Biology subjects

Heise, C.

Publications and source records attributed to Heise, C..

5 recordsLinked to original sources

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↗

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

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↗

bioRxiv2019

Photoswitchable microtubule stabilisers optically control tubulin cytoskeleton structure and function

Small molecule inhibitors provide a versatile method for studies in microtubule cytoskeleton research, since tubulin is not readily amenable to functional control using genetics. However, traditional chemical inhibitors do not allow spatiotemporally precise applications on the length and time scales appropriate for selectively modulating microtubule-dependent processes. We have synthesised a panel of taxane-based light-responsive microtubule stabilisers, whose tubulin hyperpolymerisation activity can be induced by photoisomerisation to their thermodynamically metastable state. These reagents can be isomerised in live cells, optically controlling microtubule network integrity, cell cycle repartition, and cell survival, and offering biological response on the timescale of seconds and spatial precision to the level of individual cells. These azobenzene-based microtubule stabilisers offer the possibility of noninvasive, highly spatiotemporally precise modulation of the microtubule cytoskeleton in live cells, and can prove powerful reagents for studies of intracellular transport, cell motility, and neurodegeneration.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/778993v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (82K):\norg.highwire.dtl.DTLVardef@22a5forg.highwire.dtl.DTLVardef@2c018dorg.highwire.dtl.DTLVardef@119e288org.highwire.dtl.DTLVardef@677229_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