An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.


Contents of Supporting Information
This PDF File containing supplementary Figures and Table S1:      Data are representative of three independent experiments.Image acquisition was performed using an Andor benchtop confocal microscope (Oxford Instruments, Belfast, UK) with a × 40 objective and image deconvolution applied.Image analysis was done using ImageJ software (National Institute of Health, Bethesda, MD, USA).Scale bars, 20 µm.Right side, intensity profile plots and quantification of the EGFP-K-RasG12V signal.Individual cell traces in gray, the mean is plotted in black (n ≥ 30 cells per condition; plot drawn with the OriginPro software; A.U., arbitrary units).The plasma membrane (PM)/ cytoplasm ratio was determined as the ratio of the mean of the 2 peak values corresponding to plasma membrane localization and the mean of the 3 lowest dispersed plateau values corresponding to cytoplasmic localization.A decrease of this ratio corresponds to a loss EGFP-K-RasG12V plasma membrane localization.Note that the high errors associated with this analysis and the high noise in the source data (gray traces) would make it difficult to draw definite conclusions based on these data alone.(D) Heatmap of the ATARiS-sensitivity scores of selected genes for all cancer cell lines used in this study.Negative values indicate a decrease of proliferation upon gene knockdown and therefore a higher dependency of the cell line on that gene.(E) PDE6D/ K-RasG12V BRET reduction depends on whether PDE6D or K-RasG12V is affected by indicated inhibitors.FTI-277 + GGTI-298 and Sildenafil impact on K-RasG12V, which is present in excess, due to the biosensor transfection ratio, leading to a lesser response than Deltaflexin3 blocking PDE6D.This effectively corresponds to two dynamic ranges for the two targets mapped onto the absolute BRET ratio scale of the biosensor.Thus, the potentiating effect of the combination treatments as compared to Deltaflexin3 alone can be recognized; n ≥ 2.

Figure S2 :
Figure S2: Confocal microscopy-based analysis of K-RasG12V plasma membrane localization after treatment, supplementing main Figure 2.

Figure S4 :
Figure S4: Data supplementing main Figure 6.Table S1: Materials and Equipment used in this study.

Figure S1 .
Figure S1.Data supplementing main Figure 2. (A) Correlation plots of PDE6D inhibitor/ PDE6D-KD values acquired with F-Rheb vs. F-Ator as probes (Data S3).Data of compounds 5 and 14 were excluded, due to their much lower affinity.(B) BRET-titration curves of the PDE6D/ K-RasG12V, PDE6D/ K-RasG12V-C185S complexes and only the biosensor tag pair Rluc8/ GFP2 without fused protein-of-interest as controls.The C185S mutation prevents prenylation; n ≥ 3.For the latter two constructs line fits were done and the BRET-value at the highest [Acceptor]/ [Donor] ratio (mean ± SEM of last two datapoint repeats) was used as BRETtop.Statistical comparisons of BRETtop values to PDE6D/ K-RasG12V were done using two-tailed Student's t-test.(C) K-RasG12V-membrane anchorage evaluated by BRET following treatment with 5 μM Mevastatin or knockdown of PDE6D or FNTA; n ≥ 3 (left).A plot was derived from these data showing the loss of the BRET ratio following gene knockdowns as compared to Mevastatin treatment set to 100 % (right).Knockdown validation in (E).(D) H-RasG12V-membrane anchorage BRET is not affected by knockdown of PDE6D; n ≥ 3. (E) Representative immunoblot data showing PDE6D knockdown efficiency in HEK293 EBNA cells; n ≥ 3. Immunoblot data showing FNTA knockdown efficiency in HEK293 EBNA cells were previously reported by us 1 .(F) Assessment of K-RasG12V-membrane anchorage by BRET following FTI-277, GGTI-298, Mevastatin, Sildenafil, Deltaflexin3 or indicated combination treatments; n ≥ 2.

Figure S2 .
Figure S2.Confocal microscopy-based analysis of K-RasG12V plasma membrane localization after treatment, supplementing main Figure 2. Left side, confocal micrographs of live MDCK cells stably expressing EGFP-K-RasG12V after a 4 h treatment with the control DMSO 0.1 %, or indicated concentrations of Mevastatin, FTI-277 and GGTI-298, Sildenafil, Deltaflexin3 or a combination of Deltaflexin3 and Sildenafil.Data are representative of three independent experiments.Image acquisition was performed using an Andor benchtop confocal microscope (Oxford Instruments, Belfast, UK) with a × 40 objective and image deconvolution applied.Image analysis was done using ImageJ software (National Institute of Health, Bethesda, MD, USA).Scale bars, 20 µm.Right side, intensity profile plots and quantification of the EGFP-K-RasG12V signal.Individual cell traces in gray, the mean is plotted in black (n ≥ 30 cells per condition; plot drawn with the OriginPro software; A.U., arbitrary units).The plasma membrane (PM)/ cytoplasm ratio was determined as the ratio of the mean of the 2 peak values corresponding to plasma membrane localization and the mean of the 3 lowest dispersed plateau values corresponding to cytoplasmic localization.A decrease of this ratio corresponds to a loss EGFP-K-RasG12V plasma membrane localization.Note that the high errors associated with this analysis and the high noise in the source data (gray traces) would make it difficult to draw definite conclusions based on these data alone.

Figure S4 .
Figure S4.Data supplementing main Figure 6.(A) Results of xenograft experiments of MDA-MB-231 in mice.Tumor volume (left) and body weight (right) development over time during daily treatment with vehicle control or Deltaflexin3.Per treatment group, n = 10 animals were analyzed.