Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology

PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from D-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma. Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations. Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma

[5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerization in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes─themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization

Hyaluronan-based extracellular matrix under conditions of homeostatic plasticity

Neuronal networks are balanced by mechanisms of homeostatic plasticity, which adjusts synaptic strength via molecular and morphological changes in the pre- and post-synapse. Here, we wondered whether the hyaluronic acid-based extracellular matrix (ECM) of the brain is involved in mechanisms of homeostatic plasticity. We hypothesized that the ECM, being rich in chondroitin sulfate proteoglycans such as brevican, which are suggested to stabilize synapses by their inhibitory effect on structural plasticity, must be remodelled to allow for structural and molecular changes during conditions of homeostatic plasticity. We found a high abundance of cleaved brevican fragments throughout the hippocampus and cortex and in neuronal cultures, with the strongest labelling in perineuronal nets on parvalbumin-positive interneurons. Using an antibody specific for a brevican fragment cleaved by the matrix metalloprotease ADAMTS4, we identified the enzyme as the main brevican-processing protease. Interestingly, we found ADAMTS4 largely associated with synapses. After inducing homeostatic plasticity in neuronal cell cultures by prolonged network inactivation, we found increased brevican processing at inhibitory as well as excitatory synapses, which is in line with the ADAMTS4 subcellular localization. Thus, the ECM is remodelled in conditions of homeostatic plasticity, which may liberate synapses to allow for a higher degree of structural plasticity

Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision

Photoswitchably bioactive reagents, known as “photopharmaceuticals”, promise powerful applications in high-precision biological research. Yet most photoswitch scaffolds cannot be quantitatively bidirectionally photoisomerised, so they suffer from residual background activity that can confound experiments. We rationally designed photopharmaceuticals using the emerging near-quantitative photoswitch pyrrole hemithioindigo (PHTubs), to isomer-specifically inhibit the cytoskeletal protein tubulin. These PHTub reagents allow simultaneous visible-light imaging and photoswitching in live cells, where they could be used for cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is, as far as we know, the first use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resolution biological control in live cells. This work opens up new horizons for high-precision microtubule research using PHTubs; and shows the cellular applicability of the near-quantitative photoswitch pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biological targets. </div

Hyaluronan-based extracellular matrix under conditions of homeostatic plasticity

Neuronal networks are balanced by mechanisms of homeostatic plasticity, which adjusts synaptic strength via molecular and morphological changes in the pre- and post-synapse. Here, we wondered whether the hyaluronic acid-based extracellular matrix (ECM) of the brain is involved in mechanisms of homeostatic plasticity. We hypothesized that the ECM, being rich in chondroitin sulfate proteoglycans such as brevican, which are suggested to stabilize synapses by their inhibitory effect on structural plasticity, must be remodelled to allow for structural and molecular changes during conditions of homeostatic plasticity. We found a high abundance of cleaved brevican fragments throughout the hippocampus and cortex and in neuronal cultures, with the strongest labelling in perineuronal nets on parvalbumin-positive interneurons. Using an antibody specific for a brevican fragment cleaved by the matrix metalloprotease ADAMTS4, we identified the enzyme as the main brevican-processing protease. Interestingly, we found ADAMTS4 largely associated with synapses. After inducing homeostatic plasticity in neuronal cell cultures by prolonged network inactivation, we found increased brevican processing at inhibitory as well as excitatory synapses, which is in line with the ADAMTS4 subcellular localization. Thus, the ECM is remodelled in conditions of homeostatic plasticity, which may liberate synapses to allow for a higher degree of structural plasticity

Isoquinoline-Based Biaryls as a Robust Scaffold for Microtubule Inhibitors

We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicine domain inhibitors comprise a structurally diverse class of compounds offering highly desirable cytotoxic and vascular disrupting bioactivities. Current research on colchicine domain inhibitors is focused on improving in vivo robustness and tolerability: properties that are inextricably linked to the scaffold structure employed. The isoquinoline-based biaryl scaffold we now report offers high-potency tubulin inhibition with excellent robustness and druglikeness, allowing solubility, in vivo tolerability and facile late-stage structural diversification through a tolerant synthetic route. We have confirmed the tubulin-binding properties and mechanism of these isoquinoline-based biaryls through a series of cellular tests and established safe in vivo dosing parameters in mice. By addressing several problems facing the current families of inhibitors, we thus expect that this new scaffold will find a range of powerful in vivo applications towards translational use in cancer therapy. </div

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

Optical methods to modulate microtubule dynamics show promise for reaching the micron- and millisecond-scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under-developed. We introduce “STEpos” as GFP-orthogonal, light-responsive epothilone-based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen-bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron- and second-scale spatiotemporal precision. They substantially improve potency, solubility, and ease-of-use compared to previous optical microtubule stabilisers, and the structure-photoswitching-activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high-precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience