Ligand reaction-based fluorescent peptide probes for the detection of Cu2+ and glutathione

Copper is a critical element in both human and animal metabolic processes. Its role includes supporting connective tissue cross-linking, as well as iron and lipid metabolism; at the same time, copper is also a toxic heavy metal that can cause harm to both the environment and human health. Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine combined with sulfhydryl groups. Its properties include acting as an antioxidant and facilitating integrative detoxification. GSH is present in both plant and animal cells and has a fundamental role in maintaining living organisms. GSH is the most abundant thiol antioxidant in the human body. It exists in reduced and oxidized forms within cells and provides significant biochemical functions, such as regulating vitamins such as vitamins D, E, and C, and facilitating detoxification. A fluorescent probe has been developed to detect copper ions selectively, sensitively, and rapidly. This report outlines the successful work on creating a peptide probe, TGN (TPE-Trp-Pro-Gly-Cln-His-NH₂), with specific Cu²⁺ detection capabilities, and a significant fluorescence recovery occurred with the addition of GSH. This indicates that the probe can detect Cu²⁺ and GSH concurrently. The detection limit for Cu²⁺ in the buffer solution was 264 nM (R² = 0.9992), and the detection limit for GSH using the TGN-Cu²⁺ complex was 919 nM (R² = 0.9917). The probe exhibits high cell permeability and low biotoxicity that make it ideal for live cell imaging in biological conditions. This peptide probe has the capability to detect Cu²⁺ and GSH in biological cells.     

Synthesis and evaluation of new iRGD peptide analogs for tumor optical imaging

Recently, a disulfide-based cyclic RGD peptide called iRGD, that is, c(CRGDKGPDC), has been reported to interact with both integrin and neuropilin-1 receptors for cellular and deep tissue penetration to improve the imaging sensitivity and therapeutic efficacy. In this study, two new near-infrared fluorescent iRGD conjugates, that is, Ac-Cys(IRDye®800CW)-iRGD (1), and its dual labeling analog DOTA-Cys(IRDye®800CW)-iRGD (2) were synthesized via the specific mercapto-maleimide reaction for tumor imaging. Both 1 and 2 showed significant tumor localization in optical imaging of MDA-MB-435 tumor-bearing mice. The potential of such iRGD compounds in tumor-targeted imaging and drug delivery deserves further exploration.     

Synthesis and evaluation of indium-111-labeled imidazothiadiazole sulfonamide derivative for single photon emission computed tomography imaging targeting carbonic anhydrase-IX

Carbonic anhydrase-IX (CA-IX) is a zinc enzyme overexpressed in the hypoxic regions of many types of solid tumors; therefore, in vivo imaging of CA-IX may contribute to cancer diagnosis. In this study, we newly designed and synthesized an ¹¹¹In-labeled CA-IX imaging agent based on an imidazothiadiazole sulfonamide (IS) scaffold conjugated with a chelating moiety, DO3A ([¹¹¹In]DO3A-IS1), and evaluated its utility for imaging of CA-IX high-expressing tumors. [¹¹¹In]DO3A-IS1 was successfully synthesized at a 76% radiochemical yield by reacting its precursor with ¹¹¹InCl₃ in acetate buffer. In in vitro assays, [¹¹¹In]DO3A-IS1 showed marked stability in murine plasma and greater binding to CA-IX high-expressing (HT-29) cells (118 ± 21% initial dose/mg protein) than CA-IX low-expressing (MDA-MB-231) cells (1.4 ± 0.3% initial dose/mg protein). Moreover, in an in vivo biodistribution assay, [¹¹¹In]DO3A-IS1 showed marked accumulation in the HT-29 tumor (8.71 ± 1.41% injected dose/g at 24 h postinjection). In addition, in a single photon emission computed tomography (SPECT) study, [¹¹¹In]DO3A-IS1 clearly and selectively visualized the HT-29 tumor as compared with the MDA-MB-231 tumor. These results indicate that [¹¹¹In]DO3A-IS1 may serve as a useful SPECT imaging agent with the novel scaffold targeting CA-IX.     

