Fluorescent Peptide Research Articles

Chemisch stabile Diazopeptide als selektive Sonden für Cysteinproteasen in lebenden Zellen

AbstractVon Diazopeptiden wurden schon früher berichtet, sie haben aber aufgrund ihrer hohen Reaktivität bis heute kein breites Einsatzgebiet gefunden. Wir berichten hier von der Herstellung, den Eigenschaften und Anwendungen chemisch stabiler interner Diazopeptide. Peptidylphosphoranylidenester und ‐amide reagieren mit Triflylazid hauptsächlich zu neuartigen 4,5‐disubstituierten 1,2,3‐Triazolylpeptiden. Nonaflylazid hingegen liefert Diazopeptide, die als Amide bei pH 1–14 und als Ester bei pH 1–8 chemisch stabil sind. Somit sind durch Festphasenpeptidsynthese hergestellte Diazopeptide auch während der finalen Entschützung mit 95 % Trifluoressigsäure stabil. Diazopeptide mit der Erkennungssequenz von Caspase‐3 wurden als spezifische, kovalente und irreversible Inhibitoren dieses Enzyms bei niedrigen nanomolaren Konzentrationen identifiziert. Ein fluoreszierendes Diazopeptid drang in lebende Zellen ein und ermöglichte mikroskopische Visualisierung und Quantifizierung von Apoptose durch Durchflusszytometrie. Diazopeptide stellen somit eine neue Klasse aktivitätsbasierter Sonden und Enzyminhibitoren zum Einsatz in der chemischen Biologie und medizinischen Chemie dar.

Chemically Stable Diazo Peptides as Selective Probes of Cysteine Proteases in Living Cells.

Diazo peptides have been described earlier, however, due to their high reactivity have not been broadly used until today. Here, we report the preparation, properties, and applications of chemically stable internal diazo peptides. Peptidyl phosphoranylidene-esters and amides were found to react with triflyl azide primarily to novel 3,4-disubstituted triazolyl-peptides. Nonaflyl azide instead furnished diazo peptides, which are chemically stable from pH 1-14 as amides and from pH 1-8 as esters. Thus, diazo peptides prepared by solid phase peptide synthesis were stable to final deprotection with 95 % trifluoroacetic acid. Diazo peptides with the recognition sequence of caspase-3 were identified as specific, covalent, and irreversible inhibitors of this enzyme at low nanomolar concentrations. A fluorescent diazo peptide entered living cells enabling microscopic imaging and quantification of apoptotic cells via flow cytometry. Thus, internal diazo peptides constitute a novel class of activity-based probes and enzyme inhibitors useful in chemical biology and medicinal chemistry.

Tracking and measuring local protein synthesis in vivo.

Detecting when and how much of a protein molecule is synthesized is important for understanding cell function, but current methods either cannot be performed in vivo or have poor temporal resolution. Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use different color split fluorescent proteins to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel to continually reset the time of detection of synthesis events.

Functional 20S Proteasomes in Retroviruses: Evidence in Favor.

Proteasomes are barrel-like cellular protein complexes responsible for the degradation of most intracellular proteins. Earlier, it has been shown that during assembly, hundreds of different cellular proteins are incorporated into retro-and herpes viruses. Among detected cellular proteins, there were different proteasome subunits (PS). Previous reports postulated the incorporation of 20S proteasome subunits and subunits of proteasome regulator complexes inside retroviruses. Here, we demonstrated the association of functional 20S proteasome with gammaretroviruses, betaretroviruses, and lentiviruses. Cleaved proteasome subunits β1, β2 and β5 were detected in tested viruses. Using fluorescent peptides and a cell-permeable proteasome activity probe, proteasome activity was detected in endogenous and exogenous retroviruses, including recombinant HIV-1. Taken together, our data favors the insertion of functional proteasomes into the retroviruses during assembly. The possible role of proteasomes in retroviruses is discussed.

Active Quantum Biomaterials-Enhanced Microrobots for Food Safety.

Timely disruptive tools for the detection of pathogens in foods are needed to face global health and economic challenges. Herein, the utilization of quantum biomaterials-enhanced microrobots (QBEMRs) as autonomous mobile sensors designed for the precise detection of endotoxins originating from Salmonella enterica (S. enterica) as an indicator species for food-borne contamination globally is presented. A fluorescent molecule-labeled affinity peptide functions as a specific probe, is quenched upon binding to the surface of QBEMRs. Owing to its selective affinity for endotoxin, in the presence of S. enterica the fluorescence is restored and easy to observe and quantifies optical color change to indicate the presence of Salmonella. The devised approach is designed to achieve highly sensitive detection of the S. enterica serovar Typhimurium endotoxin with exquisite selectivity through the utilization of QBEMRs. Notably, no fluorescence signal is observed in the presence of endotoxins bearing similar structural characteristics, highlighting the selectivity of the approach during food sample analysis. Technically, the strategy is implemented in microplate readers to extend microrobots-based approaches to the routine laboratory. This new platform can providefast and anticipated results in food safety.

Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria.

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Interleukin-1 receptor-dependent and -independent caspase-1 activity in retinal cells mediated by receptor interacting protein 2.

Inflammation and cell death play an important role in the pathogenesis of diabetic retinopathy. Previously we observed sustained activation of pro-inflammatory caspase-1 in retinas of diabetic animals and patients. In this study, we aimed to look at mechanisms underlying chronic caspase-1 activation in vitro and in vivo. Non-diabetic and diabetic wild type and IL-1 receptor (IL-1R1) knockout mice were used for in vivo experiments. Diabetes was induced using STZ (streptozotocin). Human Müller cells were used for in vitro studies. Cells were treated with either 5mM or 25mM glucose or interleukin-1beta (IL-1β) in the presence or absence of IL-1 receptor antagonist (IL-1ra) or siRNA against RIP2 (receptor interacting protein-2) for up to 96h. Outcome measurements to assess Müller cell functions included measurements of caspase-1 activity using a fluorescence peptide substrate, production of IL-1β by Elisa, and cell death using trypan blue exclusion assays. Our in vivo results demonstrate that caspase-1 activation progresses from an IL-1R1 independent mechanism at 10weeks of diabetes to an IL-1R1 dependent mechanism at 20weeks indicating that feedback through IL-1R1 is crucial for sustained caspase-1 activity in retinas of mice. A similar hyperglycemia-mediated caspase-1/IL-1β/IL-1R1 feedback signaling was detected in vitro in human Müller cells which was prevented by treatment with IL-1ra. Our data also indicate that hyperglycemia induces caspase-1 activation initially but IL-1β sustains caspase-1 activation via caspase-1/IL-1β/IL-1R1 feedback and we identified RIP2 as mediator for both hyperglycemia- and IL-1β-induced caspase-1 activation. Activation of caspase-1/IL-1β/IL-1R1 feedback signaling caused Müller cell death which was prevented by RIP2 knockdown. We conclude that any intervention in caspase-1/IL-1β/IL-1R1 feedback signaling presents novel therapeutic options for the treatment of diabetic retinopathy.

The α3β4 nAChR tissue distribution identified by fluorescent α-conotoxin [D11A]LvIA

α3β4, a vital subtype of neuronal nicotinic acetylcholine receptors (nAChRs), is widely distributed in the brain, ganglia, and adrenal glands, associated with addiction and neurological diseases. However, the lack of specific imaging tools for α3β4 nAChRs has hindered the investigation of their tissue distribution and functions. [D11A]LvIA, a peptide derived from marine cone snails, demonstrates high affinity and potency for α3β4 nAChRs, making it a valuable pharmacological tool for studying this receptor subtype. In this study, three fluorescent conjugates of [D11A]LvIA were synthesized using 6-TAMRA-SE (R), Cy3-NHS-ester (Cy3), and BODIPY-FL NHS ester (BDP) dyes. The electrophysiological activities were assessed in Xenopus laevis oocytes by two-electrodes voltage clamp (TEVC). [D11A]LvIA-Cy3 and [D11A]LvIA-BDP show improved selectivity and affinity, with IC50 values of 512.70 nM and 343.50 nM, respectively, and [D11A]LvIA-Cy3 exhibits better stability in cerebrospinal fluid (CSF). Utilizing [D11A]LvIA-Cy3, we successfully visualized the distribution of α3β4 nAChRs in rat trigeminal ganglia, retina, adrenal glands, and various brain regions. This novel fluorescent peptide provides a significant pharmacological tool for the exploration and visualization in-situ distribution of α3β4 nAChRs in different tissues and also assists in clarifying the function.

Abstract B054: Protease activity as a biomarker and potential target among pancreatic cancer patients

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

Fluorescent Peptides Sequester Redox Copper to Mitigate Oxidative Stress, Amyloid Toxicity, and Neuroinflammation.

Alzheimer's disease is a progressive neurodegenerative disorder that significantly contributes to dementia. The lack of effective therapeutic interventions presents a significant challenge to global health. We have developed a set of short peptides (PNGln) conjugated with a dual-functional fluorophoric amino acid (NGln). The lead peptide, P2NGln, displays a high affinity for Cu2+, maintaining the metal ion in a redox-inactive state. This mitigates the cytotoxicity generated by reactive oxygen species (ROS), which are produced by Cu2+ under the reductive conditions of Asc and Aβ16 or Aβ42. Furthermore, P2NGln inhibits both Cu-dependent and -independent fibrillation of Aβ42, along with the subsequent toxicity induced by Aβ42. In addition, P2NGln exhibits inhibitory effects on the production of lipopolysaccharide (LPS)-induced ROS and reactive nitrogen species (RNS) in microglial cells. In vitro and cellular studies indicate that P2NGln could significantly reduce Aβ-Cu2+-induced ROS production, amyloid toxicity, and neuroinflammation, offering an innovative strategy against Alzheimer's disease.

