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

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.

FIGURE 1

Schematic representation of the protease-activated retention micro-PET probe. Hepatitis C virus (HCV) tracking probe was designed using HCV NS3/4A serine protease as a marker of viral activity. A cell-penetrating probe was designed that consisted of a cell-penetrating Tat peptide, a HCV NS3/4A protease substrate, and a hydrophilic domain. For imaging, the probe was labeled by fluorescein 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 visualization using PET or optical imaging. NS, non-structural protein; TAT, transactivator of transcription of human immunodeficiency virus; FITC, fluorescein isothiocyanate; HCV, hepatitis C virus; PET, positron emission tomography.

FIGURE 2

Specific retention of TAT-ΔNS3/4A-FITC in NS3/4A-HCC36- and HCV-infected cells. HCC36 cells, NS3/4A-HCC36 cells, or JFH-1-infected HCC36 cells were incubated with 10 μM TAT-ΔNS3/4A-FITC in the absence or presence of 2 μM telaprevir (NS3/4A protease inhibitor) at 37°C for 1 h. The cells were washed with DMEM containing 10% serum three times per hour. After culturing for 1 or 8 h, the phase contrast and fluorescence of viable cells were observed under a fluorescence microscope. Scale bar: 100 μm.

FIGURE 3

Specific retention of TAT-ΔNS3/4A-FITC in NS3/4A-expressing tumors in vivo. Mice bearing established NS3/4A-HCC36 and HCC36 tumors were injected with 500 μM TAT-NS3/4A-FITC and sacrificed after 4 h. Sections of NS3/4A-HCC36 (upper panels) and HCC36 (lower panels) tumors were stained with 520 HCV protease Assay Kit (AnaSpec) to detect NS3/4A activity in tumor sections. FITC-derived fluorescence (green) and NS3/4A activity (red) were observed under a fluorescence microscope. Scale bar: 1 mm.

FIGURE 4

Specificity and half-life of TAT-ΔNS3/4A-¹²⁴I-FITC. (A) NS3/4A-HCC36 and HCC36 cells were incubated with 37 kBq of TAT-ΔNS3/4A-¹²⁴I-FITC in the presence or absence of 2 μM Telaprevir at 37°C for 1 h. The cells were washed with DMEM containing 10% serum three times per hour. The cells were collected by treatment with trypsin at the indicated times. The radioactivity of the cells was then measured with a gamma-counter. The CPM was normalized by protein concentration. (B) Kinetics of the TAT-ΔNS3/4A-¹²⁴I-FITC in vivo, BALB/c mice were intravenously injected with TAT-ΔNS3/4A-¹²⁴I-FITC, and the radioactivity in serum samples collected at the indicated times was measured using a gamma-counter. t_1/2 = 2.55 min. Error bar: standard error of triplicate determinations.

FIGURE 5

Micro-PET imaging of NS3/4A activity in vivo. (A) Mice bearing established NS3/4A-HCC36 (right hind leg) and HCC36 (left hind leg) tumors were injected with 3,700 kBq of TAT-ΔNS3/4A-¹²⁴I-FITC. Coronal and transverse images were acquired at 2, 4, and 6 h after injection of the probe. (B) Mice bearing established NS3/4A-HCC36 (right hind leg) and HCC36 (left hind leg) tumors were intraperitoneally injected with telaprevir (20 mg/kg/day for 3 days) before intravenous injection of TAT-ΔNS3/4A-¹²⁴I-FITC (3,700 kBq). Coronal images of tumor sections were acquired at 2, 4, and 6 h after injection of the probe.

FIGURE 6

Biodistribution of TAT-ΔNS3/4A-¹²⁴I-FITC in xenograft mice. Mice bearing established NS3/4A-HCC36 and HCC36 tumors were injected with 3,700 kBq of TAT-ΔNS3/4A-¹²⁴I-FITC. Selected organs and tumors were removed from the mice after 2 (white column), 4 (black column), and 6 (gray column) h. The radioactivity of individual organs was measured using a gamma-counter and normalized for sample weights. The biodistribution of TAT-ΔNS3/4A-¹²⁴I-FITC in selected organs was expressed as percentage injected dose/g tissue. Data represent mean ± SEM.

FIGURE 7

Micro-PET Imaging of NS3/4A activity in orthotopic liver implantation model. The NS3/4A-HCC36 tumors or HCC36 tumors from the ectopic tumors were harvested and transplanted into the left liver lobe of SCID mice (n = 3). After 2 weeks, the 3,700 kBq of TAT-ΔNS3/4A-¹²⁴I-FITC was intravenously injected. (A) PET imaging was performed at 4 h after injection of the probe. (B) The accumulation of radioactivity in NS3/4A-HCC36 tumors and HCC36 tumors was measured using a gamma-counter at 2 (white column), 4 (black column), and 6 (gray column) h after probe injection. Data represent mean ± SEM. Student’s t-test analysis of data. Statistical analysis was compared NS3/4A-HCC36 with HCC36. P < 0.05 was considered statistically significant.