Fumie Tanzawa

Antitumor activity and novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-C-cyano-2-deoxy-?-d-ARABINO-Pentofuranosyl) cytosine) and itsN4-palmitoyl derivative (CS-682)

We have studied the antitumor activity and the novel DNA‐self‐strand‐breaking mechanism of CNDAC (1‐(2‐C‐cyano‐2‐deoxy‐β‐d‐arabino‐pentofuranosyl)cytosine) and its N4‐palmitoyl derivative (CS‐682). In vitro, CS‐682 showed strong cytotoxicity against human tumor cells comparable with that of CNDAC; both compounds displayed a similar broad spectrum. In vivo, however, orally administered CS‐682 showed a more potent activity against human tumor xenografts than CNDAC, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 2′,2′‐difluorodeoxycytidine. Moreover, CS‐682 was effective against various human organ tumor xenografts at a wide dose range and with low toxicity, and was effective against P388 leukemic cells resistant to mitomycin‐C, vincristine, 5‐fluorouracil or cisplatin in syngeneic mice. CNDAC, an active metabolite of CS‐682, had a prolonged plasma half‐life after repeated oral administrations of CS‐682 but not after oral administrations of CNDAC itself. This difference may partially explain the higher antitumor activity of CS‐682 relative to CNDAC. In both CNDAC‐ and CS‐682‐treated carcinoma cells, CNDAC 5′‐triphosphate (CNDACTP) was generated and incorporated into a DNA strand. High performance liquid chromatography (HPLC) and mass spectrometric analysis of the nucleosides prepared by digestion of the DNA from the CNDAC‐treated cells detected ddCNC (2′‐C‐cyano‐2′,3′‐didehydro‐2′,3′‐dideoxycytidine), which was shown to be generated only when the self‐strand‐breakage of CNDACTP‐incorporated DNA occurred. The cytotoxicity of CNDAC was completely abrogated by the addition of 2′‐deoxycytidine and was low against cells with decreased deoxycytidine kinase. Our results suggest that CNDAC is converted to CNDACMP by deoxycytidine kinase and that the resulting CNDACTP incorporated into a DNA strand as CNDACMP may induce DNA‐self‐strand‐breakage. This novel DNA‐self‐strand‐breaking mechanism may contribute to the potent antitumor activity of CS‐682. Int. J. Cancer 82:226–236, 1999.

Antitumor effects of novel highly hydrophilic and non-ATP-competitive MEK1/2 inhibitor, SMK-17

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target because of its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. Here, we report derivatives of N-[2-(2-chloro-4-iodo-phenylamino)-3,4-difluorophenyl]-methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in-silico docking study suggested that SMK-17 is bound to an allosteric pocket of MEK1. The kinetic study and the kinase profiler analysis confirmed the allosteric nature of SMK-17. SMK-17 inhibited MEK1 kinase activity in a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit the phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models on oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. These findings suggest that SMK-17, an exquisitely selective, orally available MEK1/2 inhibitor, is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling both in vitro and in vivo.

A novel inhibitor stabilizes the inactive conformation of MAPK-interacting kinase 1

Mitogen-activated protein kinase (MAPK)-interacting kinases 1 (Mnk1) and 2 (Mnk2) modulate translation initiation through the phosphorylation of eukaryotic translation initiation factor 4E, which promotes tumorigenesis. However, Mnk1 and Mnk2 are dispensable in normal cells, suggesting that the inhibition of Mnk1 and Mnk2 could be effective in cancer therapy. To provide a structural basis for Mnk1 inhibition, a novel Mnk1 inhibitor was discovered and the crystal structure of Mnk1 in complex with this inhibitor was determined. The crystal structure revealed that the inhibitor binds to the autoinhibited state of Mnk1, stabilizing the Mnk-specific DFD motif in the DFD-out conformation, thus preventing Mnk1 from switching to the active conformation and thereby inhibiting the kinase activity. These results provide a valuable platform for the structure-guided design of Mnk1 inhibitors.

Abstract B133: Antitumor effects of novel high-hydrophilic and non-ATP-competitive MEK1/2 inhibitor SMK-17.

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target due to its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. We synthesized derivatives of N-[2-(2-Chloro-4-iodo-phenylamino)-3,4-difluorophenyl] -methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in silico docking study suggested that SMK-17 be bound to an allosteric pocket of MEK1. The kinetics study and kinase profiler analysis confirmed the allosteric character of SMK-17. SMK-17 inhibited MEK1 kinase activity by a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models by oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. In conclusion, we found very potent MEK inhibitor SMK-17 from our structure-activity correlation study of new derivative series of diphenyl amine sulfonamide. Kinase profiler and kinetics study revealed that SMK-17 is a non-ATP-competitive and highly selective MEK1/2 inhibitor. Moreover, SMK-17 exhibited potent antitumor activity in animal models by oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways both in vitro and in vivo. These findings suggest that SMK-17 is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling. Furthermore, we are now investigating the combination effects of SMK-17 with several drugs, and studying about prediction marker of this compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B133.