Chemical Probes Research Articles

Transcriptome-wide probing reveals RNA thermometers that regulate translation of glycerol permease genes in Bacillus subtilis.

RNA structure regulates bacterial gene expression by several distinct mechanisms via environmental and cellular stimuli, one of which is temperature. While some genome-wide studies have focused on heat shock treatments and the subsequent transcriptomic changes, soil bacteria are less likely to experience such rapid and extreme temperature changes. Though RNA thermometers (RNATs) have been found in 5' untranslated leader regions (5' UTRs) of heat shock and virulence-associated genes, this RNA-controlled mechanism could regulate other genes as well. Using Structure-seq2 and the chemical probe dimethyl sulfate (DMS) at four growth temperatures ranging from 23°C to 42°C, we captured a dynamic response of the Bacillus subtilis transcriptome to temperature. Our transcriptome-wide results show RNA structural changes across all four temperatures and reveal nonmonotonic reactivity trends with increasing temperature. Then, focusing on subregions likely to contain regulatory RNAs, we examined 5' UTRs to identify large, local reactivity changes. This approach led to the discovery of RNATs that control the expression of glpF (glycerol permease) and glpT (glycerol-3-phosphate permease); expression of both genes increased with increased temperature. Results with mutant RNATs indicate that both genes are controlled at the translational level. Increased import of glycerols at high temperatures could provide thermoprotection to proteins.

Activation of peroxymonosulfate by FeVO3-x for the degradation of carbamazepine: Vanadium mediated electron shuttle and oxygen vacancy modulated interface chemistry

Fast Fe(III)/Fe(II) circulation in heterogeneous peroxymonosulfate (PMS) activation remains as a bottleneck issue that restricts the development of PMS based advanced oxidation processes. Herein, we proposed a facile ammonia reduction strategy and synthesized a novel FeVO3-x catalysts to activate PMS for the degradation of a typical pharmaceutical, carbamazepine (CBZ). Rapid CBZ removal could be achieved within 10 min, which outperforms most of the other iron or vanadium-based catalysts. Electron paramagnetic resonance analysis and chemical probe experiments revealed SO4•−, •OH, O2•− and high valent iron (Fe(IV)) were all generated in this system, but SO4•− and Fe(IV) primarily contributed to the degradation of CBZ. Besides, X-ray photoelectron spectroscopy and X-ray adsorption spectroscopy indicated that both the generated low-valent V provides and oxygen vacancy acted as superior electron donors and accelerated internal electron transfer via the unsaturated V−O−Fe bond. Finally, the proposed system also exhibited satisfactory performance in practical applications. This work provides a promising platform in heterogeneous PMS activation.

Dynamic Formation of Brønsted Acid Sites over Supported WOx/Pt on SiO2 Inverse Catalysts─Spectroscopy, Probe Chemistry, and Calculations

Platinum-tungsten oxides are among the most studied metal–metal oxide pair catalysts for C–O hydrogenolysis reactions. The Brønsted acid density and synergy between Pt and WOx, especially in the inverse structure, are critical to reactivity and selectivity. However, a clear molecular-level understanding of the formation and dynamics of Brønsted acid sites (BAS) is lacking. Here, using in situ spectroscopic characterizations (Raman and FTIR), chemical probing (CO chemisorption and pyridine titration), density functional theory (DFT) calculations, and a model reaction (tert-butanol dehydration), we demonstrate the structural evolution of WOx species and associated BAS dynamics in various environments. In situ Raman and DFT calculations show that below monolayer coverage, the WOx species stay as isolated monomers on the SiO2 support and W3Ox trimers on Pt. The W3Ox trimers on Pt are dynamic and 10× more active toward dehydration than the WOx species on the SiO2 support. H2 plays a complex role: at low temperatures (<473 K), it creates more BAS in the form of W3O7H on Pt by reversible hydrogen spillover, and at higher temperatures (>573 K), it partially reduces the W3Ox. We further show that the inverse configuration allows changes in the BAS density via catalyst pretreatments. This study provides a strategy for tuning Brønsted acid density and regenerating sites by pretreatment and catalyst composition.

Efficient removal of recalcitrant naphthenic acids with electro-cocatalytic activation of peroxymonosulfate by Fe(III)-nitrilotriacetic acid complex under neutral initial pH condition

