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The prostate specific antigen (PSA), a serine protease with chymotrypsin-like activity, is predominantly expressed in the prostate and is considered as the most common marker in use to identify and follow the progress of prostate cancer. In addition, it is also now accepted as a marker for detecting semen in criminal cases. Here, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for detection of PSA enzymatic activity. The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane luminophore, that then undergoes efficient chemiexcitation to emit a green photon. The probe exhibits a significant turn-on response upon reaction with PSA and produces strong light emission signal with an extremely high signal-to-noise ratio. Comparison of the chemiluminescence probe with an analogous fluorescence probe showed superior detection capability in terms of response time and sensitivity. In addition, the probe was able to efficiently detect and image human semen traces on fabric, even after 3 days from sample preparation. The advantageous sensitivity and simplicity of a chemiluminescence assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer. It is expected that the new chemiluminescence probe would be broadly useful for numerous applications in which PSA detection or imaging is required.

Efficient radiolabeling reactions are important chemical tools in biomedical research especially in probe construction. Herein, three 18 F-labeled vinyl sulfones were prepared. In particular, 18 F-PEG1-VS (((2-(2-(fluoro- 18 F)ethoxy)ethyl)sulfonyl)ethane) could not only allow chemoselective labeling of bioactive molecules containing -XH (X = S, NH) groups, but also react with red blood cells both in vitro and in living mice for potential cell tracking applications. In addition, these hydrophilic agents were found to cross the blood brain barrier (BBB) efficiently and localize at the cerebellum region. In summary, 18 F-labeled vinyl sulfones provide a versatile platform for PET probe construction.

Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR 10 ) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.

Hg 2+ -sensitive carbon dots (CDs) were synthesized by microwave-assisted pyrolysis of citric acid, sodium fluoride, and urea. The CDs as a signal report unit and rhodamine B (RhB) as a reference were then encapsulated in a nanosphere of chitosan assembled by a nonsolvent-induced chitosan colloidal formation and in situ cross-linking to construct a ratiometric probe for Hg 2+ (chitosan-CDs-RhB). Interestingly, without any assistance from acids to improve the solubility of chitosan, the nanosphere containing CDs and RhB had an ultrasmall size of 9.7 nm with only approximately 1.1-nm-thick layers of chitosan enclosing one dot. In order to keep the residual functional groups on the nanosphere from compromising the fluorescence response of CDs to Hg 2+ , Co 2+ was used as a fluorescently intact metal ion to saturate the functional groups. The saturated chitosan-CDs-RhB was thus potentially developed for determining Hg 2+ in the fruit bodies and mycelia of edible and medicinal fungi. Limits of detection (LODs) of 2.24, 5.29, and 2.03 μM and recoveries in the ranges 98.3 to 101.8%, 99.5 to 104.6%, and 97.4 to 100.9% were estimated for the determination of Hg 2+ in the fruit bodies of Pleurotus ostreatus, Lentinus edodes, and Hypsizygus marmoreus, respectively. Chitosan-CDs-RhB was further developed as a fluorescent ratiometric probe for quantitatively determining intracellular Hg 2+ in fungal mycelia with a linear calibration curve of R Igreen/Ired  = - 0.145c + 1.69 within the range 0.013 to 0.356 μg g -1 . Graphical abstract An ultrasmall chitosan nanosphere encapsulating carbon dots and rhodamine B as a ratiometric probe for the determination of Hg 2+ .

Ferroptosis is an iron-dependent form of cell death resulting from loss or inhibition of cellular machinery that protects from the accumulation of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing function in normal cellular homeostasis, but certain cancers exploit and become highly dependent on specific nodes of the pathway, presumably to survive under conditions of increased oxidative stress and elevated labile ferrous iron levels. Here we introduce Ferroptosis Inducing Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe that couples Fenton-type reaction with ferrous iron to subsequent protein labeling via concomitant carbon-centered radical generation. We show that FIPC-1 induces ferroptosis in susceptible cell types and labels cellular proteins in an iron-dependent fashion. Use of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD) superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and uncovers new targets for the therapeutic modulation of ferroptosis.