Quantitative evaluation of the cell penetrating properties of an iodinated Tyr-l-maurocalcine analog

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to ¹²⁵I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9 × 10⁻⁷ μl. ¹²⁵I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of ¹²⁵I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5 min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24 h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Synthesis and evaluation of fluorescent probes for the detection of calpain activity

Two new probes for the detection of calpain I activity based on fluorescence resonance energy transfer technology have been synthesized and evaluated. The probes incorporated the cleavage site present in alpha-spectrin, a naturally occurring substrate of calpain I. The design of the internally quenched substrates is such that the calpain-sensitive bond of the peptides (between the Tyr-Gly residues) is located centrally between the donor and the quencher chromophores. The calpain assay protocol is capable of detecting enzymatic activity in the nanomolar region.     

Enzyme-targeted fluorescent small-molecule probes for bacterial imaging

Molecular imaging methods to visualize myriad biochemical processes in bacteria have traditionally been dependent upon molecular biology techniques to incorporate fluorescent biomolecules (e.g., fusion proteins). Such methods have been instrumental in our understanding of how bacteria function but are not without drawbacks, including potential perturbation to native protein expression and function. To overcome these limitations, the use of fluorescent small-molecule probes has gained much attention. Here, we highlight examples from the recent literature that showcase the utility of small-molecule probes for the fluorescence imaging of bacterial cells, including electrophilic, metabolic, and enzyme-activated probes. Although the use of these types of compounds for bacterial imaging is still relatively new, the selected examples demonstrate the exciting potential of these critical tools in the exploration of bacterial physiology.     

A novel chimeric peptide with antimicrobial activity

Beta‐lactamase‐mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co‐administration of beta‐lactam type antibiotics and beta‐lactamase inhibitors. Antimicrobial peptides are promising broad‐spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46–Tyr51 beta‐hairpin loop of beta‐lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta‐lactamase. Here, our goal was to modify this peptide for improved beta‐lactamase inhibition and cellular uptake. Motivated by the cell‐penetrating pVEC sequence, which includes a hydrophobic stretch at its N‐terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N‐terminus to facilitate uptake. Activity measurements of the peptide based on the 45–53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta‐lactamase inhibitor with a K_i value of 58 μM. Incubation of beta‐lactam‐resistant cells with peptide decreased the number of viable cells, while it had no effect on beta‐lactamase‐free cells, indicating that this peptide had antimicrobial activity via beta‐lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N‐terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta‐lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.     

Comparative analysis reveals amino acids critical for anticancer activity of peptide CIGB‐552

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB‐552, a novel cell‐penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor‐bearing mice. Studies of protein–peptide interactions have shown that COMMD1 protein is a major mediator of CIGB‐552 antitumor activity. Furthermore, a typical serine‐protease degradation pattern for CIGB‐552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB‐552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB‐552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF‐7 cells. None of the analyzed metabolites proved to be as effective as CIGB‐552 in promoting apoptosis in MCF‐7. Taking into account these results, it seemed important to examine their cell‐penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding‐dependent process, is impaired as well as metabolite–COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB‐552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5- iodoindolin-2-one (IPI-1) showed the highest binding affinity (K_d?=?68.3?nM) to recombinant human survivin, as determined by quartz crystal microbalance (QCM). In vitro studies demonstrated that the [¹²⁵I]IPI-1 binding in survivin-positive MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A cells. In addition, uptake of [¹²⁵I]IPI-1 by MDA-MB-231 cells decreased in a dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is indicative of specific binding of [¹²⁵I]IPI-1 to cellular survivin protein in vitro. Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the moderate uptake of [¹²⁵I]IPI-1 in the tumor tissue (1.37%?ID/g) at 30?min that decreased to 0.32%?ID/g at 180?min. Co-injection of S12 (2.5?mg/kg) slightly reduced tumor uptake and the tumor/muscle ratio of [¹²⁵I]IPI-1. Although further structural modifications are necessary to improve pharmacokinetic properties, our results indicate that PI derivatives may be useful as tumor-imaging probes targeting survivin.     

Synthesis and evaluation of a novel Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel ^99mTc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m (^99mTc)-labeled probe ^99mTc-SD32. The uptake of ^99mTc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. ^99mTc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that ^99mTc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that ^99mTc-SD32 would be a promising agent for in vivo hypoxia imaging.     

