A novel Zn2+ probe for fluorescent monitoring of drug and E6/E7 protein interaction in living cells

Background: The zinc-binding sites within the human papillomavirus (HPV) E6/E7 oncoproteins play a critical role in maintaining structural stability and represent promising therapeutic targets due to their cysteine-mediated Zn²⁺ coordination. Zn²⁺ dissociation from these sites serves as a key indicator of direct drug-protein interactions. However, evaluating such interactions is challenging, as E6/E7 proteins localize in the nucleus, necessitating probes capable of traversing both cellular and nuclear membranes while retaining target specificity and real-time monitoring capabilities under physiological conditions.

Results: A nuclear membrane-permeable Zn²⁺ fluorescent probe, NTAD-N2, was developed for real-time monitoring of Zn²⁺ dynamics in living SiHa cervical Cancer cells. This probe-based method was used to evaluate drug efficacy in inducing Zn²⁺ release from HPV16 E6/E7 proteins. Extracellular assays showed disulfiram surpassed ebselen in releasing Zn²⁺ from recombinant proteins. Intracellularly, ebselen induced significant Zn²⁺ release at 5 μM, while disulfiram required 60 μM. This extracellular/intracellular difference in drug efficacy suggests that the bioavailability of disulfiram was influenced by the complex intracellular microenvironment. Specificity was verified through the use of HPV-negative C33A cells as a negative control, where no Zn²⁺ release was detected. Western blot analysis demonstrated that disulfiram-induced Zn²⁺ release at 60 μM resulted from E6/E7 protein degradation, establishing that intracellular Zn²⁺ accumulation originated specifically from E6/E7 protein destabilization.

Significance: This study established a novel approach to evaluate zinc-site-targeting drug candidates in live-cell systems, bridging the gap between molecular targeting and cellular efficacy. Furthermore, it underscores the therapeutic potential of disrupting zinc coordination in viral oncoproteins, offering a framework for developing precision interventions against HPV-associated malignancies. This strategy may accelerate the discovery of metalloprotein-targeted therapies for viral infections and related cancers.