Solvatochromic and pH-Sensitive Fluorescent Membrane Probes for Imaging of Live Cells.

Membrane trafficking is essential for all cells, and visualizing it is particularly useful for studying neuronal functions. Here we report the synthesis, characterization, and application of several membrane- and pH-sensitive probes suitable for live-cell fluorescence imaging. These probes are based on a 1,8-naphthalimide fluorophore scaffold. They exhibit a solvatochromic effect, and one of them, ND6, shows a substantial fluorescence difference between pH 6 and 7. The solvatochromic effect and pH-sensitivity of those probes are explained using quantum chemical calculations, and molecular dynamics simulation confirms their integration and interaction with membrane lipids. For live-cell fluorescence imaging, we tested those probes in a cancer cell line (MCF7), cancer spheroids (MDA-MB-468), and cultured hippocampal neurons. Confocal imaging showed an excellent signal-to-noise ratio from 400:1 to about 1300:1 for cell membrane labeling. We applied ND6 during stimulation to label nerve terminals via dye uptake during evoked synaptic vesicle turnover. By ND6 imaging, we revealed cholesterol's multifaced role in replenishing synaptic vesicle pools. Our results demonstrate these fluorescent probes' great potential in studying membrane dynamic and synaptic functions in neurons and other secretory cells and tissues.

Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells.

BACKGROUND: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvß3 integrins for in vitro assays. OBJECTIVES: The main objective of this study is to efficiently prepare rhodamine B derivative, devoid of spirolactone formation that would be suitable for bioconjugation and subsequent bioimaging. METHODS: Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine dye (ROSA) in good yield. The ROSA dye was then coupled to a αvß3 integrin binding sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was used to image integrins on cancer cells. RESULTS: The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of 87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and straightforward. Imaging using standard filters on multimode plate reader and confocal microscope was performed. The αvß3 integrins present on the surface of live WM-266-4 (melanoma) and MCF- 7 (breast cancer) cells were successfully imaged. CONCLUSION: We successfully derivatized rhodamine B to create an inexpensive, stable and convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties and it is suitable for bioconjugation and many imaging applications.

Imaging Matrix Metalloproteinase Activity Implicated in Breast Cancer Progression.

Proteolysis has been cited as an important contributor to cancer initiation and progression. One can take advantage of tumor-associated proteases to selectively deliver imaging agents. Protease-activated imaging systems have been developed using substrates designed for hydrolysis by members of the matrix metalloproteinase (MMP) family. We presently describe approaches by which one can optically image matrix metalloproteinase activity implicated in breast cancer progression, with consideration of selective versus broad protease probes.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Glycosylation modulates melanoma cell α2ß1 and α3ß1 integrin interactions with type IV collagen.

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2ß1 and α3ß1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3ß1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2ß1 integrin interaction with α1(IV)382-393 but right in the middle of α3ß1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no ß-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.

Monitoring and Inhibiting MT1-MMP during Cancer Initiation and Progression.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion. Numerous substrates and binding partners have been identified for MT1-MMP, and its role in collagenolysis appears crucial for tumor invasion. However, development of MT1-MMP inhibitors must consider the substantial functions of MT1-MMP in normal physiology and disease prevention. The present review examines the plethora of MT1-MMP activities, how these activities relate to cancer initiation and progression, and how they can be monitored in real time. Examination of MT1-MMP activities and cell surface behaviors can set the stage for the development of unique, selective MT1-MMP inhibitors.

Peptoids and peptide-peptoid hybrid biopolymers as peptidomimetics.

Peptoids (oligomers of N-substituted glycine residues) and peptide-peptoid hybrid polymers (peptomers) are interesting classes of compounds mimicking structure and function of biologically active peptides. The oligomeric peptidomimetics such as peptoids are particularly important compounds since they provide access to an enormous molecular diversity, by variation of the building blocks. The modular structure of peptoids, ease of synthesis, and high compatibility with existing peptide chemistry synthetic protocols, make peptoids and peptoid-containing peptidomimetics ideal tools for structure-activity and drug discovery related studies.