There a few reports of rhodamine-based fluorescent sensors for selective detection of only Al 3+ , due to the challenge of identifying a suitable ligand for binding Al 3+ ion. The use of fluorophore moieties appended to a polymer backbone for sensing applications is far from mature. Here, we report a new fluorescent probe/monomer 4 and its ROMP derived polymer P for specific detection of Al 3+ ions. Both monomer 4 and its polymer P exhibit high selectivity toward only Al 3+ with no interference from other metal ions, having a limit detection of 0.5 and 2.1 µM, respectively. The reversible recognition of monomer 4 and P for Al 3+ was also proved in presence of Na 2 EDTA by both UV-Vis and fluorometric titration. The experimental data indicates the behavior of 4 and P toward Al 3+ is pH independent in medium conditions. In addition, the switch-on luminescence response of 4 at acidic pH (0 < 5.0), allowed us to specifically stain lysosomes (pH ~ 4.5-5.0) in live cells.

Bioorthogonal chemistry is bridging the divide between static chemical connectivity and the dynamic physiologic regulation of molecular state, enabling in situ transformations that drive multiple technologies. In spite of maturing mechanistic understanding and new bioorthogonal bond-cleavage reactions, the broader goal of molecular ON/OFF control has been limited by the inability of existing systems to achieve both fast (i.e., seconds to minutes, not hours) and complete (i.e., >99%) cleavage. To attain the stringent performance characteristics needed for high fidelity molecular inactivation, we have designed and synthesized a new C 2 -symmetric trans -cyclooctene linker (C 2 TCO) that exhibits excellent biological stability and can be rapidly and completely cleaved with functionalized alkyl-, aryl-, and H-tetrazines, irrespective of click orientation. By incorporation of C 2 TCO into fluorescent molecular probes, we demonstrate highly efficient extracellular and intracellular bioorthogonal disassembly via omnidirectional tetrazine-triggered cleavage.

Integration of multiple agent therapy (MAT) into one probe is promising for improving therapeutic efficiency for cancer treatment. However, MAT probe, if entering the cell as a whole, may not be optimal for each therapeutic agent (with different physicochemical properties), to achieve their best performance, hindering strategy optimization. A peptide-conjugated-AIEgen (FC-PyTPA) is presented: upon loading with siRNA, it self-assembles into FC siRNA -PyTPA. When approaching the region near tumor cells, FC siRNA -PyTPA responds to extracellular MMP-2 and is cleaved into FC siRNA and PyTPA. The former enters cells mainly by macropinocytosis and the latter is internalized into cells mainly through caveolae-mediated endocytosis. This two-part strategy greatly improves the internalization efficiency of each individual therapeutic agent. Inside the cell, self-assembly of nanofiber precursor F, gene interference of C siRNA , and ROS production of PyTPA are activated to inhibit tumor growth., (© 2020 Wiley-VCH GmbH.)

Solvochromatic effects are most frequently associated with solution-phase phenomena. However, in the gas phase, the absence of solvent leads to intramolecular solvation that can be driven by strong forces including hydrogen bonds and ion-dipole interactions. Here we examine whether isomerization of a single residue in a peptide results in structural changes sufficient to shift the absorption of light by an appended chromophore. By carrying out the experiments inside a mass spectrometer, we can easily monitor photodissociation yield as a readout for chromophore excitation. A series of peptides of different lengths, charge states, and position and identity of the isomerized residue were examined by excitation with both 266 and 213 nm light. The results reveal that differences in intramolecular solvation do lead to solvochromatic shifts in many cases. In addition, the primary product following photoexcitation is a radical. Ion-molecule reactions with this radical and adventitious oxygen were monitored and also found to vary as a function of isomeric state. In this case, differences in intramolecular solvation alter the availability of the reactive radical. Overall, the results reveal that small changes in a single amino acid can influence the overall structural ensemble sufficient to alter the efficiency of multiple gas-phase reactions.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

