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

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.

Structural Insights into Substrate Recognition by Clostridium difficile Sortase.

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26-PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.

Synthesis and preclinical characterization of [18F]FPBZA: a novel PET probe for melanoma.

INTRODUCTION: Benzamide can specifically bind to melanoma cells. A 18F-labeled benzamide derivative, [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy) ethoxy)ethoxy]benzamide ([18F]FPBZA), was developed as a promising PET probe for primary and metastatic melanoma. METHODS: [18F]FPBZA was synthesized via a one-step radiofluorination in this study. The specific uptake of [18F]FPBZA was studied in B16F0 melanoma cells, A375 amelanotic melanoma cells, and NB-DNJ-pretreated B16F0 melanoma cells. The biological characterization of [18F]FPBZA was performed on mice bearing B16F0 melanoma, A375 amelanotic melanoma, or inflammation lesion. RESULTS: [18F]FPBZA can be prepared efficiently with a yield of 40-50%. The uptake of [18F]FPBZA by B16F0 melanoma cells was significantly higher than those by A375 tumor cells and NB-DNJ-pretreated B16F0 melanoma cells. B16F0 melanoma displayed prominent uptake of [18F]FPBZA at 2 h (7.81±0.82%ID/g), compared with A375 tumor and inflammation lesion (3.00±0.71 and 1.67±0.56%ID/g, resp.). [18F]FPBZA microPET scan clearly delineated B16F0 melanoma but not A375 tumor and inflammation lesion. In mice bearing pulmonary metastases, the lung radioactivity reached 4.77±0.36%ID/g at 2 h (versus 1.16±0.23%ID/g in normal mice). CONCLUSIONS: Our results suggested that [18F]FPBZA PET would provide a promising and specific approach for the detection of primary and metastatic melanoma lesions.