SPHINX31 is a potent inhibitor of serine/arginine-rich protein kinase 1 (SRPK1; IC50: 5.9 nM).

SRPK1:5.9 nM (cell free)

SPHINX31抑制了PC3细胞中丝/精氨酸富集剪接因子1（SRSF1）的磷酸化，该因子是SRPK1的底物（EC50：360nM），并在视网膜色素上皮（RPE）细胞中增加了抗血管生成VEGF-A165b剪接变体的表达[1]。对于MLL突变的AML细胞系，SPHINX31抑制生长，其IC50远低于其他AML细胞系，超过一个数量级。SPHINX31对正常小鼠造血干-祖细胞（HSPCs）的集落形成潜能没有影响。1.5、3和6μMSPHINX31并不影响正常人脐血CD34+细胞的集落形成能力[2]。

在体内，SPHINX31（每只眼2μg）抑制了小鼠脉络膜新生血管模型中的血管生长和巨噬细胞浸润[1]。向DBA2J小鼠注射0.8mg/kg SPHINX31（i.p.）后，24小时内血浆中的浓度达到0.225±0.036μM。在接受MOLM-13、THP-1细胞或第一次传导的患者源AMLs的异种移植RAIL小鼠中，SPHINX31（0.8或2.0 mg/kg）显著减少了白血病细胞的生长，并且随剂量增加显著延长了接受MOLM-13、THP-1和源于患者的MLL-X AMLs的小鼠的存活时间[2]。

Cells were transduced with gRNA vectors or treated with SPHINX31 and stained at the indicated time points with anti-mouse CD11b PE/Cy5 and anti-human CD11b PE or anti-human CD13 FITC. Data were analyzed by using LSRFortessa and FlowJo. Apoptosis levels were measured in human and/or mouse AML cells transduced with dual gRNA vectors (against SRPK1 and 3' BCL2 enhancer) and/or treated with 1 or 3?μM SPHINX31 at indicated time points, by using Annexin V. Data were analyzed by using LSRFortessa instruments. Cell cycle stages were measured in human and/or mouse AML cells transduced with dual gRNA vectors against SRPK1 and/or treated with 1 or 3?μM SPHINX31 at indicated time points, using Propidium Iodide. Data were analyzed using LSRFortessa instruments [2].

For in vivo experiments, 6–10-week-old female Rosa26Cas9/+ mice were treated triweekly for two weeks with either vehicle or 2?mg/kg SPHINX31. Four weeks post-treatment, bone marrow cells from these mice were freshly extracted (as mentioned above) and blocked with anti-mouse CD16/32 and 10% mouse serum. For the identification of LK/LSK, LT-HSC, myeloid and B-cell subpopulations, staining was performed using CD4 PE/Cy5, CD5 PE/Cy5, CD8a PE/Cy5, CD11b PE/Cy5, B220 PE/Cy5, TER-119 PE/Cy5, GR-1 PE/Cy5, SCA-1 Pacific Blue, CD150 PE/Cy7, CD34 FITC and CD117 APC-eFluor780. In each of the multi-colour flow cytometry experiments, we included the fluorescence minus one (FMO) controls. FMO controls provide a measure of spillover in a given channel. This allows for correct gating and selects only the stained cells in the experimental sample. Flow cytometry analysis was performed using a LSRFortessa instrument and resulting data were subsequently analyzed using FlowJo [2].