Multicomponent Peptide Stapling as a Diversity-Driven Tool for the Development of Inhibitors of Protein–Protein Interactions

Stapled peptides are chemical entities in-between biologics and small molecules, which have proven to be the solution to high affinity protein–protein interaction antagonism, while keeping control over pharmacological performance such as stability and membrane penetration. We demonstrate that the multicomponent reaction-based stapling is an effective strategy for the development of α-helical peptides with highly potent dual antagonistic action of MDM2 and MDMX binding p53. Such a potent inhibitory activity of p53-MDM2/X interactions was assessed by fluorescence polarization, microscale thermophoresis, and 2D NMR, while several cocrystal structures with MDM2 were obtained. This MCR stapling protocol proved efficient and versatile in terms of diversity generation at the staple, as evidenced by the incorporation of both exo- and endo-cyclic hydrophobic moieties at the side chain cross-linkers. The interaction of the Ugi-staple fragments with the target protein was demonstrated by crystallography.     

Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein−Chromophore Interactions

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M⁻¹ cm⁻¹, is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.     

Fluorescent Mesoporous Nanoparticles for β-Lactamase Screening Assays

We present a sensitive and rapid screening method for the determination of β-lactamase activity of antibiotic-resistant bacteria, by designing a pH-sensitive fluorescent dye-doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β-lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye-doped mesoporous nanoparticles not only enhanced the β-lactamase-catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β-lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β -lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β-lactamase activity was as low as 7.8×10⁻⁴ U/mL, which was determined within two hours.     

Versatile Fluorescent Probes for Imaging the Superoxide Anion in Living Cells and In Vivo

The superoxide anion (O₂^.−) is widely engaged in the regulation of cell functions and is thereby intimately associated with the onset and progression of many diseases. To ascertain the pathological roles of O₂^.− in related diseases, developing effective methods for monitoring O₂^.− in biological systems is essential. Fluorescence imaging is a powerful tool for monitoring bioactive molecules in cells and in vivo owing to its high sensitivity and high temporal-spatial resolution. Therefore, increasing numbers of fluorescent imaging probes have been constructed to monitor O₂^.− inside live cells and small animals. In this minireview, we summarize the methods for design and application of O₂^.−-responsive fluorescent probes. Moreover, we present the challenges for detecting O₂^.− and suggestions for constructing new fluorescent probes that can indicate the production sites and concentration changes in O₂^.− as well as O₂^.−-associated active molecules in living cells and in vivo.     

Development of Enantioselective Fluorescent Sensors for Chiral Recognition

Novel chiral compounds have been synthesized for the enantioselective fluorescent recognition of alpha-hydroxycarboxylic acids and amino acids. By introducing dendritic branches to the chiral receptor units, the fluorescence signals of the receptors are significantly amplified because of the light-harvesting effect of the dendritic structure. This has greatly increased the sensitivity of the sensors in the fluorescent recognition. Highly enantioselective fluorescent responses have also been ac…     

Development of a New Clean-up Method for the Determination of 5-methyl-tetrahydrofolate in Milk Samples Using a Sol-Gel β-Lactoglobulin Column

The specific forms of folic acid, a vitamin of the B-group, are generally determined in foods after sample clean-up using affinity columns containing folate binding protein covalently bound to a solid support material. The new approach described in the present paper is to isolate the target analyte by using the less costly milk protein β-lactoglobulin, entrapped in the pores of a sol-gel glass. Sol-gel β-lactoglobulin columns were included in a clean-up procedure developed to determine 5-methyl-tetrahydrofolate in milk samples. Concentrations of 5-methyl-tetrahydrofolate in milk samples with different fat contents ranged from 31 to 70 ng/ml. Repeated use of the β-lactoglobulin columns for sample preparation resulted in a decrease of analyte recovery. However, due to the high column to column reproducibility good results were obtained when the standard addition method was performed with columns which had already been used for the same number of sample clean-ups.     

Nonaqueous Emulsions – A Versatile Tool for New Types of Functional Nanoparticles

Summary : The preparation of functional polymer latex particles is usually carried out in aqueous heterogeneous systems, i.e. for example in emulsion or mini-emulsion polymerization. Due to the presence of water, moisture sensitive reactions like step growth polymerizations or metal catalyzed reactions can not be accomplished without side reactions and / or decomposition. In order to avoid these side reactions, different nonaqueous emulsion systems have been developed. According to the desired polymerization procedure, these systems consist of a nonpolar organic phase surrounded by a perfluorinated solvent or of a polar organic phase which is dispersed in a nonpolar organic solvent. Both emulsions are stabilized by amphipolar block copolymers and result in long time stable particle dispersions. The resulting dispersions yield particles with narrow size distributions and – depending on the reaction conditions – diameters down to tens of nanometers. This technique allows the formation of particles consisting of numerous different classes of polymers, e.g. polyurethanes, polyesters, polyolefins etc. and the formation of more complex morphologies such as core shell structures.     

Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.      

Rational Design of the β-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.     

