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.
Highlights
Calmodulin (CaM) is a small (17 kDa) dumbbell-shaped signaling adapter, with hundreds of protein interactions and widespread functions in cellular signaling (Tidow and Nissen, 2013)
We previously showed that it selectively inhibits the functional membrane organization of oncogenic K-Ras
We previously showed that the K-Ras directed effect of ophiobolin A (OphA) is abolished if a lysine mutant of CaM is expressed to rescue the knockdown of endogenous CaM (Najumudeen et al, 2016)
Summary
Calmodulin (CaM) is a small (17 kDa) dumbbell-shaped signaling adapter, with hundreds of protein interactions and widespread functions in cellular signaling (Tidow and Nissen, 2013). Ca2+/CaM classically recognizes with high, nanomolar affinity approximately 20-residue long peptides with bulky hydrophobic and basic residues that become encased in the hydrophobic pocket formed by the two lobes. This leads to a significant conformational change of CaM with loss of the central helical structure (Tidow and Nissen, 2013). A Covalent CaM-Inhibitor Tool Compound possess a polybasic N- or C-terminus with a single lipid modification, which can bind to either or both of the hydrophobic pockets on the N- and C-lobes (Grant et al, 2020b). CaM has been pursued as a cancer drug target in the 1980s due to its significant role in activating CDKs in the cell cycle (Hait and Lazo, 1986). CaM inhibitors have been demonstrated to block tumor growth, such as, for example, in multiple myeloma cell line xenografts (Yokokura et al, 2014)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