This work investigated the activation of peroxymonosulfate by electrochemical (EC) system assisted with Fe(III)-nitrilotriacetic acid (NTA) complex for degradation of persistent naphthenic acids (NAs) under neutral initial pH conditions. As NAs are a complicated mixture, 1-adamantanecarboxylic acid (ACA) was selected as the model NA compound for degradation experiment. The addition of NTA is to chelate with Fe(III), gaining stability under neutral pH condition to facilitate the circulation of Fe(II)/Fe(III) by the electrochemical process to activate PMS. The EC/Fe(III)-NTA/PMS system was explored with applicable pH range of 3–9 and an optimized molar ratio 1: 2 for Fe: NTA. Results of quenching and chemical probe experiment together with results of electron paramagnetic resonance (EPR) analysis revealed the main reactive species of the system, including •OH, SO4•−, 1O2 and possibly Fe(IV). With the addition of NTA, the yields of •OH, SO4•−, 1O2 were enhanced. Results of mass spectrometry analysis and DFT calculations indicated the formation of 9 degradation byproducts of ACA via three primary degradation pathways such as hydroxyl substitution, carbonyl substitution, and decarboxylation. Furthermore, the EC/Fe(III)-NTA/PMS system could achieve excellent removal efficiency of ACA with different anions such as Cl-, HCO3-, NO3- and H2PO4- in the background. The practical applicability of the system was also verified with the high removal of commercial NAs mixture standard. Overall results have indicated the EC/Fe(III)-NTA/PMS system could be utilized for efficient reclamation of authentic oil and gas industrial wastewater under natural pH conditions.

A Disposable Soft Magnetic Ribbon Impedance-Based Sensor for Corrosion Monitoring

We present a new approach for the real-time monitoring of chemical corrosion based on radio-frequency (RF) impedance technology and soft ferromagnetic ribbons. The impedance (Z) of a commercial METGLAS® 2714A ribbon was measured in real time for 5 μL of drop-casted HNO3 of various concentrations. Variations in the concentration of the drop-casted acid were assessed by considering the difference in Z (η) with and without the acid treatment. We found a large and linear increase in η (from ~5 to ~35 mΩ) and a large linear decrease in measurement time (from ~240 to 70 s) with increases in acid concentration (from 0.9 to 7.4 Molar), which is promising for the development of disposable chemical sensors for the strength estimation of corrosive chemicals and for the monitoring of time-dependent chemical corrosion processes. Since the ribbon used is commercially available at a low cost and as the measurement system is quick and low power-consuming, the proposed sensor can be used as an easy, quick, and low-cost chemical probe in industry and for environmental hazard management purposes.

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Computational studies on glycosaminoglycan recognition of sialyl transferases.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

Accelerating antiviral drug discovery: lessons from COVID-19.

During the coronavirus disease 2019 (COVID-19) pandemic, a wave of rapid and collaborative drug discovery efforts took place in academia and industry, culminating in several therapeutics being discovered, approved and deployed in a 2-year time frame. This article summarizes the collective experience of several pharmaceutical companies and academic collaborations that were active in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral discovery. We outline our opinions and experiences on key stages in the small-molecule drug discovery process: target selection, medicinal chemistry, antiviral assays, animal efficacy and attempts to pre-empt resistance. We propose strategies that could accelerate future efforts and argue that a key bottleneck is the lack of quality chemical probes around understudied viral targets, which would serve as a starting point for drug discovery. Considering the small size of the viral proteome, comprehensively building an arsenal of probes for proteins in viruses of pandemic concern is a worthwhile and tractable challenge for the community.

Role variations of MnOx on monoclinic BiVO4 (110)/(040) facets for enhanced Photo-Fenton reactions

Compared with traditional Fenton reaction, peroxymonosulfate based advanced oxidation processes (PMS-AOPs) are more effective to remove the organic pollutants in wastewater in a wider pH range. Herein, selective loading of MnOx on monoclinic BiVO4 (110) or (040) facets were achieved by photo-deposition method with addition of different Mn precursors and electron/hole trapping agents. MnOx has good chemical catalysis activity for PMS activation, which can also enhance photogenerated charge separation, thus leading to enhanced activities than naked BiVO4. The BPA degradation reaction rate constants of MnOx(040)/BiVO4 and MnOx(110)/BiVO4 system are 0.245 min−1 and 0.116 min−1, which are 6.45 and 3.05 times larger than that of naked BiVO4, respectively. The roles of MnOx on different facets are different, which will promote OER process on (110) facets and utilize the dissolved O2 to produce O2•− and 1O2 more effectively on (040) facets. 1O2 is the dominated reactive oxidation species of MnOx(040)/BiVO4, while SO4•− and •OH play more important roles on MnOx(110)/BiVO4, which are proved by quenching experiments and chemical probe identifications, thus mechanism in MnOx/BiVO4-PMS-light system is proposed. The good degradation performance of MnOx(110)/BiVO4 and MnOx(040)/BiVO4 and mechanism theory may promote the application of photocatalysis in PMS based wastewater remediation.

Chemical probes based on GOLD: The discovery and elucidation of mechanism

AbstractThis review discusses the use of fluorescent probes for identifying, isolating, and monitoring cells and organelles. Fluorescent probes have advantages over antibodies in live‐cell staining due to their small size and ability to penetrate the cellular membrane without fixatives or permeabilizers. However, different cell types exhibit different staining mechanisms based on their genetic profiles and the composition of proteins, enzymes, and lipids. The authors introduce a gating‐oriented live‐cell distinction mechanism where membrane transporters such as solute carriers and ATP are responsible for importing and exporting fluorescent probes. The mechanism for known and unknown transporter identification is elucidated through dose‐dependency, inhibitor tests, and genetic techniques such as clustered regularly interspaced short palindromic repeat modulation and screening. This review provides insights into cellular staining and its potential for understanding the gene profile of different cell types, as well as guiding the identification of membrane transporters.

Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.

Overview of Chemical Methods to Probe RNA Structure with Radionucleotides.

Structural features of RNA play an important role in its capability to perform various functions in biological systems. To probe structural features, chemical probes are used to conjugate or cleave RNA at solvent-accessible sites, differentiating between flexible and constrained regions. These conjugates or cleaved products are then detected using reverse transcription (RT), where enzymatic RNA-dependent DNA primer extension is abruptly halted at the conjugation site or cleavage site. Here, we provide an overview of methods to probe RNA structure in vitro using radioactively labeled DNA primers, which provide a highly sensitive method to visualize RT stop sites with gel electrophoresis. © 2023 Wiley Periodicals LLC.

Discovery of chemical probes that perturb protein complexes using size exclusion chromatography and chemical proteomics

Most human proteins lack small-molecule ligands, rendering these proteins "undruggable." In this issue of Molecular Cell, Lazear etal.1 develop a novel chemical proteomic screening platform and discover new chemical probes targeting previously undruggable protein complexes.

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Insights into atomic‐electronegativity‐controlled excited state intramolecular proton transfer behavior for the novel oxazolonapthoimidazo[1,2‐a]‐pyridine (ONIP) compound: A time‐dependent density functional theory study

AbstractIn this work, we mainly focus on probing into the effects of atomic electronegativity on photo‐induced hydrogen bond (i.e., O–H···N) effects and excited state intramolecular proton transfer (ESIPT) processes for a novel fluorescent chemical probe ONIP derivatives (i.e., ONIP‐O, ONIP‐S, and ONIP‐Se). First, insights into optimized structural geometries and related infrared vibrational spectra, we clarify that the hydrogen bond O–H···N interaction could be strengthened via photoexcitation. Further, via predicting hydrogen bonding energy EHB, we quantificationally present that the S1‐state hydrogen bonding strengthening behavior should be more distinct with lower atomic electronegativity. When it comes to vertical photo‐induced excitation, the intramolecular charge transfer process happens and the charge redistribution plays roles in promoting ESIPT behavior. It is worth mentioning the energy gap between highest occupied molecular orbital and lowest unoccupied molecular orbital show that low atomic electronegativity should be more favorable to ESIPT process. Finally, based on the restricted optimization method, we construct the potential energy curves along with hydrogen bond wire for ONIP‐O, ONIP‐S, and ONIP‐Se. By exploring the conformation of potential energy curves and potential energy barriers, we disclose the detailed atomic‐electronegativity‐controlled ESIPT dynamical behaviors for ONIP system.

Pharmacological Progress of Mitophagy Regulation.

With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the identification of potent therapeutic agents. Mitochondria boast a central role in different integral biological processes and mitochondrial dysfunction is associated with multiple pathologies. It is, therefore, prudent to target mitochondrial quality control mechanisms by using pharmacological approaches. However, there is a scarcity of biologically active molecules, which can interact with mitochondria directly. Currently, the chemical compounds used to induce mitophagy include oligomycin and antimycin A for impaired respiration and acute dissipation of mitochondrial membrane potential by using CCCP/FCCP, the mitochondrial uncouplers. These chemical probes alter the homeostasis of the mitochondria and limit our understanding of the energy regulatory mechanisms. Efforts are underway to find molecules that can bring about selective removal of defective mitochondria without compromising normal mitochondrial respiration. In this report, we have tried to summarize and status of the recently reported modulators of mitophagy.

Targeting TAF1 with BAY-299 induces antitumor immunity in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype with poor prognoses and limited therapeutic options. The TATA-box binding protein associated factor 1 (TAF1) is an essential protein involved in the transcriptional regulation of cancer development and progress. However, the therapeutic potential and underlying mechanism of targeting TAF1 in TNBC remain unknown. Here, using chemical probe BAY-299, we identify that TAF1 inhibition leads to the induction of endogenous retrovirus (ERVs) expression and double-stranded RNA (dsRNA) formation, resulting in the activation of interferon responses and cell growth suppression in a subset of TNBC, resembling anti-viral mimicry effect. This correlation between TAF1 and interferon signature was validated in three independent breast cancer patient datasets. Furthermore, we observe heterogeneous responses to TAF1 inhibition across a set of TNBC cell lines. By integrating transcriptome and proteome data, we demonstrate that high levels of proliferating cell nuclear antigen (PCNA) protein serve as a predictive biomarker associated with suppressive tumor immune responses in various cancers, which may limit the efficiency of TAF1 inhibition.

Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn-on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM-OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD-DFT calculation. Moreover, the substrate (PM-OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image-based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn2+ in the Detection of 2'-OH Adducts.

Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn2+ cations in the modulation of SSIV readthrough efficiency.

Discovery of a Potent and Selective CDKL5/GSK3 Chemical Probe That Is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/β affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.