Beside the previously reported Topo I inhibition

Beside the previously reported Topo I inhibition, time and concentration dependent CRM1 inhibitory properties of klavuzon derivatives have been shown as a second mechanism of action in this work. Especially, compounds 3 and 20 are the most effective derivatives and they completely inhibit CRM1 activity at 50 nM concentrations in 6h of incubation. Compared to traditional two-dimensional cell cultures, 3D cell cultures are considered as a much better way to represent in vivo environment. Hence, the most active derivative, abt 737 mg 20, has been tested in HuH-7 derived 3D spheroids and it has been found that compound 20 inhibited the growth of 3D spheroids and it caused significant cell death at 10 and 30μM concentrations in 48h. Therefore, compound 20 can be a good lead compound for further studies.
Author contributions
Acknowledgements This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, 113Z146). The authors would like to acknowledge networking support by the COST Action BM1204. We also would like to thank Mehmet S. Yıldız due to his help in MTT assays. We thank to Biotechnology and Bioengineering Research Central Research Laboratories at Izmir Institute of Technology for their help in bioactivity studies.
Introduction Even though high-dose multiagent chemotherapy and administration of targeted agents induce high remission rates in patients with previously untreated non-Hodgkin lymphoma (NHL), relapse and drug resistance within a few years is common that contributes to rather short overall survival [1], [2]. Therefore, discovering new therapeutic agents for NHL with low toxicity and that produce better outcomes than current therapies is clearly an ongoing challenge. Many types of proteins, including tumor suppressors, negative regulators of the cell cycle, and specific drug targets, must localize to the cell nucleus to function properly [3]. In eukaryotic cells, the chief mediator of protein export from the nucleus to the cytoplasm is chromosomal region maintenance 1 (CRM1), also called exportin 1. CRM1 is a member of the importin-β superfamily of nuclear export receptors (karyopherins), which can interact with leucine-rich nuclear export signals (NESs) [4], [5], [6], [7], [8]. Mechanistic studies have demonstrated the function of the CRM1 nuclear export pathway to many NES-containing signaling molecules, including p53 [9], [10], IκB-α [11], surviving [12], [13], [14], and others [3]. Given the critical roles of these exported molecules in the proliferation and survival of cancer cells, including NHL cells, CRM1 may be a therapeutic target for NHL. The well-known CRM1 inhibitor LMB binds covalently to Cys528 of CRM1 via a Michael-type addition reaction and abrogates the interaction between CRM1 and its cargo protein [15], [16], [17]. In addition, other CRM1 inhibitors with structures similar to [18], [19] or obviously different [20], [21], [22] from that of LMB also target Cys528 of CRM1. LMB has a high level of activity against a broad range of cancer cell lines in vitro[19]. However, researchers did not recommend further clinical evaluation of LMB after a phase 1 trial of it because of its toxic effects (e.g., profound anorexia and malaise) and lack of efficacy at tolerable doses [23]. Previous studies have reported on the use of new CRM1 inhibitors with much lower toxicity than that of LMB [19], [22], but to the best of our knowledge, none of them are presently in clinical trials. Inhibition of CRM1 was not the cause of LMB's toxicity in the phase 1 trial described above, which is promising in terms of the development of anticancer drugs targeting CRM1. Recently, Karyopharm Therapeutics (Natick, MA, USA) developed novel, orally bioavailable small-molecule selective inhibitors of nuclear export (SINEs), which are candidate CRM1 inhibitors that specifically and irreversibly bind to the reactive site Cys528 residue in CRM1 and block its function [24]. These candidate CRM1 inhibitors were active in in vitro and in vivo human CRM1 inhibition assays [24], [25], [26], [27], [28], [29], [30], [31] and thus have become candidate therapeutic agents for malignant tumor. In the study described here, our objective was to evaluate the therapeutic efficacy of the novel SINEs KPT-185 and KPT-276 against NHL in vitro and in vivo and elucidate the mechanism of CRM1 inhibitor-mediated antitumor activity.