Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Abstract

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.

Antipsychotic Drug Fluphenazine against Human Cancer Cells

“…Furthermore, a recent study aimed to investigate if fluphenazine and its mustard derivative ( Figure 4 ) could synergistically target two cancer-associated targets: calmodulin and the tumor suppressor protein phosphatase 2A (PP2A). These proteins are modulators of the Ras- and MAPK signaling pathways, affecting cell viability and other mechanisms related to cancer cell stemness [ 29 ]. The authors used 3D spheroids of different cell lines (breast cancer cells MDA-MB-231 (K-Ras-G13D), bronchioalveolar cancer cells NCI-H358 (K-Ras-G12C), and melanoma cells A375 (B-RAF-V600E)).…”

“…The authors used 3D spheroids of different cell lines (breast cancer cells MDA-MB-231 (K-Ras-G13D), bronchioalveolar cancer cells NCI-H358 (K-Ras-G12C), and melanoma cells A375 (B-RAF-V600E)). They found that the combination of fluphenazine (a calmodulin inhibitor) and DT-061 (a PP2A activator) synergistically decreased the formation of spheroids and increased apoptosis in MDA-MB-231 cells, with the mustard derivative showing a more cytotoxic potential [ 29 ]. MDA-MB-231 is a triple-negative breast cancer cell line and is representative of one of the most aggressive types of breast cancer.…”

Markov state modelling reveals heterogeneous drug-inhibition mechanism of Calmodulin

“…Antipsychotic phenothiazines make up a class of drugs which are known CaM modulators [18][19][20][21]. By binding to CaM, the drugs effectively inhibit target-protein association.…”

“…Trifluoperazine (TFP, Fig 1C) is a potent member of this family of drugs whose interaction with CaM is well-studied [22][23][24][25]. Apart from antipsychotic purposes, TFP has also been investigated as treatment for various cancer forms [20]. Crystallographic structures of TFP bound to CaM revealed that the drug is capable of binding to CaM with varying stoichiometries at both inter-and intra-lobe binding sites (Fig 1D).…”

Markov State Modelling Reveals Heterogeneous Drug-Inhibition Mechanism of Calmodulin

“…1C) is a potent member of this family of drugs whose interaction with CaM is well-studied [22][23][24][25]. Apart from antipsychotic purposes, TFP has also been investigated as treatment for various cancer forms [20]. Crystallographic structures of TFP bound to CaM revealed that the drug is capable of binding to CaM with varying stoichiometries at both interand intra-lobe binding sites (Fig.…”