He Chenchen, Duan Shaofeng, Dong Liang, Wang Yifen, Hu Qingting, Liu Chunjing, Forrest Marcus L, Holzbeierlein Jeffrey M, Han Suxia, Li Benyi
Department of Radiation Oncology, The First Affiliated Hospital, Xi'An Jiaotong University School of Medicine, Xi'An, China.
Department of Urology, The University of Kansas Medical Center, Kansas City, Kansas.
Prostate. 2017 Aug;77(11):1187-1198. doi: 10.1002/pros.23377. Epub 2017 Jun 20.
Our previous studies demonstrated that the class IA PI3K/p110β is critical in castration-resistant progression of prostate cancer (CRPC) and that targeting prostate cancer with nanomicelle-loaded p110β-specific inhibitor TGX221 blocked xenograft tumor growth in nude mice, confirming the feasibility of p110β-targeted therapy for CRPCs. To improve TGX221's aqueous solubility, in this study, we characterized four recently synthesized TGX221 analogs.
TGX221 analog efficacy were examined in multiple prostate cancer cell lines with the SRB cell growth assay, Western blot assay for AKT phosphorylation and cell cycle protein levels. Target engagement with PI3K isoforms was evaluated with cellular thermal shift assay. PI3K activity was determined with the Kinase-Glo Plus luminescent kinase assay. Cell cycle distribution was evaluated with flow cytometry after propidium iodide staining.
As expected, replacing either one of two major functional groups in TGX221 by more hydrophilic groups dramatically improved the aqueous solubility (about 40-fold) compared to TGX221. In the CETSA assay, all the analogs dramatically shifted the melting curve of p110β protein while none of them largely affected the melting curves of p110α, p110γ, or Akt proteins, indicating target-specific engagement of these analogs with p110β protein. However, functional evaluation showed that only one of the analogs BL140 ubiquitously inhibited AKT phosphorylation in all CRPC cell lines tested with diverse genetic abnormalities including AR, PTEN, and p53 status. BL140 was superior than GSK2636771 (IC 5.74 vs 20.49 nM), the only p110β-selective inhibitor currently in clinical trials, as revealed in an in vitro Kinase-Glo assay. Furthermore, BL140 exhibited a stronger inhibitory effect than GSK2636771 on multiple CRPC cell lines including a MDV3100-resistant C4-2B cell subline, indicating BL140 elimination of MDV3100 resistance. Mechanistic studies revealed that BL140 blocked G phase cell cycle entry by reducing cyclin D1 but increasing p27 protein levels.
These studies suggested that BL140 is a promising p110β-specific inhibitor with multiple superb properties than GSK2636771 worthy for further clinical development.
我们之前的研究表明，IA类磷脂酰肌醇-3激酶（PI3K）/p110β在去势抵抗性前列腺癌（CRPC）进展中起关键作用，并且用负载纳米胶束的p110β特异性抑制剂TGX221靶向治疗前列腺癌可阻断裸鼠体内异种移植瘤的生长，证实了针对CRPC进行p110β靶向治疗的可行性。为提高TGX221的水溶性，在本研究中，我们对四种最近合成的TGX221类似物进行了特性分析。
采用磺酰罗丹明B（SRB）细胞生长测定法、AKT磷酸化的蛋白质印迹测定法和细胞周期蛋白水平的蛋白质印迹测定法，在多种前列腺癌细胞系中检测TGX221类似物的疗效。通过细胞热位移测定法评估与PI3K亚型的靶点结合情况。采用Kinase-Glo Plus发光激酶测定法测定PI3K活性。碘化丙啶染色后，通过流式细胞术评估细胞周期分布。
正如预期的那样，与TGX221相比，用更具亲水性的基团取代TGX221中两个主要官能团之一，可显著提高其水溶性（约40倍）。在细胞热位移测定法（CETSA）中，所有类似物均显著改变了p110β蛋白的熔解曲线，而它们均未对p110α、p110γ或Akt蛋白的熔解曲线产生较大影响，表明这些类似物与p110β蛋白具有靶点特异性结合。然而，功能评估显示，在所有检测的具有包括雄激素受体（AR）、磷酸酶和张力蛋白同源物（PTEN）及p53状态等不同基因异常的CRPC细胞系中，只有一种类似物BL140能普遍抑制AKT磷酸化。在体外Kinase-Glo测定中显示，BL140优于目前正在进行临床试验的唯一p110β选择性抑制剂GSK2636771（IC50分别为5.74和20.49 nM）。此外，BL140对多种CRPC细胞系包括对MDV3100耐药的C4-2B细胞亚系的抑制作用比GSK2636771更强，表明BL140消除了MDV3100耐药性。机制研究表明，BL140通过降低细胞周期蛋白D1但增加p27蛋白水平来阻断G期细胞周期进入。
这些研究表明，BL140是一种有前景的p110β特异性抑制剂，具有比GSK2636771更多的优异特性，值得进一步进行临床开发。