Yttrium (III) complexes are interesting due to the similarity of their chemistry with gadolinium complexes that are used as contrast agents in nuclear magnetic resonance (NMR) spectroscopy or imaging (MRI). While most of the paramagnetic Gd 3+ -based MRI contrast agents are T 1 or T 2 relaxation-based sensors such as Gd 3+ -complexes for zinc or pH detection, a number of diamagnetic Y 3+ -complexes rely on changes in the chemical shift for potential quantitative MRI in biological milieu. 89 Y, however, is a challenging nucleus to work with in conventional NMR or MRI due to its inherently low sensitivity and relatively long T 1 relaxation time. This insensitivity problem in 89 Y-based complexes can be circumvented with the use of dissolution dynamic nuclear polarization (DNP) which allows for several thousand-fold enhancement of the NMR or MRI signal relative to thermal equilibrium signal. Herein, we report on the feasibility of using hyperpolarized 89 Y-complexes with phosphonated open-chain ligands, 89 Y-EDTMP and 89 Y-DTPMP, as potential chemical shift-based pH NMR sensors. Our DNP-NMR data show that hyperpolarized 89 Y-DTPMP has an apparent pK a  ~ 7.01 with a 4 ppm-wide chemical shift dispersion with the signal disappearing at pH below 6.2. On the other hand, pH titration data on hyperpolarized 89 Y-EDTMP show that it has an apparent pK a of pH 6.7 and a 16-ppm wide chemical shift dispersion at pH 5-9 range. In comparison, the previously reported hyperpolarized pH NMR sensor 89 Y-DOTP has a pK a of 7.64 and ~ 10-ppm wide chemical shift dispersion at pH 4-9 range. Overall, our data suggest that hyperpolarized 89 Y-EDTMP is better than hyperpolarized 89 Y-DOTP in terms of pH sensing capability at the physiological range., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

RNA regulation is influenced by the dynamic changes in conformational accessibility on the transcript. Here we discuss the initial validation of a cell-free antisense probing method for structured RNAs, using the Tetrahymena group I intron as a control target. We observe changes in signal that qualitatively match prior traditional DMS footprinting experiments. Importantly, we have shown that application of this technique can elucidate new RNA information given its sensitivity for detecting rare intermediates that are not as readily observed by single-hit kinetics chemical probing techniques. Observing changes in RNA accessibility has broad applications in determining the effect that regulatory elements have on regional structures. We speculate that this method could be useful in quickly observing those interactions, along with other phenomena that influence RNA accessibility including RNA-RNA interactions and small molecules., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sirtuins are NAD + -dependent protein deacylases that remove acyl modifications from acyl-lysine residues, resulting in essential cellular signaling. Recognized for their role in lifespan extension, humans encode seven sirtuin isoforms (Sirt1-7), and loss of sirtuin deacylase activity is implicated in many aging-related diseases. Despite being intriguing therapeutic targets, cellular studies of sirtuins are hampered by the lack of chemical probes to measure sirtuin activity independent of sirtuin protein levels. Here, we use a modular, peptide-based approach to develop activity-based probes (ABPs) that directly measure Sirt1 activity in vitro and in cell lysates. ABPs were synthesized containing four elements: (1) thioacetyl-lysine for mechanism-based affinity towards only active sirtuins, (2) either histone H3 lysine-14 (H3K14) or p53 sequences for Sirt1 specificity, (3) a diazirine for covalent labeling upon UV irradiation, and (4) an alkyne for bioorthogonal conjugation to a fluorophore for gel-based detection of active Sirt1. Compared to the H3K14 ABP, the p53 ABP showed increased sensitivity and selective labeling of active Sirt1. Acyl-lysine peptide competition, pharmacological inhibition, and inhibitory post-translational modification of Sirt1 resulted in the loss of p53 ABP labeling both in vitro and in HEK293T cell lysates, consistent with the ABP measuring decreased Sirt1 activity. Furthermore, the p53 ABP measured subcellular Sirt1 activity in MCF7 breast cancer cells. The development of a Sirt1-selective ABP that detects Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches demonstrates the utility of a modular, peptide-based approach for selective-targeting of the sirtuin protein family and provides a framework for further development of sirtuin-selective chemical probes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Surface-enhanced Raman scattering (SERS) nanoprobes based on Au@Ag core@shell nanoparticles coated with poly(allylamine) were functionalized with small targeting molecules to evaluate simultaneously the level of expression of two cancer-related markers, both in cells and in tissues. The Au@Ag nanoparticles provide a high SERS signal enhancement in the visible range when combined with resonant Raman-active molecules. The poly(allylamine) coating plays a dual key role in (i) protecting the metal surface against the complex biological medium, leading to a stable signal of the Raman-active molecules, and (ii) enabling specific biofunctionalization through its amine functions. Using small targeting molecules linked to the polymer coating, two different nanoprobes (duplex approach) were designed. Each was able to specifically target a particular cancer-related marker: folate receptors (FRs) and sialic acid (SA). We demonstrate that the level of expression of these targeted markers can be evaluated following the SERS signal of the probes incubated on cells or tissues. The potential overexpression of folate receptors and of sialic acid was evaluated and measured in breast and ovarian cancerous tissue sections. In addition, FR and/or SA overexpression in the tumor region can be visualized with high contrast with respect to the healthy region and with high spatial accuracy consistent with histology by SERS imaging of the nanoprobe signal. Owing to the unique spectral signature of the designed nanoprobes, this approach offers an efficient tool for the spatially resolved, in situ measurement of the expression level of several cancer-related markers in tumors at the same time.Graphical abstract.

