Synthesis of Fluorescent Probes Targeting Tumor-Suppressor Protein FHIT and Identification of Apoptosis-Inducing FHIT Inhibitors

Abstract

For the early diagnosis of cancer, leading to a better chance of full recovery, marker genes whose expression is already altered in precancerous lesions are desirable, and the tumor-suppressor gene FHIT is one candidate. The gene product, FHIT protein, has a unique dinucleoside triphosphate hydrolase (AP3Aase) activity, and in this study, we designed and synthesized a series of FHIT fluorescent probes utilizing this activity. We optimized the probe structure for high and specific reactivity with FHIT and applied the optimized probe in a screening assay for FHIT inhibitors. Screening of a compound library with this assay identified several hits. Structural development of a hit compound afforded potent FHIT inhibitors. These inhibitors induce apoptosis in FHIT-expressing cancers via caspase activation. Our results support the idea that FHIT binders, no matter whether inhibitors or agonists of AP3Aase activity, might be promising anticancer agents.

Development of Nucleoside Diphosphate-Bearing Fragile Histidine Triad-Imaging Fluorescence Probes with Well-Tuned Hydrophobicity for Intracellular Delivery

“…To confirm that the observed enzyme activity was due to FHIT, we examined the effect of an FHIT inhibitor (Figure C). We found that 10 μM 8Br-HA potently inhibited FHIT activity in HCC827 lysate but had no effect in H460 lysate, suggesting that TG-mADP can detect FHIT activity in HCC827 cell lysate. The residual enzyme activity in the presence of the inhibitor may be due to enzymes other than FHIT and is consistent with little or no FHIT expression in H460 cells .…”

“…We previously developed TG-CMP as an FHIT fluorescence probe for chemical screening . However, TG-CMP failed to detect FHIT activity in HCC827 cell lysate (Figure S1), in which FHIT is reported to be expressed (Figure A), whereas the FRET-type probe reported by Marx and co-workers could detect FHIT activity in cell lysate .…”

“…Although evidence is accumulating that the catalytic activity of FHIT is not required for tumor suppressor function, binding of Ap 3 A to FHIT appears to be essential, though the mechanism involved remains unclear. , Recent work showed that FHIT is trafficked from the nucleolus to the cytosol in response to stress and interferes with translation through binding with ribosomes . So far, there have been two reports describing FHIT fluorescence probes: Marx and co-workers reported a FRET-based fluorescence probe, FI-15 , and used it to search for FHIT inhibitors in a chemical library. , In addition, our group developed “Turn-ON” type FHIT fluorescence probes, Tokyo Green (TG)-cytidine monophosphate (CMP), TG-AMP, and their derivatives, in 2021, and succeeded in identifying potent hydroxamic acid-based FHIT inhibitors . However, these probes failed to detect FHIT activity in living cells, probably due to poor membrane permeability.…”

“…In this study, we have significantly improved the reaction efficiency of our previous FHIT probe, TG-AMP, , by introducing an additional phosphate group as an FHIT recognition site and by increasing the lipophilicity sufficiently to enable efficient transfer into living cells. The developed probe could detect FHIT activity not only in cell lysates but also in living cells.…”

“…16,17 In addition, our group developed "Turn-ON" type FHIT fluorescence probes, Tokyo Green (TG)-cytidine monophosphate (CMP), TG-AMP, and their derivatives, in 2021, and succeeded in identifying potent hydroxamic acid-based FHIT inhibitors. 18 However, these probes failed to detect FHIT activity in living cells, probably due to poor membrane permeability. Here, we focused on increasing the membrane permeability of our probes to enable them to intracellularly detect FHIT activity, because such probes could be useful in fluorescence-guided surgery to delineate the boundary between precancerous (early stage) lesions and normal tissue, leading to a great reduction in recurrence rate.…”