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.

Background Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound—ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. Methods Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017–2020) was analyzed. Results GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). Conclusions This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy.

Alzheimer’s disease (AD) is the most common cause of dementia, the chronic and progressive deterioration of memory and cognitive abilities. AD can be pathologically characterised by neuritic plaques and neurofibrillary tangles, formed by the aberrant aggregation of β-amyloid and tau proteins, respectively. We tested the hypothesis that VEGF isoforms VEGF-A165a and VEGF-A165b, produced by differential splice site selection in exon 8, could differentially protect neurons from neurotoxicities induced by β-amyloid and tau proteins, and that controlling expression of splicing factor kinase activity could have protective effects on AD-related neurotoxicity in vitro. Using oxidative stress, β-amyloid, and tau hyperphosphorylation models, we investigated the effect of VEGF-A splicing isoforms, previously established to be neurotrophic agents, as well as small molecule kinase inhibitors, which selectively inhibit SRPK1, the major regulator of VEGF splicing. While both VEGF-A165a and VEGF-A165b isoforms were protective against AD-related neurotoxicity, measured by increased metabolic activity and neurite outgrowth, VEGF-A165a was able to enhance neurite outgrowth but VEGF-A165b did not. In contrast, VEGF-A165b was more effective than VEGF-A165a in preventing neurite “dieback” in a tau hyperphosphorylation model. SRPK1 inhibition was found to significantly protect against neurite “dieback” through shifting AS of VEGFA towards the VEGF-A165b isoform. These results indicate that controlling the activities of the two different isoforms could have therapeutic potential in Alzheimer’s disease, but their effect may depend on the predominant mechanism of the neurotoxicity—tau or β-amyloid.

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical (•NO2). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO’s substrate hydrogen peroxide (H2O2) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on •NO2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Background Microtubule‐associated protein tau (MAPT) undergoes alternative splicing at exon 10 which encodes one of its four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau isoforms with all four repeats (4R) whilst exon 10 exclusion leads to isoforms with three repeats (3R). The 1:1 ratio between 3R and 4R isoforms is tightly regulated by splicing machinery and crucial to normal function. Kinases such as CLKs regulate the activity of splicing factors and their interaction with target transcripts, including MAPT. Disruption to this balance leads to the formation of neurofibrillary tangles (NFTs) and neurodegeneration but the mechanism of this is not yet wholly understood. Methods Differentiated Neuro2a and SH‐SY5Y cells were used as neuronal models as these cell lines show an imbalance of MAPT isoforms with bias towards 4R and 3R respectively. Splicing factor SRSF1 was transiently knocked down to confirm its role in MAPT splicing. Novel kinase inhibitors were used to reduce the activity of CLKs and determine the effect on MAPT splicing. The expression ratio of 4R and 3R isoforms was measured by RT‐PCR. Okadaic acid (OA) was used to produce a neurotoxic model and investigate the use of kinase inhibitors for neuronal protection. As a functional measure β3‐tubulin fluorescent marker was used to quantify OA dependent reduction in neurite outgrowth. Results 4R:3R tau expression significantly decreased by 65% in Neuro2a cells with knockdown of SRSF1 (students t‐test, p<0.001). CLK inhibition increased 4R:3R by 50% in SH‐SY5Y cells compared to control (one‐way ANOVA, p<0.05). SHSY5Y cells neurite outgrowth was significantly reduced by 60% and 85% with 1nM and 5nM OA treatment respectively, demonstrating a concentration dependent effect (one‐way ANOVA, p<0.001). At 1nM OA, neurite outgrowth was rescued by co‐treatment with a novel CLK inhibitor (two‐way ANOVA, p<0.0001). Conclusions Novel CLK inhibitors can modulate the expression of MAPT splicing isoforms and exert a functional effect on neurite outgrowth. This suggests these compounds could have a therapeutic effect by rectifying the balance of 4R:3R tau expression.