A Covalent Calmodulin Inhibitor as a Tool to Study Cellular Mechanisms of K-Ras-Driven Stemness

Abstract

Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5–4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40–260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.

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

“…CaM can act as a trafficking chaperone of K-Ras by shielding the hydrophobic farnesyl tail from the aqueous environment of the cytoplasm, thus effectively increasing the diffusibility of K-Ras (22,40). In order to detect an effect of the CaM inhibitors on the K-Ras/ CaM interaction in cells, we established a BRET assay (22), where we genetically fused Rluc8 to the N-terminus of K-RasG12V and GFP2 to the N-terminus of CaM.…”

“…Both the covalently reacting CaM inhibitor OphA and the non-covalent, highly potent calmidazolium reduced the BRET signal when tested across a wider concentration range, in agreement with their CaM inhibiting activity ( Figure 2B ). In order to robustly distinguish the magnitude of these effects, we analyzed the dose response data using a normalized area under the curve measure, the DSS 3 BRET -score (22,41). These data revealed that addition of the electrophile in Flu-M significantly increased the intracellular activity against CaM/ K-RasG12V as compared to its non-covalent counterpart Flu by more than a factor of two ( Figure 2C ).…”

“…These data revealed that addition of the electrophile in Flu-M significantly increased the intracellular activity against CaM/ K-RasG12V as compared to its non-covalent counterpart Flu by more than a factor of two ( Figure 2C ). Yet the potency remained below that of OphA, which however has itself significant off-target activities that may be associated with its broad toxicity spectrum (22).…”

“…By genetically fusing BRET-donor enabling Rluc8 and the acceptor GFP2 to Ras proteins, we established BRET-assays, which can measure the loss of the functional membrane organization of Ras after inhibition of its trafficking chaperone CaM (22). Tight packing of BRET-luminophore labelled RasG12V in plasma membrane nanoclusters leads to high BRET-levels, similar to what we previously showed (42,43).…”

“…We previously showed that the natural product ophiobolin A (OphA), a covalent CaM inhibitor, can block stemness properties of cancer cells and K-Ras membrane organization (21). We recently developed less toxic functional analogues of OphA, which confirmed this potential (22). This lends an exciting new rational to the targeting of CaM, which justifies renewed interest in CaM inhibitor development (23).…”