Bridged polymacrocyclic ligands featured by structurally different cages offer the possibility of coordinating multiple trivalent lanthanide ions, giving rise to the exploitation of their different physicochemical properties, e.g., multimodal detection for molecular imaging purposes. Intrigued by the complementary properties of optical and MR-based image capturing modalities, we report the synthesis and characterization of the polymetallic Ln(III)-based chelate comprised of two DOTA-amide-based ligands (DOTA-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bridged via 1,10-diaza-18-crown-6 (DA18C6) motif. The DOTA-amide moieties and the DA18C6 were used to chelate two Eu(III) ions and one Tb(III) ion, respectively, resulting in a multinuclear heterometallic complex Eu 2 LTb . The bimetallic complex without Tb(III), Eu 2 L , displayed a strong paramagnetic chemical exchange saturation transfer (paraCEST) effect. Notably, the luminescence spectra of Eu 2 LTb featured mixed emission including the characteristic bands of Eu(III) and Tb(III). The advantageous features of the complex Eu 2 LTb opens new possibilities for the future design of bimodal probes and their potential applicability in CEST MR and optical imaging.

Untargeted metabolomics indicates that the number of unidentified small-molecule metabolites may exceed the number of protein-coding genes for many organisms, including humans, by orders of magnitude. Uncovering the underlying metabolic networks is essential for elucidating the physiological and ecological significance of these biogenic small molecules. Here we develop a click-chemistry-based enrichment strategy, DIMEN (deep interrogation of metabolism via enrichment), that we apply to investigate metabolism of the ascarosides, a family of signaling molecules in the model organism C. elegans . Using a single alkyne-modified metabolite and a solid-phase azide resin that installs a diagnostic moiety for MS/MS-based identification, DIMEN uncovered several hundred novel compounds originating from diverse biosynthetic transformations that reveal unexpected intersection with amino acid, carbohydrate, and energy metabolism. Many of the newly discovered transformations could not be identified or detected by conventional LC-MS analyses without enrichment, demonstrating the utility of DIMEN for deeply probing biochemical networks that generate extensive yet uncharacterized structure space.

Chemical probes have been instrumental in microbiology since its birth as a discipline in the 19th century when chemical dyes were used to visualize structural features of bacterial cells for the first time. In this review article we will illustrate the evolving design of chemical probes in modern chemical biology and their diverse applications in bacterial imaging and phenotypic analysis. We will introduce and discuss a variety of different probe types including fluorogenic substrates and activity-based probes that visualize metabolic and specific enzyme activities, metabolic labeling strategies to visualize structural features of bacterial cells, antibiotic-based probes as well as fluorescent conjugates to probe biomolecular uptake pathways.

Objectives: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium., Materials and Methods: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability., Results: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein., Conclusion: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies., Competing Interests: Funding was provided by the Presbyterian Health Foundation (RAT). This does not alter our adherence to PLOS ONE policies on data or materials sharing.

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC 50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

Selective profiling of steviol-catalyzing UDP-glycosyltransferases in plants was accomplished with a probe metabolically synthesized from two substrate-derived components comprising an alkynylated sugar receptor (steviol) module and a diazirine-modified sugar donor (UDP-glucose) module, thereby illustrating a facile approach for harnessing biosynthetic enzymes of natural glycosides in plants for synthetic biology.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [ 125 I]I- para -iodobenzoate (PIB) label and a residualizing [ 99m Tc]Tc(CO) 3 label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [ 125 I]I-PIB-Ec1 and [ 99m Tc]Tc(CO) 3 -Ec1 in TNBC tumors. [ 125 I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [ 99m Tc]Tc(CO) 3 -Ec1, which resulted in significantly ( p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.

