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

Abstract

<div> <div> <div> <p>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 (<b>PHTubs</b>), to isomer-specifically inhibit the cytoskeletal protein tubulin. These <b>PHTub</b> 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 <b>PHTubs</b>; 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. </p> </div> </div> </div>

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

“…We had initially produced SBTub3P, the phosphate prodrug of previous lead compound SBTub3, however its aqueous solubility was only moderate (<2 mg/mL) potentially due to aggregate formation by π-stacking of the planar compound. We synthesised SBTubA4P hoping that the out-of-plane central methoxy group would reduce π-stacking, which combined with the hydrophilicity of the three extra methoxy groups would give better solubility, as we have seen in other contexts 6,19 . Indeed, SBTA4P dissolved to at least 10 mM in water, which was to prove important in later assays.…”

“…(1) Compounds must permeate through two glia cell layers to reach mitotically active neural stem cells (neuroblasts). This forces the use of high bath concentrations and potent compounds: however, since surface and surrounding cells are exposed to far higher concentrations than central cells, only potent compounds with extremely high photoswitchability of bioactivity ("FDR"; discussed in 19 ) can be used: otherwise outer cells die, and morphology and physiology are lost. Indeed, we could use high concentrations of high-FDR SBTub2M (30 µM) in brain explants without noticeable toxicity.…”

“…enzymatic hydrolysis of cages). In the MT field, recent photoswitchable analogues of taxane 12 , epothilone 13 and colchicinoid 5,[14][15][16][17][18][19] MT inhibitors have all been applied to cellular studies. These photopharmaceuticals have enabled noninvasive, reversible optical control over MT dynamics and MT-associated downstream effects, with cell-specific spatial precision and sub-second-scale temporal precision, and have been brought to bear on research in embryology 20 , neuroscience 21 , and cytoskeleton 5 .…”

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

“…Diffusion occurs at different rates for different classes of drug and of photoswitch. For example, small colchicinoid SBTs seem to diffuse much faster (in 2D cell culture, timescale under 1 min) than small colchicinoid HTIs [7,15]; while larger taxol-or epothilone-based photoswitches seem to diffuse slowly (in 2D cell culture, timescale of minutes) although this apparent slowness might also be due to their stabilising mechanism. Note 5.…”

“…Intriguingly, these compounds seem to show much slower diffusion than comparable small molecules. [15,23,24] Their solubility is moderate at best; and they display at best only ca. 10-fold ratio of bioactivity between the isomers, whereas PSTs and SBTubs can have ratios ≥200 making the latter much simpler to use.…”