Abstract PR1: In silico designed covalent peptidomimetic inhibitors (KPT-SINE) of CRM1 modulate tumor suppressor protein nuclear export and induce apoptosis in cancer cells
Authors: Senapedis William T. , Baloglu Erkan , Froim Doriana , McCauley Dilara , Chook Yuh Min , Kauffman Michael , Shacham Sharon , Landesman Yosef , Kalid Ori , Shechter Sharon , St Martin Jean Richard , Kashyap Trinayan , Klebanov Boris , Plamondon Louis , Sandanayaka Vincent 
Published: August 2014

Abstract
Chromosome region maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to nuclear accumulation of tumor suppressor (TSP) and growth regulatory proteins (GRP). Through CRM1 inhibition, nuclear localization of these proteins restores cell cycle checkpoints and genome surveying functions culminating in apoptosis of tumor cells. Conversely, CRM1 inhibition of normal cells induces reversible cell cycle arrest. We developed a structural model of CRM1 suited for binding diverse small molecules and used it to guide the discovery and development of KPT-SINE (selective inhibitors of nuclear export). The model predicted that a switching motion of Met545 and Met583 in CRM1 was required for inhibitor accommodation. We found that published CRM1 inhibitors display efficient mimicry of cargo Nuclear Export Signals (NES) and a thiol reactive warhead proximal to Cys528 in CRM1. This was further confirmed by Yuh Min Chook who solved the X-ray structures of CRM1. In silico screening yielded 402 hits (from a library of ˜250,000 drug-like compounds) with 19 molecules active in vitro (IC50&lt;30μM) and guided the optimization of KPT-SINE. Lead KPT-SINE irreversibly bound and inhibited CRM1 nuclear export and were cytotoxic to a broad range of tumor cells and xenografts. Through comparisons of molecular and cellular markers in both cancer and normal cells we concluded that KPT-SINE treatment ultimately induced cancer cell death.
Methods: CRM1 Model: The CRM1 model was developed using the novel consensus Induced Fit Docking approach (cIFD). In vitro/vivo evaluation: Compounds were tested in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cells transfected with mutant CRM1-Cys528Ser were treated with KPT-SINE in order to determine covalent binding to Cys528. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. Multiple KPT-SINE derivatives were tested for: 1) Nuclear localization of TSPs, 2) Effects on cell cycle and viability, 3) Effects on TSP protein expression, and 4) Mouse xenograft model efficacy.
Results: KPT-SINE, potent inhibitors in the cell-based microscopy assay (EC50=20-130nM), were robust inhibitors of cancer cell proliferation and inducers of apoptosis. KPT-SINE inhibition of CRM1 started within 30 minutes and reached a maximum in 8 hrs. Additionally, KPT-SINE effects were blocked by transient transfection of mutant CRM1-Cys528Ser, supporting the role of Cys528. We also identified nuclear accumulation of p53, FOXO1a, FOXO3a, APC, IκB, p27, PP2Aα, and p21, which was followed by cell cycle arrest and death of cancer cells while normal cells undergo a reversible block. Both solid (MDA-MB-468) and hematological (Z-138) mouse xenografts treated with KPT-SINE at 25 mg/kg QoDx3 PO weekly for 4 weeks inhibited tumor growth by more than 70%. Immunohistochemical analysis of the tumor molecular markers from KPT-SINE treated animals exhibited nuclear localization of TSPs, a decrease in proliferation (Ki-67), and an increase in apoptosis (TUNEL).
Conclusions: We described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs at the molecular level through to potent in vivo efficacy in xenograft models. KPT-SINE, which were discovered and optimized using a structure-based approach, are due for Phase I clinical trials Mid-2012.
This proffered talk is also presented as Poster A2.
Citation Format: William T. Senapedis, Jr., Erkan Baloglu, Doriana Froim, Dilara McCauley, Yuh Min Chook, Michael Kauffman, Sharon Shacham, Yosef Landesman, Ori Kalid, Sharon Shechter, Jean Richard St Martin, Trinayan Kashyap, Boris Klebanov, Louis Plamondon, Vincent Sandanayaka. In silico designed covalent peptidomimetic inhibitors (KPT-SINE) of CRM1 modulate tumor suppressor protein nuclear export and induce apoptosis in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR1.