Biology subjects

Thorn-Seshold, J.

Publications and source records attributed to Thorn-Seshold, J..

9 recordsLinked to original sources

bioRxiv2025

Logic-gating the HaloTag system with Conditional-Halo-ligator 'CHalo' reagents

HaloTag proteins spontaneously ligate onto any chemical reagent featuring a chloroalkane motif (CA). We introduce the conditional CHalo motif, which ligates to HaloTag only after uncaging by light or enzymes. (1) Photo-triggered CHalo fluorogenic reagents allow spatiotemporally-specific labeling; (2) photo-triggered CHalo heterodimerisers can photocontrol protein recruitment; and (3) enzyme-triggered CHalo reagents can durably record diverse enzyme activities, and multiplexing them should allow quantitative ratiometric recording of multiple activities in parallel. CHalo thus permits manifold extensions to the HaloTag technology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/676741v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d18cd6org.highwire.dtl.DTLVardef@175694forg.highwire.dtl.DTLVardef@154ffd5org.highwire.dtl.DTLVardef@167fec2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

The delivery of nano-formulated drugs to solid tumours is selectively increased by co-application of the vascular disrupting agent CA4P

Improving the efficacy of existing cytotoxic chemotherapeutics requires increasing drug delivery to tumours while minimising systemic toxicity. Formulating these drugs as nanoparticles can reduce their exposure to healthy tissues, but broadly applicable strategies to enhance tumoral accumulation are lacking. Here, we show that co-administering small molecule vascular disrupting agents together with nanoparticle formulations (e.g. diagnostic reporters, or clinical drugs irinotecan and doxorubicin) increases their tumoral uptake by up to threefold, without raising systemic exposure. In a syngeneic mouse model of triple-negative breast cancer, this enhancement diminished when co-treatments were repeated, limiting its therapeutic benefit. However, since most solid tumour types are susceptible to vascular disrupting agents, this approach may be a broadly applicable strategy to improve the selectivity of drug delivery: with particular relevance for single dose use in diagnostic or research settings. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/669501v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@a5b07corg.highwire.dtl.DTLVardef@1e5dcd2org.highwire.dtl.DTLVardef@49134org.highwire.dtl.DTLVardef@1d90aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Cellularly-Retained Fluorogenic Probes for Sensitive Cell-Resolved Bioactivity Imaging

Here, we develop a general design for high-quality fluorogenic activity probes to quantify biological processes in live cells, by creating a scaffold that efficiently generates cell-retained bright fluorescent soluble products upon reaction with biochemical targets. Live cell probes must be designed to be membrane-permeable; but that often means that their fluorophore products are similarly permeable, resulting in rapid signal loss from the activating cell: which limits their cell-by-cell resolution as well as their sensitivity for quantifying low-turnover processes. Current strategies to retain fluorescent products within cells usually disrupt native biology: e.g. by non-specific alkylation or solid precipitation. Here, scanning charge- and polarity-based approaches to trigger cell retention, we developed a bright fluorogenic rhodol scaffold Trappable Green (TraG) that balances all key requirements for signal integration (rapid probe entry, but effective product retention, across a variety of cell lines) and is easily adaptable to quantify many target types (shown here with probes for GSH, TrxR, and H2O2). The simple and rugged TraG scaffold can now permit straightforward elaboration to a range of cell-retained enzyme activity probes, that enable more accurate cell-resolved imaging as well as higher-sensitivity integration of low-turnover processes, without the drawbacks of alkylation or precipitation-based strategies. O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/649302v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d5a99forg.highwire.dtl.DTLVardef@730dd8org.highwire.dtl.DTLVardef@191ea65org.highwire.dtl.DTLVardef@b393d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Imaging membrane damage in ferroptosis and necrosis by wash-free fluorogenic chemical probes

Selectively labelling cells with damaged membranes is needed in contexts as simple as identifying dead cells in culture, or as complex as imaging membrane barrier functionality in vivo. The commonly used dyes are permanently coloured/fluorescent dyes that are simply excluded by intact membranes, but to achieve good image contrast therefore requires removing their extracellular signal by washing or background subtraction, which are not possible in vivo. Here, we develop fluorogenic probes which sensitively and selectively reveal damaged cells, without needing washing steps since their fluorescence turns on from near-zero background. From a set of novel fluorogenic probes impermeabilised by sulfonations along different vectors, we identify a specific disulfonated fluorogenic scaffold that enters cells only upon membrane damage, where it is enzymatically activated to mark them. The esterase probe iPS-FS2 is a reliable tool to reveal live cells that have been permeabilised by biological, biochemical, or physical membrane damage; and it can be used in multicolour microscopy. We confirm the modularity of this approach by also adapting it for redox-unmasked cell-excluded probes with improved hydrolytic stability. This scaffold-based design thus provides tools for wash-free in vivo imaging of membrane damage, which is relevant across many pathologies. The insightss gained from these probes should also be translatable to damage-targeted prodrugs, for selective therapy of membrane-compromised cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/543437v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1594016org.highwire.dtl.DTLVardef@7e7053org.highwire.dtl.DTLVardef@1008cb0org.highwire.dtl.DTLVardef@1148bd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Prior flavivirus immunity skews the yellow fever vaccine response to expand cross-reactive antibodies with increased risk of antibody dependent enhancement of Zika and dengue virus infection

