Förster Resonance Energy Transfer Based Biosensor for Targeting the hNTH1–YB1 Interface as a Potential Anticancer Drug Target

Abstract

The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human Endonuclease III (hNTH1). In the present study, we used Förster Resonance Energy Transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid in vitro highthroughput screening for potential inhibitors of the hNTH1-YB1 complex. Two pilot screens were carried out allowing the selection of several promising compounds exhibiting IC50 values in the low micromolar range. Interestingly, two of these compounds bind to YB1 and sensitize drug-resistant breast tumor cells to the chemotherapeutic agent, cisplatin. Taken together, these findings demonstrate that the hNTH1-YB1 interface is a druggable target for the development of new therapeutic strategies for the treatment of drug-resistant tumors. Moreover, beyond this study, the simple design of our biosensor defines an innovative and efficient strategy for the screening of inhibitors of therapeutically relevant protein-protein interfaces.

YB-1 as an Oncoprotein: Functions, Regulation, Post-Translational Modifications, and Targeted Therapy

“…In addition, YB-1 binds to the DNA repair proteins MSH2, DNA polymerase δ, Ku80, and WRN75. A recent study demonstrated that human endonuclease III (hNTH1) is a DNA repair protein and bifunctional DNA glycosylase that binds to YB-1 in the nucleus, and the hNTH1-YB-1 interface plays a role in the response to cisplatin in MCF7 cells [ 71 ]. YB-1 can be PARylated (Poly(ADP-ribosylation)) by PARP1 during the process of interacting with damaged DNA [ 72 ].…”

Focus on DNA Glycosylases—A Set of Tightly Regulated Enzymes with a High Potential as Anticancer Drug Targets

“…A direct interaction between NTH1 and the multifunctional YB1 protein has indeed been reported to occur in tumor cells in response to genotoxic stress. Through this interaction, YB1 has been shown to stimulate the AP-lyase activity of NTH1 [106,162]. Several reports have demonstrated that the level of nuclear expression of YB1 is predictive of drug resistance and poor prognosis [163][164][165][166][167][168] and the increased abundance of the NTH1-YB1 complex in tumor cells after exposure to genotoxic stress, including cisplatin treatment, could explain the increased resistance of these cells to anticancer agents [169,170].…”

“…shed light on the mode of inhibition of these promising molecules and the molecular contacts underlying this unexpected specificity. [193] (A), the DNA glycosylase activity [197] (B) of DNA glycosylases, or of the interaction interface (C) between a DNA glycosylase (here, NTH1) and its cellular partner (YB1) [162]. FRET: Förster resonance energy transfer.…”

“…Different technical solutions have been found to select inhibitors of the DNA glycosylase activity. Mancuso and colleagues added APE1 [193] (A), the DNA glycosylase activity [197] (B) of DNA glycosylases, or of the interaction interface (C) between a DNA glycosylase (here, NTH1) and its cellular partner (YB1) [162]. FRET: Förster resonance energy transfer.…”

“…In a recent study conducted by Senarisoy et al, an alternative approach was proposed to restore sensitivity of drug-resistant tumor cells, by targeting the interaction of NTH1 with one of its cellular partners, YB1, instead of targeting the catalytic activities of NTH1 [162]. YB1 is a transcription factor that has been shown to bind to NTH1 and stimulate its AP-lyase activity.…”