(489.24) Detection of the Pin1-ligand interactions using 19F NMR spectroscopy

Poster Board Number: A94

Xiao-Ru Chen (Texas Aamp;M University), Tatyana Igumenova (Texas Aamp;M University)

Pin1 is a peptidyl-prolyl cis-trans isomerase with specificity towards the pSer/pThr-Pro protein motifs. Through its cis-trans isomerization activity, Pin1 regulates the stability of oncogenic and tumor suppressor proteins. The involvement of Pin1 in cancer development makes it an attractive target for therapeutic intervention. To that end, a wide variety of small-molecule inhibitors were developed to specifically target Pin1. A major challenge that remains in the Pin1 field is the high percentage of false-positive readouts. Hence, there is a need to establish a robust and facile binding assay to directly validate Pin1 as the inhibitor target. We present a 19F Nuclear Magnetic Resonance (NMR)-based approach that directly reports on the binding of inhibitors and substrates to Pin1, and enables identification of which Pin1 domain, WW or PPIase, harbors the binding site. The method relies on the introduction of 19F labels into the strategic positions of Pin1. Upon fluorination, Pin1 remains well-folded and catalytically active. The ligand binding process is monitored by the changes in the position and linewidth of the Pin1 19F peaks in the NMR spectra. Moreover, quantitative information about the affinity of the Pin1-ligand interactions is readily available from the chemical shift-based binding curves. This approach is demonstrated for several small-molecule inhibitors and the bivalent peptide substrates of Pin1.