Pharmacological modulation of cannabinoid type 2 receptor (CB 2 R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB 2 R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB 2 R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB 2 R specificity was demonstrated by competition experiments in living cells expressing CB 2 R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

The analysis of protein antigens as biomarkers in clinical samples is particularly helpful for the early diagnosis of diseases. However, this is difficult to accomplish owing to the presence of the antigens in trace amounts as well as the complexity of the matrixes in clinical samples. In this study, a lab-on-membrane platform that can be combined with paper spray ionization mass spectrometry was developed for the in situ high-throughput sensitive detection of the prostate-specific antigen (PSA). The sensitivity of the proposed platform was enhanced via two strategies: (1) the synthesis of a biotin-streptavidin scaffold caused an increase in the capturing efficiency of PSA by a factor of 5 and (2) the immobilization of a large number of mass tag molecules on the gold nanoparticles allowed for the amplification of the mass spectrometry signals. The limit of detection was approximately 3.0 pg mL -1 . The selectivity to PSA was guaranteed by using an antibody-aptamer pairing sandwich immunoassay, and PSA detection was unaffected even when other protein antigens (carcinoembryonic antigen and carbohydrate antigen 125) were present. The modified membranes maintained their performance for at least 30 days when stored at 4 °C. Finally, analysis of human serum samples confirmed that the PSA concentration as determined using the proposed platform was consistent with that determined with a conventional chemiluminescent immunoassay. Thus, this PSA analyzing platform is suitable for prostate cancer diagnosis in clinical settings.

Molecular recognition is a specific non-covalent and frequently reversible interaction between two or more systems based on synthetically predefined character of the receptor. This phenomenon has been extensively studied over past few decades, being of particular interest to researchers due to its widespread occurrence in biological systems. In fact, a straightforward inspiration by biological systems present in living matter and based on, e.g., hydrogen bonding is easily noticeable in construction of molecular probes. A separate aspect also incorporated into the molecular recognition relies on the direct interaction between host and guest with a covalent bonding. To date, various artificial systems exhibiting molecular recognition and based on both types of interactions have been reported. Owing to their rich optoelectronic properties, chromophores constitute a broad and powerful class of receptors for a diverse range of substrates. This review focuses on BODIPY and porphyrin chromophores as probes for molecular recognition and chiral discrimination of amino acids and their derivatives.

Purpose: The purpose of this study was to develop and evaluate two α v β 6 -targeted fluorescent imaging agents. The integrin subtype α v β 6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. α v β 6 -targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH 2 -PEG 28 -A20FMDV2-K16R-PEG 28 (1), a peptide that has high affinity and selectivity for the integrin α v β 6 : (a) 5-FAM-X-PEG 28 -A20FMDV2-K16R-PEG 28 (2), and (b) IRDye800-PEG 28 -A20FMDV2-K16R-PEG 28 (3)., Procedures: Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for α v β 6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ 6 (α v β 6 +) and DX3puro (α v β 6 -) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ 6 /DX3puro and BxPC-3 (α v β 6 +) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model., Results: Peptides were obtained in > 99% purity. IC 50 values were 28 nM (2) and 39 nM (3). Rapid α v β 6 -selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the α v β 6 positive subcutaneous and orthotopic tumors, with negligible uptake in the α v β 6 -negative tumor. Blocking studies with a known α v β 6 -targeting peptide demonstrated α v β 6 -specific binding of 3., Conclusion: Two fluorescence imaging agents were developed. The α v β 6 -specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the α v β 6 -positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

Purpose: We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC., Procedures: We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC., Results: EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC., Conclusions: This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

