High-throughput fluorescence polarization screen for inhibitors of VICKZ1 RNA binding

The VICKZ(Igf2bp) protein family consists of RNA binding proteins (RBPs) that have important roles during development, helping stabilize, localize, and translationally regulate mRNAs in embryonic cells. Thers hree VICKZ paralogs, after birth;VICKZ1&3 expression is dramatically down-regulated, In many types of cancers, however, VICKZ1&3 is expressed and has been correlated with many pro-oncogenic processes, including proliferation, apoptosis, resistance to drugs, tumor progression, and metastasis. VICKZ1 binds a number of specific mRNAs .One such pro-oncogenic mRNA is Kras. We have seen that Inhibition of VICKZ1 expression in mouse models inhibits tumor metastases.

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 fluorescently-labeled fragments of Kras mRNA, we identified with the FP a 200-NT sequence in its 3’UTR that binds VICKZ1. Using this fragment, we scanned over 100,000 compounds that would prevent VICKZ1 binding. This highly robust assay has yielded approximately 7reproducible hits dose-dependent (IC50around the tens of uM) that pass quality control by mass spectrometer. This molecules did not have an effect on a control RBP (La protein) binding to its target.

These hits are being validated with orthogonal assays. Such as electrophoretic mobility shift assay(EMSA) and Microscale thermophoresis (MST). To evaluate and confirm the hits.

Incubation of cells with the lead compound demonstrated dose-dependent inhibition of wound healing, reduction of VICKZ1 target RNAs, and downregulation of ERK phosphorylation (a readout of K-ras signaling).And no toxicity was observed (cell proliferation unaffected)