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

Jennifer Batson,Claire Allen,Brian Gibbons,Jingxue Zhang,Stefan Knapp,David O Bates,Renate Griffith,Tom Hawtrey,Joana Da Rocha,Clara Redondo,Jules C Hancox,Apirat Chaikuad,Stephen F Wearmouth,Roya Babaei-Jadidi,Chunyun Du,Cynthia Tallant,Jonathan C Morris,Hamish D Toop

doi:10.1021/acschembio.6b01048

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.

Highlights

Vascular endothelial growth factor A (VEGF-A) is a key angiogenic factor in new blood vessel formation[1] and a critical driver of neovascular eye diseases[2] including diabetic retinopathy, exudative age related macular degeneration, and cancer progression.[3]
Serine/arginine (SR) proteins such as serine arginine-rich splicing factor 1 (SRSF1) regulate VEGF-A splicing by binding to the mRNA in a phosphorylation-dependent manner.[9−11] Specific splice factor kinases are responsible for the phosphorylation of the SRSF proteins and can be used to modulate the splicing events[10,11] For example, serine/arginineprotein kinase 1 (SRPK1) regulates splicing of pro-angiogenic
This inhibitor demonstrated that inhibition of SRPK allows for modulation of the VEGF-A

Summary

■ RESULTS AND DISCUSSION

We recently identified a 3-(trifluoromethyl)anilide scaffold (SPHINX) with moderate potency but high selectivity for SRPK1 (Figure 1a,c).[16]. The level of inhibition of lesion size (∼50%) is similar to that seen with inhibitors of VEGF (e.g., VEGF-TRAP, 30%),[28] recombinant VEGF-A165b protein (43%),[29] or intraocular injection of SRPK1 inhibitors[6,19,21] Extraction of protein from the retinae of the animals demonstrated VEGF-A165b expression in the retinae, which was greater in treated than in nontreated eyes measured both by Western blot (Supporting Information Figures 7A,B) and by isoform specific ELISA, where a switch in splicing was seen toward antiangiogenic VEGF (Supporting Information Figure 7C−E). These compounds have antiangiogenic properties in vivo These compounds 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 (

■ METHODS

■ ACKNOWLEDGMENTS

■ REFERENCES