The potassium ion (K + ) plays significant roles in many biological processes. To date, great efforts have been devoted to the development of K + sensors for colorimetric, fluorescent, and photoacoustic detection of K + separately. However, the development of molecular K + probes for colorimetric detection of urinary K + , monitoring K + fluxes in living cells by fluorescence imaging, and photoacoustic imaging of K + dynamics in deep tissues still remains an open challenge. Herein, we report the first molecular K + probe (NK2) for colorimetric, fluorescent, and photoacoustic detection of K + . NK2 is composed of 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as the chromophore and phenylazacrown-6-lariat ether (ACLE) as the K + recognition unit. Predominate features of NK2 include a short synthetic procedure, high K + selectivity, large detection range (5-200 mM), and triple-channel detection manner. NK2 shows good response to K + with obvious color changes, fluorescence enhancements (about threefold), and photoacoustic intensity changes. The existence of other metal ions (including Na + , Mg 2+ , Ca 2+ , Fe 2+ ) and pH changes (6.5-9.0) have no obvious influence on K + sensing of NK2. Portable test strips stained by NK2 can be used to qualitatively detect urinary K + by color changes for self-diagnosis of diseases induced by high levels of K + . NK2 can be utilized to monitor K + fluxes in living cells by fluorescent imaging. We also find its excellent performance in photoacoustic imaging of different K + concentrations in the mouse ear. NK2 is the first molecular K + probe for colorimetric, fluorescent, and photoacoustic detection of K + in urine, in living cells, and in the mouse ear. The development of NK2 will broaden K + probes' design and extend their applications to different fields. Graphical abstract.

A new type of ultrathin fiber microprobe for selective electroporation is reported. The microprobe is 10 cm long and has a diameter of 350 µm. This microprobe is a low cost tool, which allows electroporation of an arbitrary selected single cell or groups of cells among population with use of a standard microscope and cell culture plates. The microprobe in its basic form contains two metal microelectrodes made of a silver-copper alloy, running along the fiber, each with a diameter of 23 µm. The probe was tested in vitro on a population of normal and cancer cells. Successful targeted electroporation was observed by means of accumulation of trypan blue (TB) dye marker in the cell. The electroporation phenomenon was also verified with propidium iodide and AnnexinV in fluorescent microscopy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Ryszard Buczyński, Dariusz Pysz and Ryszard Stępień are inventors of patents owned by Institute of Electronic Materials Technology in Warsaw in technical field of the publication]., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

We report a label-free and "signal-on" homogeneous photoelectrochemical cytosensing system for ultrasensitive detection of cancer cells, which is a truly homogeneous PEC cytosensing system without the photoactive material immobilization and target recognition probe modification, providing a new avenue in early and accurate cancer diagnosis and clinical analysis.

As the second most common transition metal in the human body, zinc is of great interest to research but has few viable routes for its direct structural study in biological systems. Herein, Zn valence-to-core X-ray emission spectroscopy (VtC XES) and Zn K-edge X-ray absorption spectroscopy (XAS) are presented as a means to understand the local structure of zinc in biological systems through the application of these methods to a series of biologically relevant molecular model complexes. Taken together, the Zn K-edge XAS and VtC XES provide a means to establish the ligand identity, local geometry, and metal-ligand bond lengths. Experimental results are supported by correlation with density-functional-theory-based calculations. Combining these theoretical and experimental approaches will enable future applications to protein systems in a predictive manner.

Significant advancement of chemoproteomics has contributed to uncovering the mechanism of action (MoA) of small-molecule drugs by characterizing drug-protein interactions in living systems. However, cell-membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels, due to their low abundance and unique biophysical properties associated with multiple transmembrane domains, can present challenges for proteome-wide mapping of drug-receptor interactions. Herein, we describe the development of novel tetrafunctional probes, consisting of (1) a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive group, (3) a hydrazine-labile cleavable linker, and (4) biotin for enrichment. In live cell labeling studies, we demonstrated that the ACT-based probe showed superior reactivity and selectivity for labeling on-target GPCR by mass spectrometry analysis compared with control probes including diazirine-based probes. By leveraging ACT-based cleavable probes, we further identified a set of representative ionotropic receptors, targeted by CNS drugs, with remarkable selectivity and precise binding site information from mouse brain slices. We anticipate that the robust chemoproteomic platform using the ACT-based cleavable probe coupled with phenotypic screening should promote identification of pharmacologically relevant target receptors of drug candidates and ultimately development of first-in-class drugs with novel MoA.

We report on using the synthetic aminoadamantane-CH 2 -aryl derivatives 1 - 6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5 . Study of 1 - 6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6 ) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5 , respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6 . In the active M2 S31N blockers 1 and 6 , the phenyl and isoxazolyl head groups achieve a deeper binding position and high k on /low k off and high k on /high k off rate constants, compared to inactive 2 - 5 , which have much lower k on and higher k off . Compounds 1 - 5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high k on and low k off rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1 - 5 or 1 - 4 and 6 , respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2 - 4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.

