxic concentrations in in vitro studies.

References

(1) R.P. Hulse et al. (2014). Neurobiology of Disease 71: 245-259.

(2) M.V.R. Gammons et al. (2013). Physiology and Pharmacology 54: 5797-5806

(3) W. Chen et al. (2007). J Peripher Nerv Syst. 12: 121-130