Development of Potent, Selective SRPK1 Inhibitors as Potential Topical Therapeutics for Neovascular Eye Disease

Abstract

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform

“…By searching in-house profiling data on a panel of known kinase inhibitors, we encountered the FDA-approved ALK inhibitor Alectinib, which exhibited a significant capacity to target SRPK1 in a cell-based KiNativ profiling assay using HeLa cells (Figures 1A and 1B; Table S1; Patricelli et al, 2007). As expected, the compound potently inhibited the kinase activity of SRPK1 with an half maximal inhibitory concentration (IC 50 ) of 11 nM by an in vitro kinase activity assay (Z-Lyte) (Figure 1C), which is significantly more potent than original SRPIN340 (IC 50 = 367 nM; Fukuhara et al, 2006) and comparable with the recent published SRPK1 inhibitor SPHINX31 (IC 50 = 6.0 nM; Batson et al, 2017). These observations raised an intriguing possibility that the anti-tumor effect of Alectinib might have benefited from its activities against SRPK1 and other kinases besides blocking ALK, which is currently under investigation in our laboratories.…”

“…As shown earlier, the VEGF pre-mRNA is predominantly spliced to generate the VEGF-A165a isoform in most cell types, and inhibition of SRPK1 is able to switch VEGF splicing to produce the VEGF-A165b isoform (Amin et al, 2011; Batson et al, 2017). This switch is of high biological importance because VEGF-A165a is a major ligand to bind and induce VEGFR2 phosphorylation, thereby promoting angiogenesis, whereas VEGF-A165b is able to bind VEGFR2 but fails to induce its phosphorylation (Bates et al, 2002; Woolard et al, 2004), therefore functioning as an antagonist for VEGF-A165a to block angiogenesis (Manetti et al, 2011; Rennel et al, 2008).…”

“…Three chemical scaffolds have been described with inhibitory activities against SRPK1 (Batson et al, 2017; Morooka et al, 2015; Fukuhara et al, 2006; Siqueira et al, 2015; Székelyhidi et al, 2005; Gammons et al, 2013), and, among them, SPHINX31 was recently derived from SPHINX and reported to have much improved potency (Batson et al, 2017). Although high-throughput screening has been commonly used to generate lead compounds in kinase inhibitor projects, we chose to query existing kinase inhibitor libraries and known FDA-approved drugs for their potential abilities to target SRPK1, an approach that offers advantages in expediting structure-activity relationship (SAR) studies and optimizing pharmacological properties.…”

“…Consistently, multiple studies have linked elevated SRPK1 expression to VEGF-A165a expression, increased likelihood of metastasis, and poor prognosis in cancer patients (Mavrou et al, 2015; Li et al, 2014; Gammons et al, 2014). These findings thus suggest a promising avenue for cancer intervention, which has led to the development of small-molecule inhibitor for SRPKs (Batson et al, 2017). …”

Vascular endothelial growth factor-A165b ameliorates outer-retinal barrier and vascular dysfunction in the diabetic retina

“…Inhibitors of SRPK1 have been shown to be anti-angiogenic in animal models of choroidal neovascularization [28], prostate [60] and colorectal cancer [61] and malignant melanoma [62]. These inhibitors are being developed as a new class of anti-angiogenic agents for age-related macular degeneration [63], and if they can up-regulate VEGF-A 165 b in DR, then the results shown here indicate that they could be potential novel therapeutics, having the advantage over existing anti-angiogenic therapies of being neuroprotective for RGC cells and protective for RPE cells, at the same time as reducing extravasation and preventing new vessel growth.…”