High-Throughput Fluorescence Polarization Screen for Inhibitors of VICKZ1 RNA Binding

Introduction VICKZ (Igf2bp) protein family consists of RNA binding proteins (RBPs) that have important roles during development in many embryonic cell types. Three VICKZ paralogs have been identified in mammals; after birth, VICKZ1&3 is dramatically down-regulated and is almost non-detectable in adults. In many types of cancers, VICKZ1&3 is re-expressed and has been correlated with many pro-oncogenic processes. VICKZ1 has been shown to promote proliferation, invasion and chemo-resistance in malignancies by regulating post-transcriptional processes of RNA in the cell, VICKZ1 binds a number of specific mRNAs. One such mRNA is Kras.

Material and method Fluorescence Polarization This assay is based on changes in fluorescence polarizationas a result of binding of a protein to a labeled RNA. When polarized light excites a fluorophore, such as fluorescein-labeled K-ras RNA, the relatively small flRNA usually undergoes rotational diffusion more rapidly than the time required for light emission. Therefore, the position of the flRNA at the time of light emission is largely randomized, resulting in depolarization of the emitted light. In contrast, when protein, such as VICKZ1 binds to the flRNA, the larger size and volume of the protein–flRNA complex causes rotation to be slower, increasing the likelihood that the protein–flRNA

complex will be in the same plane at the time of light emission as it was at the time of

excitation. Therefore, the emitted light remains highly polarized.

Results and discussion In collaboration with the Israel National Center for Personalized Medicine (INCPM), I have performed a high-throughput Fluorescence polarization (FP) screen for small molecule inhibitors of VICKZ1 RNA binding. By comparing 5’ fluorescently-labeled fragments of Kras mRNA, we identified by FP a 200nt sequence in its 3’UTR that binds VICKZ1. Using this fragment as a probe, we scanned over 100,000 compounds for those that would prevent VICKZ1 binding. This highly robust assay has yielded approximately 7 reproducible hits whose inhibition is dose-dependent (IC50 in the tens of uM)and specific for VICKZ1 (no effect on a control RBP - La protein - binding to its target - Bcl RNA).

These hits are currently being validated with orthogonal assays such as electrophoretic mobility shift assay (EMSA), Microscale thermophoresis (MST), and isothermal titration calorimetry (ITC). Binding of IGF2BP1 to Kras RNA was inhibited 8 - 10 fold at high concentrations (25-50 uM) of the lead compound. Incubation of cells with the lead compound demonstrated dose-dependent inhibition of wound healing, reduction of VICKZ1 target RNAs as determined by real time PCR, and downregulation of ERK phosphorylation (a readout of K-ras signaling). No toxicity was observed (cell proliferation unaffected) in any of the cell lines tested.

Conclusion We have performed a screen for small molecule inhibitors of VICKZ1 binding to Kras mRNA., we have compounds that inhibit binding in a FP. The lead compound was confirmed and demonstrated biological effectiveness with no toxicity. Analogs of the confirmed hit are currently being screened to select molecules that can work at even lower concentrations. These analogs will be tested in in vivo models.