One Scaffold,Different Organelle Sensors: pH-Activable Fluorescent Probes for Targeting Live Microglial Cell Organelles**

Targeting live cell organelles is essential for imaging, understanding, and controlling specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds are used to target specific cell organelles. Here, we have designed a modular one-step synthetic strategy using a common reaction intermediate to develop new lysosomal, mitochondrial, and nucleus-targeting pH-activable fluorescent probes that are all based on a single boron dipyrromethane scaffold. The divergent cell organelle targeting was achieved by synthesizing probes with specific functional group changes to the central scaffold resulting in differential fluorescence and pK_a. Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH-activable fluorescent probes in live primary microglial cells with pK_a values ranging from ∼3.2–6.0. We introduce a structure-organelle-relationship (SOR) framework to target nuclei ( NucShine ), lysosomes ( LysoShine ), and mitochondria ( MitoShine ) in live microglia. This work will result in future applications of SOR beyond imaging to target and control organelle-specific biochemical processes in disease-specific models.     

A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity‐Based Probes: Diubiquitin as a Case Study

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity‐based probes. We describe the use of this handle in the generation of linkage‐specific activity‐based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity‐based probes based on all seven isopeptide‐linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage‐specific manner.     

Sequence‐Specific Covalent Capture Coupled with High‐Contrast Nanopore Detection of a Disease‐Derived Nucleic Acid Sequence

Hybridization‐based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence‐specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease‐relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap‐containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast—in essence, digital—signal that enables sensitive, single‐molecule sensing of the cross‐linked duplexes.     

Comparison of Bioorthogonal β-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of β-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal β-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.     

Computer-Based Drug Design of Positive Modulators of Store-Operated Calcium Channels to Prevent Synaptic Dysfunction in Alzheimer’s Disease

Store-operated calcium entry (SOCE) constitutes a fine-tuning mechanism responsible for the replenishment of intracellular stores. Hippocampal SOCE is regulated by store-operated channels (SOC) organized in tripartite complex TRPC6/ORAI2/STIM2. It is suggested that in neurons, SOCE maintains intracellular homeostatic Ca2+ concentration at resting conditions and is needed to support the structure of dendritic spines. Recent evidence suggests that positive modulators of SOC are prospective drug candidates to treat Alzheimer’s disease (AD) at early stages. Although STIM2 and ORAI2 are definitely involved in the regulation of nSOC amplitude and a play major role in AD pathogenesis, growing evidence suggest that it is not easy to target these proteins pharmacologically. Existing positive modulators of TRPC6 are unsuitable for drug development due to either bad pharmacokinetics or side effects. Thus, we concentrate the review on perspectives to develop specific nSOC modulators based on available 3D structures of TRPC6, ORAI2, and STIM2. We shortly describe the structural features of existing models and the methods used to prepare them. We provide commonly used steps applied for drug design based on 3D structures of target proteins that might be used to develop novel AD preventing therapy.     

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.     

"DNA traffic lights": concept of wavelength-shifting DNA probes and application in an aptasensor

Add it and see it: The concept of "DNA traffic lights" for wavelength-shifting DNA probes has a great potential in the application of biosensors, for example, in DNA aptamers. A visual color change in the DNA aptasensor fluorescence from green to red occurs after specific target binding.     

Induced‐Fit Docking Approach Provides Insight into the Binding Mode and Mechanism of Action of HIV‐1 Integrase Inhibitors

A three‐dimensional model of a complex between HIV‐1 integrase (IN), viral DNA, and metal ions that we recently built was used as a target for a docking method (induced‐fit docking, IFD) that accurately predicts ligand binding modes and concomitant structural changes in the receptor. Six different well‐known integrase strand transfer inhibitors (INSTIs): L‐708,906, L‐731,988, S‐1360, L‐870,810, raltegravir, and elvitegravir were thus used as ligands for our docking simulations. The obtained IFD results are consistent with the mechanism of action proposed for this class of IN inhibitors, that is, metal chelating/binding agents. This study affords new insight into the possible mechanism of inhibition and binding conformations for INSTIs. The impact on our hypothesis of specific mutations associated with IN inhibitor resistance was also evaluated. All these findings might have implications for integrase‐directed HIV‐1 drug discovery efforts.     

Templated Chemistry for Sequence‐Specific Fluorogenic Detection of Duplex DNA

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple‐helix formation at adjacent positions on a specific purine‐rich target sequence of duplex DNA. One fluorescein‐labeled probe contains an α‐azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double‐stranded DNA, at least for homopurine–homopyrimidine target sites, under native and nondenaturing conditions.     

Probes and nano-delivery systems targeting NAD(P)H:quinone oxidoreductase 1: a mini-review

The two-electron cytoplasmic reductase NAD(P)H:quinone oxidoreductase 1 is expressed in many tissues. NAD(P)H:quinone oxidoreductase 1 is well-known for being highly expressed in most cancers. Therefore, it could be a target for cancer therapy. Because it is a quinone reductase, many bioimaging probes based on quinone structures target NAD(P)H:quinone oxidoreductase 1 to diagnose tumours. Its expression is higher in tumours than in normal tissues, and using target drugs such as β-lapachone to reduce side effects in normal tissues can help. However, the physicochemical properties of β-lapachone limit its application. The problem can be solved by using nanosystems to deliver β-lapachone. This mini-review summarizes quinone-based fluorescent, near-infrared and two-photon fluorescent probes, as well as nanosystems for delivering the NAD(P)H:quinone oxidoreductase 1-activating drug β-lapachone. This review provides valuable information for the future development of probes and nano-delivery systems that target NAD(P)H:quinone oxidoreductase 1.     

Solution NMR studies of an intrinsically unstructured protein within a dilute, 75 kDa eukaryotic protein assembly; probing the practical limits for efficiently assigning polypeptide backbone resonances

This paper describes an efficient NMR strategy for assigning the backbone resonances of an intrinsically unstructured protein (IUP), p21-KID, bound to its biological target, Cdk2/cyclin A. In order to overcome the challenges associated with the high molecular weight (75 kDa) and low solubility of the ternary complex (0.2 mM), we used perdeuteration, TROSY, and high-sensitivity cryogenic NMR probes at high magnetic-field strengths (i.e. 16.4, 18.8 and 21.1 Tesla). p21-KID was also prepared by using specific amino acid isotope labels. Most importantly, we studied binary, subcomplexes that allowed resonance assignments to be made in stages. We show that subdomains of p21-KID folded within binary complexes into the same conformations as observed in the ternary, Cdk2/cyclin A complex. This is a general feature of IUPs, which often adopt highly extended conformations when bound to other proteins. This strategy is suitable for studies of IUPs within considerably larger biomolecular assemblies as long as the IUP can be uniformly and selectively isotope labeled.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.     

Guidelines for the Synthesis of Small‐Molecule Irreversible Probes Targeting G Protein‐Coupled Receptors

Irreversible probes have been proven to be useful pharmacological tools in the study of structural and functional features in drug receptor pharmacology. They have been demonstrated to be particularly valuable for the isolation and purification of receptors. Furthermore, irreversible probes are helpful tools for the identification and characterization of binding sites, thereby supporting the advancement of rational drug design. In this Minireview, we provide insight into universal strategies and guidelines to successfully synthesize irreversible probes that target G protein‐coupled receptors (GPCRs). We provide an overview of commonly used chemoreactive and photoreactive groups, and make a comparison of their properties and potential applications. Furthermore, there is a particular focus on synthetic approaches to introduce these reactive groups based on commercially available reagents.     

Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.