Human pathogenic flaviviruses pose a significant health concern and vaccination is the most effective instrument to control their circulation. How pre-existing immunity to antigenically related viruses modulates immunization outcome remains poorly understood. In this study, we evaluated the effect of vaccination against tick-borne encephalitis virus (TBEV) on the epitope immunodominance and immunogenicity of the yellow fever 17D vaccine (YF17D) in a cohort of 250 human vaccinees. Following YF17D vaccination, all study participants seroconverted and generated protective neutralizing antibody titers. At day 28, TBEV pre-immunity did not affect the polyclonal neutralizing response which largely depended on the IgM fraction. We found that sera from TBEV-immunized individuals enhanced YF17D vaccine virus infection via antibody-dependent enhancement (ADE). Upon vaccination, individuals with TBEV pre-immunity had higher concentrations of cross-reactive IgG antibodies with limited neutralizing capacity against YF17D whereas vaccinees without prior flavivirus exposure showed a non-cross-reacting response. Using a set of recombinant YF17D envelope protein mutants displaying different epitopes, we identified quaternary epitopes as the primary target of neutralizing antibodies. Sequential immunizations redirected the IgG response towards the pan-flavivirus fusion loop epitope (FLE) with the potential to mediate enhancement of dengue and Zika virus infections whereas TBEV naive individuals elicited an IgG response directed towards neutralizing epitopes without an enhancing effect. We propose that the YF17D vaccine effectively conceals the FLE and primes a neutralizing IgG response in individuals with no prior flavivirus exposure. In contrast, the response in TBEV-experienced recipients favors weakly-neutralizing, cross-reactive epitopes potentially increasing the risk of severe dengue and Zika disease due to ADE.

immunology↗

bioRxiv2022

Cyclic dichalcogenides extend the reach of bioreductive prodrugs to harness the thioredoxin system: applications to seco-duocarmycins

Small molecule prodrug approaches that can activate cancer therapeutics selectively in tumors are urgently needed. Here, we developed the first antitumor prodrugs designed for activation by the thioredoxin (Trx) oxidoreductase system. This critical cellular disulfide redox axis is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidised nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of a series of 10 "off-to-on" reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular activity dependent on reducing enzyme systems in 177 cell lines, to establish broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well tolerated in vivo in mice, indicating low systemic release of their duocarmycin cargo, and in vivo anti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising initial results indicating effective tumoral drug release, presumably by in situ bioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase type, validates its ability to access in vivo compatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

biochemistry↗

bioRxiv2021

A high-throughput neutralization assay for yellow fever serodiagnostics

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks, for surveillance and to evaluate vaccine efficacy in population-wide studies. All this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming and limited in throughput, classical plaque reduction neutralization test (PRNT) is still considered gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika and dengue viruses amenable for differential diagnostics. IMPORTANCEFast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance and monitoring of vaccine efficacy. Although classical PRNT still remains gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as a low throughput and an overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing large number of samples in a highly standardized manner and has the potential to be implemented in clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika and dengue viruses opening new avenues for differential diagnostics.

microbiology↗

bioRxiv2021

Photoswitchable epothilone-based microtubule stabilisers allow GFP imaging-compatible, optical control over the microtubule cytoskeleton

Optical methods to modulate microtubule stability and dynamics are promising approaches to reach the micron- and millisecond-scale resolution needed to decrypt the diverse roles of the microtubule cytoskeleton in biology. However, such optical methods have until now focussed nearly exclusively on microtubule destabilisation. Here, we introduce "STEpos" as light-responsive epothilone reagents, designed to photoswitchably bind to tubulin and stabilise lateral contacts in the microtubule lattice. Using a novel styrylthiazole photoswitch, designed to allow the hydrogen-bonding that is key to epothilone potency, we have created the first set of GFP-orthogonal photoswitchable microtubule stabilisers. The STEpos can photocontrol microtubule polymerisation, cell division, and cellular microtubule dynamics with micron- and second-scale spatiotemporal precision. STEpos offer substantial improvements of potency, solubility, and ease-of-use compared to the only previous photopharmaceuticals for microtubule stabilisation. The intriguing structure-photoswitching-activity relationship insights from this work will also assist future developments of improved STEpo reagents, and we anticipate that these will contribute greatly to high-precision cytoskeleton research across the fields of biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

cell biology↗

bioRxiv2021

In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential for in vivo translation across a variety of research fields and for all eukaryotes. However, photopharmacologys reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or to in vivo applications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes. Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminations in vivo to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar, in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing general in vivo photopharmacology one step closer to productive realisation.

cell biology↗