Cancer Cell Biology and Drug Discovery Group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
PLoS One. 2022 May 26;17(5):e0268635. doi: 10.1371/journal.pone.0268635. eCollection 2022.
Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.
吩噻嗪类药物（PTZ）被开发为单胺神经递质受体，特别是多巴胺受体的抑制剂。由于这种活性，它们已被使用了几十年作为抗精神病药物。此外，它们具有显著的抗癌特性，并且已经进行了多次重新利用的尝试。然而，它们不完全了解的多药理学具有挑战性。在这里，我们研究了吩噻嗪类药物氟奋乃静（Flu）及其芥末衍生物（Flu-M）协同作用于两个与癌症相关的靶点的潜力，即钙调蛋白（CaM）和肿瘤抑制蛋白磷酸酶 2A（PP2A）。这两种蛋白质都已知可以调节 Ras 和 MAPK 通路、细胞活力和癌细胞干性的特征。一致地，我们表明，钙调蛋白抑制剂和 PP2A 激活剂 DT-061 的组合协同抑制 MDA-MB-231（K-Ras-G13D）、NCI-H358（K-Ras-G12C）和 A375（B-raf-V600E）癌细胞的 3D 球体形成，并增加 MDA-MB-231 中的细胞凋亡。我们推断，这些活性仍然存在于 PTZ 中，PTZ 是 PP2A 激活剂开发的起点，而几种 PTZ 是已知的 CaM 抑制剂。我们表明，氟奋乃静和氟奋乃静-M 在体外和细胞中都保留了 CaM 抑制活性，而芥末衍生物在细胞中的活性更高。与 Ras 质膜组织的 CaM 依赖性一致，芥末衍生物有力地降低了致癌 Ras 的功能性膜组织，而 DT-061 几乎没有影响。与 DT-061 一样，两种 PTZ 都能强烈降低 c-MYC 水平，这是 PP2A 激活的标志。在 MIA PaCa-2 细胞中与 KRAS-G12C 特异性抑制剂 AMG-510 进行基准测试表明，氟奋乃静-M 的效力高于 DT-061 和 CaM 抑制剂的组合对 MAPK 输出的影响，以及对细胞增殖的强烈影响。虽然我们的研究是有限的，但我们的结果表明，保留 CaM 抑制和 PP2A 激活特性但失去其神经副作用的改良 PTZ 衍生物可能是进一步作为抗癌药物的有趣选择。
