Investigating Karyopherin B1: small molecule interactions for cancer therapy

The advent of gene expression profiling studies has allowed for the identification of genes with potential as disease markers and therapeutic targets. Our laboratory identified the eukaryotic nuclear importer protein Karyopherin B1 (KpnB1), to be up-regulated in different cancer cell lines, including cervical and oesophageal as well as transformed cells. Inhibition of KpnB1 in these cells using small interfering RNA (siRNA) resulted in significant cancer cell death via apoptosis, suggesting KpnB1 is essential for cancer cell survival. Within our laboratory, we established that candidate small molecules targeted against KpnB1 identified using a rational drug design approach. The outcome of this research is for examine inhibitors of KpnB1 for potential as future anti-cancer agents using. Based on the long-term goal of this research, this particular project was aimed at investigating a small molecule inhibitor identified in our laboratory, known as Inhibitor of Nuclear Import-43 (INI-43) for its potential to bind to the nuclear importer, KpnB1. Using conventional assays as well as cutting edged techniques including circular dichrosim (CD) and isothermal titration calorimeter (ITC), an examination of INI-43 and its interactions with KpnB1 was made. In vitro analysis showed that INI-43 exhibits cytotoxic effects on cervical cancer cells with an IC₅₀ of ≈10μM and induces apoptotic cell death. The NFAT dual luciferase assay measured nuclear import of KpnB1 associated proteins, showing that INI-43 inhibits nuclear import/activity of NFAT in a dose dependent manner. Confocal microscopy of exogenous FRFP-KpnB1 as well as endogenous KpnB1 in the presence of INI-43 showed a change in the localisation of KpnB1 upon drug treatment. Both FRFP-KpnB1 and endogenous KpnB1 appear to be prevented from entering the nucleus, and is retained in the peri-nuclear space and the cytoplasm suggesting that INI-43 inhibits KpnB1 movement into the nucleus. To investigate KpnB1-INI-43 interactions, purified KpnB1 was prepared and used in biophysical techniques. Purified KpnB1 protein was prepared using GST-tagged purification methods and the tagged protein confirmed by mass spectrometry. Purified GST-KpnB1 was used in drug binding studies including circular dichrosim (CD) isothermal titration calorimetry (ITC). CD showed a drug concentration dependant shift in the spectra at around 233nm, indicative of drug protein interaction possibly occurring in a region of KpnB1 containing aromatic amino acids. The purified GST-KpnB1 was used in ITC, which confirmed an interaction between KpnB1 and INI-43, a relatively weak interaction. In conclusion, our data shows that the small molecule, INI-43 kills cancer cells, likely by interfering with KpnB1 associated nuclear import pathways. We show that INI-43 interferes with the nuclear localisation of KpnB1 itself and biophysical assays provide evidence for possible KpnB1-INI-34 interactions. Small molecules such as INI-43 present as promising tools to studying the potential of KpnB1 as an anticancer target