Identification of Novel Kinase Inhibitors and Assessment of their Potency

Background – Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and therefore plays a crucial role in many disease pathologies, including age-related macular degeneration and cancer. VEGF exists in two classes of isoform, the proangiogenic VEGFxxxa isoforms and antiangiogenic VEGFxxxb isoforms, where xxx is the number of amino acids. These isoforms are generated through alternative splicing, which is mediated by SRSF1. When SRSF1 is phosphorylated by SRPK1, production of proangiogenic isoforms is promoted (1). Inhibition of SRPK1 leads to a reduction in phosphorylated SRSF1, a switch to antiangiogenic isoforms and a reduction in blood vessel growth.

We have developed a group of novel SRPK1 inhibitors, based on parent compound SPHINX (Sr PHosphorylation Inhibitor X) - (5-methyl-N-[2-(morpholin-4-yl)-5-(tri-fluoromethyl)phenyl] furan-2-carboxamide).

Purpose – To assess the potency of novel kinase inhibitors (SPHINXs), and their effects on downstream signalling, with the aim to induce alternative splicing of VEGF to anti-angiogenic isoforms.

Methods – Differential scanning fluorimetry (DSF) was used to initially evaluate affinity for SRPK1. 10μM compound was added to 0.5μM kinase in SYPRO orange containing assay buffer (500 mM NaCl, 10 mM Hepes) and run on a Light cycler 480 PCR machine - from 25 to 95 degrees at a rate of 3 degrees/min with continuous quantification to generate a melting curve. Compounds with a ΔTm of 2°C or greater were considered hits and passed to the next stage of screening.

Kinase assays were used to determine IC50, with compounds run in serial dilution from 10μM to 1pM to generate a concentration-response curve. Compounds were incubated with SRPK1 kinase and an SRPK1 substrate peptide before addition of ATP, followed by addition of a luciferase reagent to generate a luminescent signal.

Cytotoxicity was determined in human prostate cancer cells (PC3), using a cellular proliferation reagent to estimate the number of live cells with and without treatment.

Western blotting and ELISAs were used to assess production of different VEGF isoforms, also in PC3 cells.

Results – All of the compounds with ΔTm of 2°C in the DSF assay had IC50 values <50nM; two thirds of these <10nM. Cytotoxicity studies showed that EC50 of these compounds is variable (concentration required to have a 50% effect on proliferation and survival),ranging from1µM to30µM. Two compounds with high EC50 (>15µM) were taken into further testing. Western blot analysis showed that treatment with these two compounds produced a dose dependent increase in the ratio of VEGF165b to total VEGF (EC50 779.6nM). This was supported by ELISA data showing a 3.3-3.7 fold increase in the anti-angiogenic isoform relative to the pro-angiogenic isoform at 1µM.

Conclusions – Our screening cascade is able to highlight potent inhibitors of SRPK1, in a high throughput approach. Novel SPHINX compounds were able to potently inhibit SRPK1 activity, and produce downstream effects on splicing of VEGF isoforms in a dose dependent manner.

(1) Gammons MVR et al. (2013) Investigative Ophthalmology &Visual Science 54 6052-62