Scheme 1.
Structure of compounds employed in this study.
Shown are the calmodulin inhibitors calmidazolium (used as chloride salt and shown without counterion) and W7, the PP2A activator DT-061, and the phenothiazines trifluoperazine, fluphenazine and fluphenazine mustard. Note the relatedness between DT-061 and phenothiazines, which all feature the tricyclic moiety characteristic of phenothiazines.
Fig 1.
Cellular assessment of CaM inhibitors and PP2A activators suggests their synergistic potential against Ras pathway dependent cancer cell lines.
(A) Heatmap of ATARiS gene sensitivity scores of two KRAS dependent cell lines (NCI-H358 and MDA-MB-231) and a BRAF dependent cell line (A375). Negative values (shaded red) indicate sensitivity of the cell line proliferation to the knockdown of shown genes, whereas positive scores (shaded blue) indicate insensitivity. (B) IC50 values for indicated compounds in Ras pathway mutant cell lines NCI-H358, MDA-MB231 and A375 grown as 3D spheroids under serum free conditions. Compounds were tested either as single agent at a concentration range of 0.2 μM– 10 μM (calmidazolium), 0.6 μM– 40 μM (DT-061), 0.2 μM– 40 μM (trifluoperazine) and 0.6 μM– 40 μM (ARS-1620) or in combination, applying the whole concentration range of calmidazolium combined with 1 μM or 2 μM of DT-061, as indicated. Data represent mean values ± SD, n = 2. Statistical comparisons were done using the average values across all three cell lines. (C) Bliss synergism scores for combinatorial effects of calmidazolium and DT-061 in KRAS- or BRAF-mutant cancer cell lines. Data represent mean values ± SD, n = 2. Positive scores indicate synergistic drug interactions whereas negative scores would denote antagonistic drug interactions. A score of zero would indicate no antagonistic or synergistic effect. (D) Flow cytometric analysis of apoptosis in MDA-MB-231 cells. MDA-MB-231 cells were treated with DT-061 (10 μM) or calmidazolium (5 μM) or their combination for 24 h and then labelled with Annexin V-FITC and 7AAD. Benzethonium chloride and DMSO treated cells were used as positive and negative controls, respectively, for setting the gates. Percentage of total apoptotic cells (early and late stage) from two biological repeats (mean ± SD) is presented. Statistical analysis was performed using Fisher’s exact test comparing the total number of viable and apoptotic cells from two biological repeats. (E) Effect of inhibitors on cellular apoptosis studied using Caspase-3/7Glo assay. MDA-MB-231 cells were treated with DT-061 (5 μM), calmidazolium (5 μM), combination of DT-061 and calmidazolium (5 μM each), and staurosporine (1 μM) for 24 h. The caspase-3/7 activity was measured as a luminescence read-out and normalized to the vehicle control, 0.05% (v/v) DMSO. Data represent mean values ± SD, n = 2. (F) Western blot analysis of PARP-cleavage activity in MDA-MB-231 cells treated with DMSO (0.2% (v/v)), DT-061 (20 μM), calmidazolium (5 μM), combinations of DT-061 (10 μM) and calmidazolium (5 μM), and staurosporine (1 μM) for 24 h. GAPDH detection was used as loading control.
Fig 2.
Assessment of the in vitro inhibition of CaM and in cellulo disruption of K-Ras/ CaM binding.
(A) CaM inhibition was assessed using 100 nM CaM and displacement of 10 nM F-PMCA CaM-binding peptide by inhibitors using fluorescence polarization measurements. Data represent mean values ± SD, n = 2. (B) Dose-response analysis of indicated inhibitors at 0.1–80 μM using the Rluc8-K-RasG12V/ GFP2-CaM BRET assay in HEK-293 EBNA cells. The acceptor/ donor plasmid ratio was 9/1. Data represent mean values ± SD, n ≥ 2. (C) DSS3 BRET i.e. normalized area ’under’ the inhibition curve values (hence, here actually above the curve) of dose response data shown in (B). Statistical analysis was performed using unpaired t-test.
Table 1.
CaM-binding affinity of compounds from fluorescence anisotropy data in Fig 2A (mean ± SD, n = 2).
Fig 3.
BRET assay reveals high potency of fluphenazine mustard against Ras-membrane organization in cells.
(A) Testing of indicated compounds at 20 μM for 24 h exposure in KRasG12V (A) and H-RasG12V (B) nanoclustering-BRET assays. Controls are FTI-277 (1 μM), OphA (2.5 μM), calmidazolium (20 μM) and DT-061 (20 μM). The acceptor/donor plasmid ratio of GFP2- and Rluc8-tagged RasG12V was 4/1. Data represent mean values ± SD, n ≥ 2. (C) DSS3 BRET values derived from dose response data for calmidazolium, DT-061, Flu, Flu-M (0.1 μM– 80 μM) and OphA (0.3 μM– 20 μM) using K-RasG12V and H-RasG12V nanoclustering-BRET assays. The acceptor/donor plasmid ratio was 4/1. Data represent mean values ± SD, n ≥ 2.
Fig 4.
PP2A activity, MAPK signaling and anti-proliferative effects of Flu-M.
(A) Expression levels of c-MYC was measured in MDA-MB-231 after treatment with 10 μM or 20 μM of inhibitors for 24 h. Vinculin was used as loading control. A representative blot from 4 biological repeats is presented. (B) Expression of c-MYC relative to vinculin levels was analyzed. 0.1% (v/v) and 0.2% (v/v) of DMSO was used as control for 10 μM and 20 μM compound treatments, respectively. Data represent mean ± SD of 4 biological repeats. Statistical analysis was performed using unpaired t-test. (C) MAPK signaling output was measured in MIA PaCa-2 cells after 2 h treatment of trametinib (1 μM), AMG-510 (1 μM), calmidazolium (20 μM), DT-061 (20 μM), combination of DT-061 and calmidazolium (each 10 μM) and fluphenazine mustard (20 μM) in the absence of serum followed by EGF stimulation (50 ng/ml) for 10 min. 0.2% (v/v) DMSO was used as vehicle control. Representative blot from three independent biological repeats. (D) Densitometric quantification of the pERK/ERK ratio of all three biological repeats (mean values ± SD) normalized to control. Statistical analysis was performed using unpaired t-test. (E) DSS3 measuring the anti-proliferative effects of AMG-510 (0.003–40 μM), fluphenazine (0.6–80 μM) and fluphenazine mustard (0.6–80 μM). Results represent mean values ± SD, n = 3.