The characterization of specific metabolite-protein interactions is important in chemical biology and drug discovery. For example, nuclear receptors (NRs) are a family of ligand-activated transcription factors that regulate diverse physiological processes in animals and are key targets for therapeutic development. However, the identification and characterization of physiological ligands for many NRs remains challenging, because of limitations in domain-specific analysis of ligand binding in cells. To address these limitations, we developed a domain-specific covalent chemical reporter for peroxisome proliferator-activated receptors (PPARs) and demonstrated its utility to screen and characterize the potency of candidate NR ligands in live cells. These studies demonstrate targeted and domain-specific chemical reporters provide excellent tools to evaluate endogenous and exogenous (diet, microbiota, therapeutics) ligands of PPARs in mammalian cells, as well as additional protein targets for further investigation.

The high temperature requirement A (HTRA) family of serine proteases mediates protein quality control. These proteins process misfolded proteins in several diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). While their structures and activation mechanisms have been studied, the precise details of the regulation of their activity under physiological conditions have not been completely elucidated, partly due to the lack of suitable chemical probes. In the present study, we developed novel activity-based probes (ABPs) targeting the HTRAs and demonstrated their utility in the monitoring and quantification of changes in enzyme activity in live cells. Using our probes, we found the activity of HTRA1 to be highly elevated in an AD-like cell-based model. We also observed the active HTRA2 in live cells by using a mitochondrion-targeted probe. We believe that our probes can serve as a useful tool to study the role of human HTRAs in neurodegenerative diseases.

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners., Competing Interests: Declaration of Interests All authors are (or were at the time of their involvement with the construction of the NIBR MoA Box) employees of Novartis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Precise evaluation of telomerase activity is highly crucial for early cancer diagnosis. In this study, a sensitive catalytic hairpin assembly-dynamic light scattering (CHA-DLS) assay for telomerase activity detection is developed by using the diameter change of gold nanoparticle (AuNP) probes. The telomerase substrate primer can be extended in the presence of telomerase, producing a telomerase extension product (TEP) with telomeric repeat units (TTAGGG) n at its 3'-end. The TEP can specifically trigger the CHA process and form tremendous AuNPs-H1/H2 nanostructures, resulting in a significant increase in the diameter measured by DLS. Telomerase activity from different cancer cell lines (MCF-7, Huh7, and 5637) was detected using the proposed strategy, the diameter of AuNP probes increased with the number of cancer cells, and this method can accurately detect telomerase activity down to 6 MCF-7 cells, 10 Huh7 cells, and three 5637 cells. Moreover, the CHA-DLS biosensor was successfully applied in urine specimens from healthy individuals and different cancer patients, which can distinguish bladder cancer patients from healthy people and other cancer patients, indicating that the noninvasive method has a great potential for application in early diagnosis of bladder cancer.

5-Carboxylcytosine (5caC) plays a vital role in the dynamics of DNA demethylation, and sequencing of its sites will help us dig out more biological functions of 5caC. Herein, we present a novel chemical method to efficiently label 5caC distinguished from other bases in DNA. Combined with bisulfite sequencing, 5caC sites can be located at single-base resolution, and the efficiency of 5caC labeling is 92% based on the Sanger sequencing data. Furthermore, dot blot assays have confirmed that 5caC-containing DNA isolated from HeLa cells was successfully labeled using our method. We expect that our strategy can be further applied to selectively tagging other carboxyl-modified bases and mapping their sites in RNA.

The importance of glial cells in the modulation of neuronal processes is now generally accepted. In particular, enormous progress in our understanding of astrocytes and microglia physiology in the central nervous system (CNS) has been made in recent years, due to the development of genetic and molecular toolkits. However, the roles of satellite glial cells (SGCs) and macrophages-the peripheral counterparts of astrocytes and microglia-remain poorly studied despite their involvement in debilitating conditions, such as pain. Here, we characterized in dorsal root ganglia (DRGs), different genetically-modified mouse lines previously used for studying astrocytes and microglia, with the goal to implement them for investigating DRG SGC and macrophage functions. Although SGCs and astrocytes share some molecular properties, most tested transgenic lines were found to not be suitable for studying selectively a large number of SGCs within DRGs. Nevertheless, we identified and validated two mouse lines: (i) a CreERT2 recombinase-based mouse line allowing transgene expression almost exclusively in SGCs and in the vast majority of SGCs, and (ii) a GFP-expressing line allowing the selective visualization of macrophages. In conclusion, among the tools available for exploring astrocyte functions, a few can be used for studying selectively a great proportion of SGCs. Thus, efforts remain to be made to characterize other available mouse lines as well as to develop, rigorously characterize and validate new molecular tools to investigate the roles of DRG SGCs, but also macrophages, in health and disease., Competing Interests: The authors have declared that no competing interests exist.