Ion transfer mechanism of fluorescence-labeled octa-arginine on model biomembrane surfaces

The interfacial behavior of a synthesized fluorescent polycationic cell-penetrating peptide, octa-arginine covalently labeled with carboxyfluorescein (FAM-R8), was investigated through spectroelectrochemical analysis at the bare and phospholipid-modified water|1,2-dichloroethane (DCE) interfaces. The potential modulated fluorescence (PMF) analysis indicated that the adsorption of FAM-R8 from the aqueous side of the bare interface occurred in the wide potential range at lower FAM-R8 concentrations, while the ion transfer across the interface proceeded at the positive edge of the polarizable potential window as the concentration increased. The complicated interfacial reaction behavior of FAM-R8 was further observed at the biomimetic interface in the presence of a phospholipid layer, where the adsorption potential of FAM-R8 was positively shifted as compared with the bare interface. Moreover, the ion transfer process of FAM-R8 became predominant when the surface excess of FAM-R8 was substantially increased at the biomimetic interface. The FAM-R8 concentration and its interaction with the phospholipid layer were found to play an important role for the membrane permeation mechanism.

Enhancing peptide and PMO delivery to mouse airway epithelia by chemical conjugation with the amphiphilic peptide S10

Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs, and the endosomal entrapment phenomenon which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer (PMO) improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogenous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a PEG linker was the most beneficial strategy compared to direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

A Novel Enzyme-Based Electrochemical Biosensor for Sensitive Detection of Hydrogen Peroxide Based on the Fluorescent Peptide Self-Assembled Nanomaterials

A Novel Enzyme-Based Electrochemical Biosensor for Sensitive Detection of Hydrogen Peroxide Based on the Fluorescent Peptide Self-Assembled Nanomaterials

Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 – implications for post COVID-19 conditions

BackgroundSevere COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC).MethodsThe IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10.ResultsForty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals.ConclusionsThis work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

Ru(II)-Catalyzed C(sp2)-H Activation Annulation: Synthesis of Fluorescent Benzoisoquinolonyl Acetate/Peptides from N-Arylamides and Ethyne at Room Temperature.

Benzoisoquinolones are aryl ring extended isoquinolinone derivatives, which are constituents of alkaloid natural products. This report describes the synthesis of novel benzoisoquinolone amino acid/peptide derivatives from the respective N-aryl amino esters/peptides through Ru-catalyzed C(sp2)-H annulation at room temperature. The N-terminal amide acts as an intrinsic directing group and coordinates with the active Ru(II) catalyst for the C-H bond activation and annulation of the aryl ring to produce benzoisoqunolone derivatives. Importantly, these benzoisoquinolinones exhibit fluorescence (QY ∼35%) in protic polar solvents, possibly due to charge transfer, and exhibit cell internalization to the cell nucleus without any significant cytotoxicity to human cell lines (HEK293T). Hence, our results are exceptional to transform standard amino acids/peptides into fluorescent peptides at room temperature in the late stage, which could be applicable for tracking specific target peptides by fluorescence microscopy.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

Novel strategies for enhanced fluorescence visualization of glioblastoma tumors based on HPMA copolymers conjugated with tumor targeting and/or cell‐penetrating peptides

AbstractNano‐sized polymer systems are often used as carriers for drugs and contrast agents to increase circulation time and solubility and to reduce possible side effects. These nanomedicines usually accumulate in tumor tissue due to the enhanced permeability and retention (EPR) effect. However, a targeting group may be attached to the polymer carrier in addition to the active substance to further increase tumor accumulation and specificity. In this study, the oligopeptide sequence RGD was chosen to target αvβ3 integrins overexpressed in the tumor vasculature and on some tumor cells. A set of polymer conjugates bearing a fluorescent dye and RGD peptide of different structures (linear, cyclic, branched) was prepared for use in tumor diagnosis, with a potential future application in navigated surgery. The accumulation of the most promising candidate, a targeted fluorescent nanoprobe, increased by 35% in glioblastoma tumors compared to the non‐targeted control, which accumulated only due to the EPR effect. However, the administration of a polymer‐bound modified cilengitide as an antiangiogenic treatment did not show a beneficial effect in the suppression of angiogenesis.