Exploring the structural requirements of collagen-binding peptides.

Collagen synthesis and tissue remodeling are involved in many diseases; therefore, collagen-specific binding agents have been developed to study collagen changes in various tissues. Based on a recently reported collagen binding peptide, which contains unnatural biphenylalanine (Bip) amino acid residue, constructs with various structure variations were synthesized to explore the contributions of unnatural Bip residue, conformational restrain, and amino acid sequence in collagen recognition. Their binding efficiency to collagens was evaluated in vitro using pure collagens. The results indicate that the C-terminal unnatural Bip residue, rather than the peptide sequence or conformational restrain, dominated the collagen I binding. Subsequent tissue binding study showed that the selected peptide did not offer preferential selectivity over collagen I in tissue, suggesting that a simple in vitro binding assay cannot adequately model the complex biological environment.

Pancreatic cancer-associated Cathepsin E as a drug activator.

Pancreatic ductal adenocarcinoma (PDAC) is challenging to treat, and better means to detect and/or treat pancreatic cancer are urgently needed to save lives. Cathepsin E (Cath E) is a proteolytic enzyme highly expressed in PDAC. In this study, a novel approach using Cath E activation of a Cath E-specific prodrug was demonstrated. Specific activation of the prodrug is expected to kill pancreatic cancer cells without harming normal pancreatic cells. A novel 5-aminolevulinic acid (5-ALA) prodrug was custom-designed to be activated selectively by endogenous Cath E within the PDAC cells. The 5-ALA prodrug was incubated with Cath E-positive and -negative tumor cells and illuminated with various doses of light. In addition, mice genetically engineered to develop PDAC were injected intravenously with the 5-ALA prodrug, and the pancreas was treated with light irradiation. One day after treatment, PDAC tissue was assessed for apoptosis. The 5-ALA prodrug was activated within the Cath E-positive tumor but not in the normal pancreatic tissue. When used in combination with light treatment, it allowed delivery of selective photodynamic therapy (PDT) to the cancerous tissues, with minimal harm to the adjacent normal tissues. With this novel Cath E activation approach, it is possible to detect pancreatic cancer cells accurately and specifically impair their viability, while sparing normal cells. This treatment could result in fewer side effects than the non-specific treatments currently in use. Cath E is a specific and effective drug activator for PDAC treatment.

A cardiac tissue-specific binding agent of troponin I.

A fluorescently labeled, biphenylalanine-rich peptide was identified as a should be cardiac troponin I-specific binding agent with preferential staining affinity to myocardium tissues and extremely low staining to other stromal components. Fluorescence images demonstrate that this new peptide is an excellent contrast agent useful for examining troponin I structural distribution and expression density within heart tissue sections. It provides a clear contrast between myocardial cells and the surrounding collagen matrix.

Detection of pancreatic cancer tumours and precursor lesions by cathepsin E activity in mouse models.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas-bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC. METHODS: Pancreas from normal, chronic pancreatitis and PDAC patients was assessed for Cath E expression by quantitative real-time PCR and immunohistochemistry. Human PDAC xenografts and genetically engineered mouse models (GEMM) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an optical imaging system. RESULTS: The specificity of Cath E expression in PDAC patients and GEMM of pancreatic cancer was confirmed by quantitative real-time PCR and immunohistochemistry. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMM in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMM before tumour formation. CONCLUSIONS: The elevated Cath E expression in PanIN and pancreatic tumours allowed in-vivo detection of human PDAC xenografts and imaging of pancreas with PanIN and PDAC tumours in GEMM. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.

Molecular imaging of Cathepsin E-positive tumors in mice using a novel protease-activatable fluorescent probe.

UNLABELLED: The purpose of this study is to demonstrate the ability of imaging Cathepsin E (Cath E) positive tumors in living animals through selective targeting of Cath E proteolytic activity using a sensitive molecular imaging agent. METHODS: A peptide-based Cath E imaging probe and a control probe were synthesized for this study. Human Cath E-positive cancer cells (MPanc96-E) were implanted subcutaneously in nude mice. Tumor-bearing mice were examined in vivo with near-infrared fluorescence (NIRF) imaging at various time points after intravenous injection of the Cath E sensing imaging probe. Excised organs and tissues of interest were further imaged ex vivo. RESULTS: Upon specific Cath E proteolytic activation, the NIRF signal of the imaging probe a was converted from an optically quenched initial state to a highly fluorescent active state. Imaging probe a was able to highlight the Cath E-positive tumors as early as 24 h post injection. Fluorescent signal in tumor was 3-fold higher than background. The confined specificity of imaging probe a to tumor associated Cath E was verified by using control imaging probe b. Both in vivo and ex vivo imaging results confirmed the superior selectivity and sensitivity of imaging probe a in Cath E imaging. CONCLUSIONS: The small animal studies demonstrated the capability of probe a for imaging Cath E-positive tumors. The developed optical probe could be applied in early diagnostic imaging and guiding subsequent surgical procedure.

Assessment of cardiovascular fibrosis using novel fluorescent probes.

Cardiovascular fibrosis resulted from pressure overload or ischemia could alter myocardial stiffness and lead to ventricular dysfunction. Fluorescently labeled collagen-binding protein CNA 35, derived from the surface component of Staphylococcus aureus, and a novel synthetic biphenylalanine containing peptide are applied to stain fibrosis associated collagen and myocytes, respectively. Detailed pathological characteristics of cardiovascular fibrosis could be identified clearly in 2 hours. This staining pair requires only simple staining and brief washing, generating less than 10 ml of waste. The image information collected by this novel fluorescent staining pair is compatible with it collected by the traditional Masson's Trichrome and Picrosirius Red staining which are widely used to stain cardiovascular fibrosis and isolated cells.

Selective detection of Cathepsin E proteolytic activity.

BACKGROUND: Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide-substrate that is only sensitive to Cath E but not Cath D. METHODS: Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH2, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence. RESULTS: Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K(cat)/K(M)) of Cath E for substrate e was 16.7 µM⁻¹S⁻¹. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B. CONCLUSIONS: This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement. GENERAL SIGNIFICANCE: This study forms the foundation of Cath E specific inhibitor development in further studies.

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector.

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.

Total solid-phase synthesis of bombesin analogs with different functional groups at the C-terminus.

Five bombesin analogs with different functional groups at the C-terminus were synthesized using a solid-phase strategy. The protocols were optimized using 4-(hydroxymethyl)benzoic acid (HMBA) resin to synthesize a common precursor followed by nucleophilic cleavage of the base sensitive peptide ester linkage. The C-terminal modifications included ethylamide, butylamide, methyl ester, propyl ester and hydrazide. Cleavage from the resin was possible with the fully protected or deprotected precursor peptide; however, higher purity of the final products was achieved when cleavage protocols were conducted after side-chain deprotection. The synthesized peptides were analyzed and characterized using reverse phase HPLC and ESI-MS. The peptides were obtained in 13-32% overall recovery, calculated from the coupling efficiency of the first amino acid residue, and in 91-97% purity.