Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.

This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of the POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-sequencing and real-time quantitative polymerase chain reaction analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by messenger RNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1 week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging and pathway analysis. Experience with protein or proteome manipulation is useful. This Protocol Extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.

Z-REX uncovers a bifurcation in function of Keap1 paralogs.

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-à-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX-a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2- driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells: (1) zKeap1b-expressing cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding following whole-cell electrophile treatment; (2) zKeap1a-expressing cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered upon whole-cell electrophile exposure; (3) 1:1 ZKeap1a:zKeap1b-co-expressing cells show no Nrf2-release from the Keap1-complex following whole-cell electrophile administration, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a's behavior during electrophilic stress. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.

Investigating a Boronate-Affinity-Guided Acylation Reaction for Labelling Native Antibodies.

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate-affinity-based Ab ligand featuring a 4-(dimethylamino)pyridine and an S-aryl thioester to label full-length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide-labelled Ab (N3 -Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N3 -Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.

The not so identical twins: (dis)similarities between reactive electrophile and oxidant sensing and signaling.

In this tutorial review, we compare and contrast the chemical mechanisms of electrophile/oxidant sensing, and the molecular mechanisms of signal propagation. We critically analyze biological systems in which these different pathways are believed to be manifest and what the data really mean. Finally, we discuss applications of this knowledge to disease treatment and drug development.

Wdr1 and cofilin are necessary mediators of immune-cell-specific apoptosis triggered by Tecfidera.

Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1-ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.

Real-Time Identification of Irrigation Water Pollution Sources and Pathways with a Wireless Sensor Network and Blockchain Framework.

Real-time identification of irrigation water pollution sources and pathways (PSP) is crucial to ensure both environmental and food safety. This study uses an integrated framework based on the Internet of Things (IoT) and the blockchain technology that incorporates a directed acyclic graph (DAG)-configured wireless sensor network (WSN), and GIS tools for real-time water pollution source tracing. Water quality sensors were installed at monitoring stations in irrigation channel systems within the study area. Irrigation water quality data were delivered to databases via the WSN and IoT technologies. Blockchain and GIS tools were used to trace pollution at mapped irrigation units and to spatially identify upstream polluted units at irrigation intakes. A Water Quality Analysis Simulation Program (WASP) model was then used to simulate water quality by using backward propagation and identify potential pollution sources. We applied a "backward pollution source tracing" (BPST) process to successfully and rapidly identify electrical conductivity (EC) and copper (Cu2+) polluted sources and pathways in upstream irrigation water. With the BPST process, the WASP model effectively simulated EC and Cu2+ concentration data to identify likely EC and Cu2+ pollution sources. The study framework is the first application of blockchain technology for effective real-time water quality monitoring and rapid multiple PSPs identification. The pollution event data associated with the PSP are immutable.

Regioselective SN2-Type Reaction for the Oriented and Irreversible Immobilization of Antibodies to a Glass Surface Assisted by Boronate Formation.

Antibodies have exquisite specificities for molecular recognition, which have led to their incorporation into array sensors that are crucial for research, diagnostic, and therapeutic applications. Many of these platforms rely heavily on surface-bound reactive groups to covalently tether antibodies to solid substrates; however, this strategy is hindered by a lack of orientation control over antibody immobilization. Here, we report a mild electrophilic phenylsulfonate (tosylate) ester-containing boronic acid affinity ligand for attaching antibodies to glass slides. A high level of antibody coupling located near the Fc region of the boronated antibody complex could be achieved by the proximal nucleophilic amino acid driven substitution reaction at the phenylsulfonate center. This enabled the full-length antibodies to be permanently tethered onto surfaces in an oriented manner. The advantages of this strategy were demonstrated through the individual and multiplex detection of protein and serum biomarkers. This strategy not only confers stability to the immobilized antibodies but also presents a different direction for the irreversible attachment of antibodies to solid supports in an orientation-controlled way.