Chuang Chih-Hung, Cheng Tian-Lu, Chen Wei-Chun, Huang Yi-Jung, Wang Hsin-Ell, Lo Yen-Chen, Hsieh Yuan-Chin, Lin Wen-Wei, Hsieh Ya-Ju, Ke Chien-Chih, Huang Kang-Chieh, Lee Jin-Ching, Huang Ming-Yii
Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan.
Front Microbiol. 2022 Nov 4;13:896588. doi: 10.3389/fmicb.2022.896588. eCollection 2022.
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 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 I in the hydrophilic domain to form a TAT-ΔNS3/4A-I-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-I-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-I-FITC probe can be used to represent NS3/4 protease activity and distribution a clinical PET imaging system allowing. This strategy may be extended to detect any cellular protease activity for optimization the protease-based therapies.
丙型肝炎病毒（HCV）NS3/4A蛋白酶是直接作用抗病毒药物的一个有吸引力的靶点。对NS3/4A蛋白酶的分布和活性进行实时追踪，对于临床诊断和疾病管理很有用。然而，尚未开发出能够系统检测NS3/4A蛋白酶活性或分布的方法。我们设计了一种蛋白酶可激活的保留探针，用于通过正电子发射断层扫描（PET）成像追踪HCV NS3/4A蛋白酶活性。设计了一种细胞穿透探针，它由一个细胞穿透性Tat肽、HCV NS3/4A蛋白酶底物和亲水结构域组成。该探针在亲水结构域用异硫氰酸荧光素（FITC）和碘进行标记，形成TAT-ΔNS3/4A-I-FITC探针。在NS3/4A底物处被切割后，非穿透性的亲水结构域被释放并积聚在细胞质中，从而实现PET或光学成像。TAT-ΔNS3/4A-FITC探针选择性地积聚在表达NS3/4A的HCC36（NS3/4A-HCC36）细胞/肿瘤以及HCV感染的HCC36细胞中。PET成像显示，TAT-ΔNS3/4A-I-FITC探针选择性地积聚在NS3/4A-HCC36异种移植瘤和肝植入的NS3/4A-HCC36肿瘤中，但在对照HCC36肿瘤中未积聚。在允许的临床PET成像系统中，TAT-ΔNS3/4A-I-FITC探针可用于表征NS3/4蛋白酶的活性和分布。这种策略可能会扩展到检测任何细胞蛋白酶活性，以优化基于蛋白酶的治疗方法。