Development of New Capillary Electrophoresis-Based Techniques for Protein Analysis

With the sequencing of human genome almost complete, human genome project enters the postgenome-sequencing era. Compared to genomics, the analysis of proteome is rather difficult since in human cells there are around 200 000 proteins, which are expressed at any time at different levels     

Fluorogenic Probe Using a Mislow–Evans Rearrangement for Real-Time Imaging of Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) mediates the biology of wound healing, apoptosis, inflammation, etc. H₂O₂ has been fluorometrically imaged with protein- or small-molecule-based probes. However, only protein-based probes have afforded temporal insights within seconds. Small-molecule-based electrophilic probes for H₂O₂ require many minutes for a sufficient response in biological systems. Here, we report a fluorogenic probe that selectively undergoes a [2,3]-sigmatropic rearrangement (seleno-Mislow-Evans rearrangement) with H₂O₂, followed by acetal hydrolysis, to produce a green fluorescent molecule in seconds. Unlike other electrophilic probes, the current probe acts as a nucleophile. The fast kinetics enabled real-time imaging of H₂O₂ produced in endothelial cells in 8 seconds (much earlier than previously shown) and H₂O₂ in a zebrafish wound healing model. This work may provide a platform for endogenous H₂O₂ detection in real time with chemical probes.     

An Activatable NIR-II Fluorescent Reporter for In Vivo Imaging of Amyloid-β Plaques

Fluorescence imaging in the second near-infrared (NIR-II) window holds great promise for in vivo visualization of amyloid-β (Aβ) pathology, which can facilitate characterization and deep understanding of Alzheimer's disease (AD); however, it has been rarely exploited. Herein, we report the development of NIR-II fluorescent reporters with a donor-π-acceptor (D-π-A) architecture for specific detection of Aβ plaques in AD-model mice. Among all the designed probes, DMP2 exhibits the highest affinity to Aβ fibrils and can specifically activate its NIR-II fluorescence after binding to Aβ fibrils via suppressed twisted intramolecular charge transfer (TICT) effect. With suitable lipophilicity for ideal blood–brain barrier (BBB) penetrability and deep-tissue penetration of NIR-II fluorescence, DMP2 possesses specific detection of Aβ plaques in in vivo AD-model mice. Thus, this study presents a potential agent for non-invasive imaging of Aβ plaques and deep deciphering of AD progression.     

Recognition of Protein Native Folds Using a Continuous Potential Function

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Development of a Protein Chip to Measure PKCβ Activity

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.     

Synthesis of Small-Molecule Fluorescent Probes for the In Vitro Imaging of Calcium-Activated Potassium Channel KCa3.1

Small-molecule probes for the in vitro imaging of K_Ca3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K_Ca3.1 channels, was chosen as the targeting component. BODIPY dyes 15 – 20 were synthesized and connected by a Cu^I-catalyzed azide–alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8 , yielding fluorescently labeled ligands 21 – 26 . The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K_Ca3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of K_Ca3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.     

Development and Validation of an HPTLC–Densitometric Method for Determination of ACE Inhibitors

A high-performance thin layer chromatographic method coupled with densitometric analysis has been developed for measurement of benazepril and cilazapril, both pure and in their commercial dosage forms. The active substances were extracted from tablets with methanol (mean recovery 102%) and chromatographed on silica gel 60 F₂₅₄ HPTLC plates in horizontal chambers with ethyl acetate–acetone–acetic acid–water, 8:2:0.5:0.5 (v/v), as mobile phase. Chromatographic separation of these ACE inhibitors was followed by UV densitometric quantitation at 215 nm. Calibration plots were constructed in the range 0.4 to 2.0 g L^–1 for benazepril (2.0–10.0 g spot^–1) and from 0.5 to 1.5 g L^–1 for cilazapril (4.0–12.0 g spot^–1) with good correlation coefficients (r 0.990). The method was used to determine benazepril and cilazapril in pharmaceutical preparations with satisfactory precision (1.4% < RSD < 5.6%) and accuracy (1.7 < RE < 5.1).     

Simultaneous Multi-Wavelength UV-Visible Detection for HPLC Using a Diode-Array Spectrophotometer—A Versatile Software Package

Abstract

A BASIC program was developed to monitor absorbance simultaneously at various wavelengths using an HP8451A Diode Array Spectrophotometer linked to a HPLC system and equipped with a flow cell. The program measures a maximum of ten wavelengths and takes a maximum of 240 measurements for each wavelength, using only 16K of memory and a dual disc drive. The program includes the options of replotting using a different ordinate scale and saving the numerical data on disc.     

Versatile Post-synthetic Modifications of Helical β-Peptide Foldamers Derived from a Thioether-Containing Cyclic β-Amino Acid

We introduce a novel cyclic β-amino acid, trans-(3S,4R)-4-aminotetrahydrothiophene-3-carboxylic acid (ATTC), as a versatile building block for designing peptide foldamers with controlled secondary structures. We synthesized and characterized a series of β-peptide hexamers containing ATTC using various techniques, including X-ray crystallography, circular dichroism, and NMR spectroscopy. Our findings reveal that ATTC-containing foldamers can adopt 12-helical conformations similar to their isosteres and offer the possibility of fine-tuning their properties via post-synthetic modifications. In particular, chemoselective conjugation strategies demonstrate that ATTC provides unique post-synthetic modification opportunities, which expand their potential applications across diverse research areas. Collectively, our study highlights the versatility and utility of ATTC as an alternative to previously reported cyclic β-amino acid building blocks in both structural and functional aspects, paving the way for future research in the realm of peptide foldamers and beyond.