Nuclear export factor chromosome region maintenance 1 (CRM1) is an attractive anticancer and antiviral drug target that spurred several research efforts to develop its inhibitor. Noncovalent CRM1 inhibitors are desirable, but none is reported to date. Here, we present the crystal structure of yeast CRM1 in complex with S109, a substructure of CBS9106 (under clinical test). Superimposition with the LFS-829 (another covalent CRM1 inhibitor) complex inspired the design of a noncovalent CRM1 inhibitor. Among nine synthesized compounds, noncovalent CRM1 inhibitor 1 (NCI-1) showed a high affinity to human and yeast CRM1 in the absence or presence of GST-bound Rasrelated nuclear protein (RanGTP). Unlike covalent inhibitors, the crystal structure showed that NCI-1 is bound in the "open" nuclear export signal (NES) groove of CRM1, simultaneously occupying two hydrophobic pockets. NCI-1 additionally inhibited the nuclear export and proliferation of cells harboring the human CRM1-C528S mutant. Our work opens up the avenue of noncovalent CRM1 inhibitor development toward a more potent, less toxic, and broad-spectrum anticancer/antiviral therapy.

Exportin-1 (also named as CRM1) plays a prominent role in autoimmune disorders and has emerged as a potential therapeutic target for colitis. Here we report on the rational structure-based discovery of a small-molecule antagonist of exportin-1, LFS-829, with low-range nanomolar activities. The co-crystallographic structure, surface plasmon resonance binding assay, and cell-based phenotypic nuclear export functional assay validated that exportin-1 is a key target of LFS-829. Moreover, we demonstrated that the C528S mutation or the knockdown on exportin-1 can abolish the cellular activities of LFS-829. Strikingly, oral administration of LFS-829 can significantly reverse the pathological features of colitis model mice. We revealed that LFS-829 can attenuate dual NF-κB signaling and the Nrf2 cytoprotection pathway via targeting exportin-1 in colitis mice. Moreover, LFS-829 has a very low risk of cardiotoxicity and acute toxicity. Therefore, LFS-829 holds great promise for the treatment of colitis and may warrant translation for use in clinical trials.

The protein chromosome region maintenance 1 (CRM1) is an important nuclear export factor and drug target in diseases such as cancer and viral infections. Several plant-derived CRM1 inhibitors including plumbagin and oridonin possess potent antitumor activities. However, their modes of CRM1 inhibition remain unclear. Here, a multimutant CRM1 was engineered to enable crystallization of these two small molecules in its NES groove. Plumbagin and oridonin share the same three conjugation sites in CRM1. In solution, these two inhibitors targeted more CRM1 sites and inhibited its activity through promoting its aggregation, in addition to directly targeting the NES groove. While the plumbagin-bound NES groove resembles the NES-bound groove state, the oridonin complex reveals for the first time a more open NES groove. The observed greater NES groove dynamics may improve cargo loading through a "capture-and-tighten" mechanism. This work thus provides new insights on the mechanism of CRM1 inhibition by two natural products and a structural basis for further development of these or other CRM1 inhibitors.

Chromosome region maintenance 1 (CRM1) exports nuclear export signal (NES) containing cargos from nucleus to cytoplasm and plays critical roles in cancer and viral infections. Biochemical and biophysical studies on this protein were often obstructed by its low purification yield and stability. With the help of PROSS server and NES protection strategy, we successfully designed three small fragments of CRM1, each made of four HEAT repeats and capable of binding to NESs in the absence of RanGTP. One of the fragments, C7, showed dramatically improved purification yield, thermostability, mechanostability, and resistance to protease digestion. We showed by isothermal titration that the protein kinase inhibitor NES binds to C7 at 1.18 μM affinity. Direct binding to C7 by several reported CRM1 inhibitors derived from plants were verified using pull-down assays. These fragments might be useful for the development of CRM1 inhibitors towards treatment of related diseases. The strategy applied here might help to tackle similar problems encountered in different fields. K E Y W O R D S

No abstract

Chromosomal region maintenance protein 1 (CRM1) is a validated anticancer drug target, and its covalent inhibitor KPT-330 has been approved for marketing. However, the development of CRM1 inhibitors, especially the noncovalent ones, is still very limited. Drug repurposing is an effective strategy to develop drug leads for new targets. In this work, we virtually screened a library of marketed drugs and identified zafirlukast as a new CRM1 inhibitor. Biochemical and structural analysis revealed that zafirlukast was a noncovalent CRM1 inhibitor that bound to four subpockets in the nuclear-export-signal (NES) groove. Methylation of the sulfonamide group rendered zafirlukast completely inactive against CRM1. Zafirlukast inhibited the growth of a variety of cancer cells and worked synergistically with the drug doxorubicin. Taken together, these works laid a solid foundation for reshaping zafirlukast as a valuable lead compound for further design of noncovalent, specific, and potent CRM1 inhibitors toward the treatment of various cancers.

No abstract

CRM1 is an important drug target in diseases such as cancer and viral infection. Plumbagin and oridonin, the herbal ingredients with known anti-cancer activities, were reported to inhibit CRM1-mediated nuclear export. However, their modes of CRM1 inhibition are unclear. Here, a multi-mutant of yeast CRM1 was engineered to enable the crystallization of these two small molecules in CRM1’s NES-binding groove. Each structure showed three inhibitor-binding sites, among which two are conserved in humans. Besides the known binding site, another site also participated in oridonin and plumbagin’s CRM1 inhibition. While the plumbagin-bound NES groove resembled the NES-bound groove state, the oridonin-bound groove revealed for the first time a more open NES groove, which may potentially improve cargo-loading through a capture-and-tighten mechanism. Our work thus provides a tool for CRM1 inhibitor crystallization, new insights of CRM1-cargo interaction, and a structural basis for further development of these or other CRM1 inhibitors.