Small-Molecule Antagonist Targeting Exportin-1 via Rational Structure-Based Discovery

Abstract

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.

Discovery and biological evaluation of a small‐molecule inhibitor of CRM1 that suppresses the growth of triple‐negative breast cancer cells

“…In the present study, we aimed to develop a potent CRM1 inhibitor based on a scaffold of sulforaphene (LFS‐01). In our previous work, we revealed that the isocyanate group of LFS‐01 17 or its synthetic analogue 22 could be covalently trapped with the cysteine residue in the nuclear export signal (NES) binding cleft of CRM1 in a covalent fashion (PDB code: 5ZPU). Therefore, to increase the binding of the noncovalent moiety of the small‐molecule compound with CRM1, we first added a phenyl ring next to the sulfoxide moiety in the parent structure of LFS‐01, which led to the formation of the compound, LFS‐06, that inhibited the nuclear export function with an IC 50 value of 1.5 μM that is 3‐fold stronger than that of LFS‐01, as was determined by the cell‐based phenotypic nuclear export functional assay described previously.…”

“…The crystallographic structure of CRM1 was obtained from the PDB bank (PDB code: 5ZPU), showing the complex structure of CRM1 binding with our previously identified sulforaphene analogue, LFS-829. 22 First, the reactive Cys539-sulfur atom was linked to the unsaturated carbon atom on the isothiocyanate of LFS-31 at the alkylation site for the initial modification of the ligand structure. Next PDBQT files were generated using AutoDockTools, and docking parameter files were written out, according to the standard protocol.…”

Crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP

“…Structural analysis by means of X-ray crystallography defined the molecular basis of CRM1 inhibition. Furthermore, it has been used as a reliable approach for the development of novel CRM1 inhibitors (Kalid et al, 2012;Sun et al, 2013;Tian et al, 2020). The crystallographic analysis of several natural and synthetic compounds bound to CRM1 defined a general mechanism of inhibition by the covalent modification of a reactive cysteine residue located in the NES-binding cleft of human CRM1 (Cys528).…”

“…The binding of inhibitor compounds, mediated by the reactive cysteine, interferes with the binding of the NES peptide and prevents the formation of a stable export complex. As the human protein failed to crystallize in complex with inhibitors, CMR1 from the yeast Saccharomyces cerevisiae ( Sc CRM1) was genetically modified to incorporate the reactive cysteine (T539C) and was used to crystallize CRM1 in complex with several inhibitor compounds (Lapalombella et al, 2012;Etchin et al, 2013;Haines et al, 2015;Hing et al, 2016;Tian et al, 2020). Recently, we developed a crystallization approach using a stabilized variant of human CRM1 ( Hs CRM1), with which we succeeded in solving the crystal structure of LMB bound to Hs CRM1 in complex with RanGTP (Shaikhqasem et al, 2020).…”

Novel Mechanistic Observations and NES-Binding Groove Features Revealed by the CRM1 Inhibitors Plumbagin and Oridonin

“…All these types of inhibitors possess Michael acceptors that can covalently conjugate to a cysteine (C528) in the NES groove . Prior structural studies have demonstrated how bacterial products (e.g., leptomycin B and ratjadone A) and synthetic nuclear export inhibitors (NEIs; e.g., KPT-185, KPT-276, CBS9106, and LFS-829) bind to CRM1, which have provided useful information for CRM1 biology and drug development. − …”