Sotuletinib (BLZ945) is an orally active, effective and specific CSF-1R inhibitor (IC50: 1 nM), >1000-fold selective against its closest receptor tyrosine kinase homologs.

CSF1R:1 nM

在骨髓衍生的巨噬细胞（BMDMs）中，Sotuletinib 特异性抑制 CSF-1 依赖的增殖，其 EC50 为 67 nM，并降低 CSF-1R 的磷酸化。Sotuletinib 阻断了巨噬细胞与胶质瘤细胞之间对彼此的生存、增殖和/或极化产生的相互作用，从而促进肿瘤发生。[1]

在携带胶质瘤的小鼠身上，Sotuletinib 通过抑制 CSF-1R 阻断肿瘤进展并显著提高存活率。Sotuletinib 还能够抑制体内患者衍生的倾向神经型肿瘤球和细胞系的原位肿瘤生长。[1] Sotuletinib (200 mg/kg, 经口) 有效减缓了小鼠乳腺肿瘤病毒驱动的多瘤病毒中间T抗原(MMTV-PyMT)乳腺癌发生模型和表达角蛋白14的人乳头状瘤病毒16型(K14-HPV-16)转基因宫颈癌模型中恶性细胞的生长。[2]

Inhibition of biochemical TrkA, TrkB and TrkC: TrkA and TrkC biochemical assays are carried out by HTRF method. The reaction mixtures contains 1 μM peptide substrate, 1 μM ATP, and either 1.8 nM TrkA or 34 nM TrkC in the reaction buffer (50 mM HEPES pH 7.1, 10 mM MgCl2, 2 mM MnCl2, 0.01% BSA, 2.5 mM DTT and 0.1 mM Na3VO4) at a final volume of 10 μL. All reactions are carried out at room temperature in white ProxiPlate? 384-well Plus plates and are quenched with 5 μL of 0.2 mM EDTA at 60 min. Five μL of the detection reagents (2.5 ng PT66K and 0.05 μg SAXL per well) are added, the plates are incubated at room temperature for 1 h and then read in EnVision reader. Compounds are diluted into assay mixture (final DMSO 0.5%), and IC50 values are determined by 12-point (from 50 to 0.000282 μΜ) inhibition curves in duplicate under the assay conditions. TrkB biochemical assay is carried out by caliper microfluidic method. The reaction mixtures contained 1 μM peptide substrate, 10 μM ATP, and 2 nM TrkB in a reaction buffer containing 100 mM HEPES, pH 7.5, 5 mM MgCl2, 0.01% Triton X-100, 0.1% BSA, 1 mM DTT, 10 μΜNa3VO4, and 10 μΜBeta-Glycerophosphate. The reactions are carried out at room temperature for 3 hrs, and the products are determined by Caliper EZ-reader. Compounds are diluted into assay mixture (final DMSO 1%), and IC50 values are determined by 12-point (from 50 to 0.000282 μΜ) inhibition curves in duplicate under the assay conditions.

Cell growth rate is determined using the MTT cell proliferation kit. Briefly, cells are plated in triplicate in 96-well plates: 1,000 cells per well for glioma cell lines, 5 x 1,000 cells per well for BMDM and CRL-2467, and 2.5 x 1,000 cells per well for HUVEC and HBMEC cell lines. For all experiments, media is changed every 48 h. Cells are grown in the presence or absence of 6.7–6,700 nM of BLZ945, or 8 μg/mL of CSF-1R neutralizing antibody. BMDM and CRL-2467 cells were supplemented with 10 ng/mL and 30 ng/ mL recombinant mouse CSF-1, respectively. Reduction of the MTT substrate is detected by colorimetric analysis using a plate reader as per the manufacturer's protocol, and measured at 595 nm and 750 nm on a spectraMax 340pc plate reader.(Only for Reference)