Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from ß cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis. This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

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.

Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides.

A novel and convenient method for the synthesis of C-terminally branched collagen-model peptides has been achieved using tricine (N-[tris(hydroxymethyl)methyl]glycine) as a branching scaffold and 1,2-diaminoethane or 1,4-diaminobutane as a linker. The peptide sequence was incorporated directly onto the linker and scaffold during solid-phase synthesis without additional manipulations. The resulting branched triple-helical peptides exhibited comparable thermal stabilities to the parent, unbranched sequence, and served as substrates for matrix metalloproteinase-1 (MMP-1). The tricine-based branch reported herein represents the simplest synthetic scaffold for the convenient synthesis of covalently linked homomeric collagen-model triple-helical peptides.

Determining the Substrate Specificity of Matrix Metalloproteases using Fluorogenic Peptide Substrates.

A continuous assay method, such as the one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors toward native substrates, a variety of fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET) triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. Results of these studies have been valuable for providing insights into (a) the relative triple-helical peptidase activities of the various collagenolytic MMPs, (b) the collagen preferences of these MMPs, and (c) the relative roles of MMP domains and specific residues in efficient collagenolysis. The present chapter provides an overview of MMP FRET triple-helical substrates and describes how to construct and utilize these substrates.

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.

Identification of activators of methionine sulfoxide reductases A and B.

The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB.

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.

Development of second generation peptides modulating cellular adiponectin receptor responses.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

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.

Structure of a proteolytically resistant analogue of (NLys)5SFTI-1 in complex with trypsin: evidence for the direct participation of the Ser214 carbonyl group in serine protease-mediated proteolysis.

Peptide-peptoid hybrids are found to be potent inhibitors of serine proteases. These engineered peptidomimetics benefit from both types of units of the biopolymeric structure: the natural inhibitor part serves as a good binding template, while the P1-positioned peptoid component provides complete resistance towards proteolysis. In this report, the mechanism of proteolytic resistance of a P1 peptoid-containing analogue is postulated based on the crystal structure of the (NLys)(5)-modified sunflower trypsin inhibitor SFTI-1 in complex with bovine trypsin solved at 1.29 Šresolution. The structural differences between the (NLys)(5)SFTI-1-trypsin complex and the native SFTI-1-trypsin complex are surprisingly small and reveal the key role of the carbonyl group of the Ser214 residue of the enzyme, which is crucial for binding of the inhibitor and plays a crucial role in proteolysis mediated by serine proteases. The incorporated NLys5 peptoid residue prevents Ser214 from forming a hydrogen bond to the P1 residue, and in turn Gln192 does not form a hydrogen bond to the carbonyl group of the P2 residue. It also increases the distance between the Ser214 carbonyl group and the Ser195 residue, thus preventing proteolysis. The hybrid inhibitor structure reported here provides insight into protein-protein interaction, which can be efficiently and selectively probed with the use of peptoids incorporated within endogenous peptide ligands.

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.

Dissociation of antimicrobial and hemolytic activities of gramicidin†S through N-methylation modification.

ß-Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug-resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of ß-sheet AMPs, we hypothesize that N-methylation of the intramolecular hydrogen bond(s)-forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model ß-sheet antimicrobial peptide, gramicidin S (GS), to study the N-methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N-methylated GS analogues by replacement of residues at the ß-strand and ß-turn regions with N-methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC50 values increased fivefold compared with that of GS, when the internal hydrogen-bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N-methylalanine in the ß-turn region. However, analogues containing N-methylleucine at ß-strand and N-methylalanine at ß-turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N-methylated GS analogues using (1)H NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N-methylation of the internal hydrogen bond-forming amide affected the conformation, backbone shape, and side chain orientation of GS.

Introduction to peptide synthesis.

A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

Effects of cyclic lipodepsipeptide structural modulation on stability, antibacterial activity, and human cell toxicity.

Bacterial infections are becoming increasingly difficult to treat due to the development and spread of antibiotic resistance. Therefore, identifying novel antibacterial targets and new antibacterial agents capable of treating infections by drug-resistant bacteria is of vital importance. The structurally simple yet potent fusaricidin or LI-F class of natural products represents a particularly attractive source of candidates for the development of new antibacterial agents. We synthesized 18 fusaricidin/LI-F analogues and investigated the effects of structure modification on their conformation, serum stability, antibacterial activity, and toxicity toward human cells. Our findings show that substitution of an ester bond in depsipeptides with an amide bond may afford equally potent analogues with improved stability and greatly decreased cytotoxicity. The lower overall hydrophobicity/amphiphilicity of amide analogues in comparison with their parent depsipeptides, as indicated by HPLC retention times, may explain the dissociation of antibacterial activity and human cell cytotoxicity. These results indicate that amide analogues may have significant advantages over fusaricidin/LI-F natural products and their depsipeptide analogues as lead structures for the development of new antibacterial agents.

A practical synthesis of N α-Fmoc protected L-threo-ß-hydroxyaspartic acid derivatives for coupling via α- or ß-carboxylic group.

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available D: ,L: -tHyAsp racemic mixture by co-crystallization with L: -Lys, followed by ion exchange chromatography yielding enantiomerically pure L: -tHyAsp and D: -tHyAsp, and their selective orthogonal protection. In this way N ( α )-Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or ß-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via ß-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.

Peptidomimetics: Fmoc solid-phase pseudopeptide synthesis.

Peptidomimetic modifications or cyclization of linear peptides are frequently used as attractive methods to provide more conformationally constrained and thus more stable and bioactive peptides. Among numerous peptidomimetic approaches described recently in the literature, particularly attractive are pseudopeptides or peptide bond surrogates in which peptide bonds have been replaced with other chemical groups. In these peptidomimetics the amide bond surrogates possess three-dimensional structures similar to those of natural peptides, yet with significant differences in polarity, hydrogen bonding capability, and acid-base character. The introduction of such modifications to the peptide sequence is expected to completely prevent protease cleavage of amide bond and significantly improve peptides' metabolic stability. In this chapter we consider Fmoc solid-phase synthesis of peptide analogs containing the amide surrogate that tend to be isosteric with the natural amide. This includes synthesis of peptidosulfonamides, phosphonopeptides, oligoureas, depsides, depsipeptides, and peptoids.