2. Academic Validation
  3. A novel Zn2+ probe for fluorescent monitoring of drug and E6/E7 protein interaction in living cells

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

  • Anal Chim Acta. 2025 Aug 15:1363:344202. doi: 10.1016/j.aca.2025.344202.
Zhenzhen Xie 1,  Lan Yu 2,  Zuoping Li 3,  Na Zhao 3,  Shiwan You 3,  Zhihao Zhao 3,  Jindi Dou 3,  Xiling Deng 4,  Shiguo Sun 5
Affiliations

Affiliations

  • 1 Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China; Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, China.
  • 2 College of Chemistry & Pharmacy, Northwest A&F University, Yangling, 712100, Shaanxi, China.
  • 3 Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China.
  • 4 Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China. Electronic address: dxl_pha@shzu.edu.cn.
  • 5 Key Laboratory of Xinjiang Phytomedicine Resources of Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, China; Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, China; College of Chemistry & Pharmacy, Northwest A&F University, Yangling, 712100, Shaanxi, China; Shenzhen Research Institute, Northwest A&F University, Shenzhen, 518000, China. Electronic address: sunsg@nwsuaf.edu.cn.
PMID: 40480729 DOI: 10.1016/j.aca.2025.344202
Abstract

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.

Keywords

E6/E7 protein; Fluorescence monitoring; Unstable zinc-binding site; Zn(2+) probe; Zn(2+) release.

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