Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

Chih-Hung Chuang; Tian-Lu Cheng; Wei-Chun Chen; Yi-Jung Huang; Hsin-Ell Wang; Yen-Chen Lo; Yuan-Chin Hsieh; Wen-Wei Lin; Ya-Ju Hsieh; Chien-Chih Ke; Kang-Chieh Huang; Jin-Ching Lee; Ming-Yii Huang

doi:10.3389/fmicb.2022.896588

Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe

Front Microbiol. 2022 Nov 4:13:896588. doi: 10.3389/fmicb.2022.896588. eCollection 2022.

Authors

Chih-Hung Chuang^{1

2

3}, Tian-Lu Cheng^{2

3

4}, Wei-Chun Chen⁵, Yi-Jung Huang^{2

3}, Hsin-Ell Wang⁶, Yen-Chen Lo⁶, Yuan-Chin Hsieh⁷, Wen-Wei Lin^{2

8}, Ya-Ju Hsieh^{2

9}, Chien-Chih Ke^{2

9}, Kang-Chieh Huang², Jin-Ching Lee^{5

10}, Ming-Yii Huang^{3

11

12

13}

Affiliations

¹ Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
² Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.
³ College of Medicine, Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁴ Department of Biomedical and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁵ Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.
⁶ Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei City, Taiwan.
⁷ School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan.
⁸ Department of Laboratory Medicine, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁹ Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan.
¹⁰ Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.
¹¹ Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
¹² Department of Radiation Oncology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
¹³ Center for Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan.

Abstract

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for direct-acting antiviral agents. Real-time tracking of the NS3/4A protease distribution and activity is useful for clinical diagnosis and disease management. However, no approach has been developed that can systemically detect NS3/4A protease activity or distribution. We designed a protease-activatable retention probe for tracking HCV NS3/4A protease activity via positron emission topography (PET) imaging. A cell-penetrating probe was designed that consisted of a cell-penetrating Tat peptide, HCV NS3/4A protease substrate, and a hydrophilic domain. The probe was labeled by fluorescein isothiocyanate (FITC) and ¹²⁴I in the hydrophilic domain to form a TAT-ΔNS3/4A-¹²⁴I-FITC probe. Upon cleavage at NS3/4A substrate, the non-penetrating hydrophilic domain is released and accumulated in the cytoplasm allowing PET or optical imaging. The TAT-ΔNS3/4A-FITC probe selectively accumulated in NS3/4A-expressing HCC36 (NS3/4A-HCC36) cells/tumors and HCV-infected HCC36 cells. PET imaging showed that the TAT-ΔNS3/4A-¹²⁴I-FITC probe selectively accumulated in the NS3/4A-HCC36 xenograft tumors and liver-implanted NS3/4A-HCC36 tumors, but not in the control HCC36 tumors. The TAT-ΔNS3/4A-¹²⁴I-FITC probe can be used to represent NS3/4 protease activity and distribution via a clinical PET imaging system allowing. This strategy may be extended to detect any cellular protease activity for optimization the protease-based therapies.

Keywords: HCV NS3/4A serine protease; TAT-ΔNS3/4A-124I-FITC probe; cellular protease activity; micro-positron emission tomography; protease-activated retention peptide.