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAP Switch ) that can be conjugated to biomolecules using click chemistry. SNAP Switch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAP Switch , we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAP Switch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) that synthesizes the macrocyclic core of the antibiotic erythromycin. Each of its six multidomain modules presumably sample distinct conformations, as biosynthetic intermediates tethered to their acyl carrier proteins interact with multiple active sites during the courses of their catalytic cycles. The spatiotemporal details underlying these protein dynamics remain elusive. Here, we investigate one aspect of this conformational flexibility using two domain-specific monoclonal antibody fragments (F ab s) isolated from a very large naïve human antibody library. Both F ab s, designated 1D10 and 2G10, were bound specifically and with high affinity to the ketoreductase domain of DEBS module 1 (KR1). Comparative kinetic analysis of stand-alone KR1 as well as a truncated bimodular derivative of DEBS revealed that 1D10 inhibited KR1 activity whereas 2G10 did not. Co-crystal structures of each KR1-F ab complex provided a mechanistic rationale for this difference. A hybrid PKS module harboring KR1 was engineered, whose individual catalytic domains have been crystallographically characterized at high resolution. Size exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS) of this hybrid module bound to 1D10 provided further support for the catalytic relevance of the "extended" model of a PKS module. Our findings reinforce the power of monoclonal antibodies as tools to interrogate structure-function relationships of assembly line PKSs.

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.

Selectivity is a key requirement of high-quality chemical probes and lead medicines; however, methods to quantify and compare the selectivity of small molecules have not been standardized across the field. Herein, we discuss the origins and use of a comprehensive, single value term to quantify selectivity, the Gini coefficient. Case studies presented include compounds that target protein kinases, small molecules that bind RNA structures, and small molecule chimeras that bind to and degrade the target RNA. With an increasing number of transcriptome- and proteome-wide studies, we submit that reporting Gini coefficients as a quantitative descriptor of selectivity should be used broadly.

Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the ubiquitin-proteasome system. Over the last 5 years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine., Competing Interests: Declaration of Interests C.M.C. is founder, shareholder, and consultant to Arvinas, Inc. and Halda, LLC, which support research in his laboratory., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Selective and sensitive molecular probes for hydrogen peroxide (H 2 O 2 ), which plays diverse roles in oxidative stress and redox signaling, are urgently needed to investigate the physiological and pathological effects of H 2 O 2 . A lack of reliable tools for in vivo imaging has hampered the development of H 2 O 2 mediated therapeutics. By combining a specific tandem Payne/Dakin reaction with a chemiluminescent scaffold, H 2 O 2 -CL-510 was developed as a highly selective and sensitive probe for detection of H 2 O 2 both in vitro and in vivo. A rapid 430-fold enhancement of chemiluminescence was triggered directly by H 2 O 2 without any laser excitation. Arsenic trioxide induced oxidative damage in leukemia was successfully detected. In particular, cerebral ischemia-reperfusion injury-induced H 2 O 2 fluxes were visualized in rat brains using H 2 O 2 -CL-510, providing a new chemical tool for real-time monitoring of H 2 O 2 dynamics in living animals., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Porphyrins have planar and conjugated structures, good optical properties, and other special functional properties. Owing to these excellent properties, in recent years, porphyrins and their analogues have emerged as a multifunctional platform for chemical sensors. The rich chemistry of these molecules offers many possibilities for metal ions detection. This review mainly discusses two types of molecular porphyrin and porphyrin composite sensors for metal ions detection, because porphyrins can be functionalized to improve their functional properties, which can introduce more chemical and functional sites. According to the different application materials, the section of porphyrin composite sensors is divided into five sub-categories: (1) porphyrin film, (2) porphyrin metal complex, (3) metal-organic frameworks, (4) graphene materials, and (5) other materials, respectively.