Synthesis and bioevaluation of radioiodinated nitroimidazole hypoxia imaging agents by one-pot click reaction

Eight radioiodinated 2-nitroimidazole derivatives for use as hypoxia imaging agents were synthesized by one-pot click reaction using four azides, two alkynes, and [¹³¹I]iodide ions and evaluated by hypoxic cellular uptake and biodistribution experiments. The results suggested that radiotracers with suitable partition coefficients (log P: −0.2–1.2) were more likely to have higher hypoxic cellular uptake. Among these eight molecules, [¹³¹I]15 ([¹³¹I]-(5-iodo-1-(2-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)ethyl)-4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazole)) had a suitable log P (0.05 ± 0.03) and contained two 2-nitroimidazole groups. The hypoxic/aerobic cellular uptake ratio of [¹³¹I]15 was 4.4 ± 0.5, and the tumor/blood (T/B) and tumor/muscle (T/M) ratios were 2.03 ± 0.45 and 6.82 ± 1.70, respectively. These results suggested that [¹³¹I]15 was a potential hypoxia imaging agent.     

Synthesis and activity of tumor-homing peptide iRGD and histone deacetylase inhibitor valproic acid conjugate

In this Letter, we present a concise strategy to prepare a conjugate of the tumor homing peptide iRGD and histone deacetylase inhibitor valproic acid, VPA–GFLG-iRGD. The conjugate VPA–GFLG-iRGD and a mixture of VPA and GFLG-iRGD have shown similar cytotoxicity against DU-145 prostate cancer cells. However, the treatment of DU-145 cells with conjugate VPA–GFLG-iRGD resulted in a decreased percentage of cells in the G2 phase, whereas the exposure of a mixture of VPA and GFLG-iRGD led to an increased percentage of cells in the G2 phase. We also found that GFLG-iRGD possessed cytotoxicity at the tested concentrations.     

Fluorogenic probes for imaging cellular phosphatase activity

The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress toward the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-infrared (near-IR) excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.     

Dual emissive bispyrene peptide probes for highly sensitive measurements of trypsin activity and evaluation of trypsin inhibitors

Peptide substrates were double labeled with pyrenes to prepare fluorescent probes for highly sensitive detection of protease activity and evaluation of protease inhibitors using pyrene monomer/excimer signals. Two proximate pyrene moieties formed excited state dimers in the probes, and these pyrene excimer formations were dissociated by tryptic digestion. The specificity constant of the optimum bispyrene peptide probe was 2.7 times higher than that of the conventional peptide-4-methylcoumarin amide. Moreover, our probe had high sensitivity with an estimated detection limit for trypsin of 4.11?pM. The half maximal inhibitory concentration and dissociation constant of the Bowman–Birk inhibitor were successfully estimated.     

Synthesis of fluorescent and low cytotoxicity phenol formaldehyde resin (PFR)@Ag composites for cell imaging and antibacterial activity

Ag nanoparticles (NPs) were loaded onto the surface of phenol formaldehyde resin (PFR) NPs without any reducing agent. The as‐synthesized PFR@Ag composites have low cytotoxicity, which makes them promising antibacterial agents. Furthermore, the good fluorescence of PFR could be used for cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Dynamic PET and SPECT imaging with radioiodinated,amyloid-reactive peptide p5 in mice: A positive role for peptide dehalogenation

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72 h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients.     

Synthesis and characterization of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, ^99mTc sestamibi and ^99mTc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. ¹²³I has a similar energy as ^99mTc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an ¹²³I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with ¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog ¹²⁷I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n = 6) and 24 h (n = 8) post injection (p.i.). Results: ¹²³I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8–111 GBq/μmol; 400–3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7′-position, with removal of the 6′,7′-double bond, and addition of a hydroxy group at 6′-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01 ± 0.48%ID/g) and high heart to liver (2.98 ± 0.93), heart to lung (4.11 ± 1.04) and heart to blood (8.37 ± 3.97) ratios. At 24 h p.i., the majority of ¹²³I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. Conclusion: ¹²³I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.     

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine,a cell‐penetrating peptide

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l )‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l )‐Trp‐Arg₈ and Br‐vindoline‐(d )‐Trp‐Arg₈ suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l )‐Trp was more active than conjugate with the (d )‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l )‐Trp‐Arg₈ exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.