Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Optically controlling MT inhibitors could be an elegant solution to this problem of specificity, since light can be applied with high spatiotemporal precision (). Small molecule approaches are particularly attractive, since genetic engineering is not required and dosing is straightforward, so their scope for practical applications to both research and medicine is extensive. Prior research toward the optical modulation of MT inhibitors has explored both photobleachable and photouncageable drugs (), and a photoactivation approach based on carbon-carbon double bond isomerization has been proposed (). However, the nonspecific toxicity of these designs may be significant, since short wavelengths and/or high light intensity are required, leading to side reactions and toxic byproducts (). Fundamentally though, such irreversibly triggered approaches cannot combat the diffusion of the active drug, so they will always suffer from limited spatiotemporal resolution. Spatially and temporally restricting bioactivity instead demands reversible, in situ switching over many off↔on cycles (). We here present a series of MT inhibitors that can be fully reversibly photoswitched by low-intensity visible light, to control microtubule structure, dynamics, and a range of MT-dependent processes in living cells and organisms, with the spatiotemporal precision of light.

Microtubules (MTs) are highly dynamic components of the cytoskeleton that play vital roles in a variety of cellular processes, including intracellular transport, cell motility, and proliferation (). As such, small molecule inhibitors that interfere with MT dynamics are indispensible tools in cell biology (). In medicine, they are one of the most clinically useful classes of chemotherapeutics, due to their antimitotic and pro-apoptotic effects (). However, the current inhibitors are nonspecific in the sense that their bioactivity cannot be either spatially or temporally directed, e.g., against selected cells and tissues, at defined times. This restricts their scope of application in research, as it prevents their use in spatially or temporally addressing the varied processes dependent on microtubule dynamics. In cancer medicine, this nonspecificity causes severe systemic side effects such as cardiotoxicity and neurotoxicity (), which limit the doses at which chemotherapeutics can be applied, thus impairing their therapeutic value (). Therefore, developing inhibitors of MT dynamics whose action can be targeted to specific cells at defined times is an important challenge ().

Lastly, we examined PSTs’ control over the MT cytoskeleton in mammalian tissue in vivo. We selected the highly water-soluble prodrug PST-1P to avoid needing a cosolvent, aiming at better in vivo compatibility. The cremaster muscle tissue of living mice was superfused () for 40 min with PST-1P, while being either illuminated at 390 nm with a low-power LED or else kept in the dark. Then, under red light conditions, animals were sacrificed and the tissue was excised, fixed, and stained for MTs. PST-1P destroyed the MT network under 390 nm illumination, but caused no disruptive effects in the dark ( Figure 6 C; Figures S5 A–S5D). This confirms the suitability of PSTs for optically defined MT depolymerization in mammalian tissue in vivo.

Immunofluorescence images of MT structure after treatment with 50 μM PST-1P applied in vivo to mouse cremaster tissue shows total MT disruption under 390 nm illumination (A), but the dark regime result (B) is identical to negative controls (C-D) showing no disruption. 3D rendered z-stacks of optical sections of 30 μm total thickness are shown. Tubulin staining is shown in yellow, nuclei are shown in red. Scale bars, 20 μm.

We next examined PST-1’s optical control over MT dynamics in vivo. We monitored mitotic progression of developing C. elegans embryo as a readout of functional microtubule dynamics. The synchronicity of several blastomeres at early developmental stages gives a useful internal control for the normal mitosis rate (). We used transgenic embryos with mCherry-tagged cell membrane marker PH and histone H2B to identify blastomeres and follow their cell-cycle progression. Embryos were bathed in PST-1, and individual cells within 8- to 32-cell stage embryos were targeted with millisecond pulses of 405 and/or 514 nm light, once the chromosomes organized into a single plate (entry to metaphase). Applying 405 nm pulses blocked the targeted cell in metaphase ( Figure 6 A; Movie S4 ), but cells targeted by a 405 + 514 nm rescue protocol divided normally ( Figure 6 B; Movie S5 ). Crucially, in both cases, neighboring cells continued mitosis unperturbed. Thus, PST-1 can achieve reversible, optical control over MT dynamics and its dependent processes in vivo, and can execute that control with single-cell spatial precision.

(C) PST-1P applied in living mouse tissue gives complete MT disruption under 390 nm light, but has no effects in the dark. The cremaster muscle in live C57BL/6 mice was superfused with 50 μM PST-1P or with PBS (“ctrl”) for 40 min while dark conditions or 390 nm were applied. Muscle was then excised, fixed, and stained for α-tubulin (yellow) and nuclei (red). 3D-rendered z-stacks of total thickness 30 μm are shown; scale bars, 15 μm. See also Figure S5

(A and B) PSTs achieve fully reversible optical control over mitosis within a living organism, with single-cell spatial precision (see full data in Movies S4 and S5 ). C. elegans embryos expressing mCherry::H2B and mCherry::PH were bathed in 40 μM PST-1, and a single cell in each embryo was illuminated at either 405 nm (blue ROI, toxic regime) or 405 + 514 nm (green ROI, rescue protocol). Cells illuminated by the toxic regime showed mitotic arrest (stationary chromosomes marked with red arrows), while in cells exposed to the rescue protocol, chromosomes continued to segregate (yellow arrows); neighboring cells continued mitosis unperturbed. Scale bars, 10 μm; time is shown relative to chromosome organization into the metaphase plate (start of illumination); two z-slices are given in (B) at t = 8 min, as the cells of interest had moved to distinct focal planes; see also Figures S4 B–S4G.

To directly visualize PSTs’ effects on MT dynamics in live cells, we imaged the end-binding protein EB3. EB3 clusters at the plus tips of growing MTs, and dissociates in phases of MT shrinkage. As its binding/unbinding kinetics are fast, EB3 imaging thus reveals MT growth dynamics (). We treated interphase cells expressing mCherry-tagged EB3 with PST-1, and imaged them while photoswitching PST-1 in situ with alternating 2 min phases of pulsed 405 nm and 514 nm illuminations. Control cells (without PST-1 treatment) showed no variation of comet behavior with the illumination phase. In contrast, under PST-1 treatment, applying 405 nm light led to the disappearance of EB3 comets in less than a second, while changing to a 514 nm phase restored comet size and dynamics—also in less than a second—and this switching was entirely reversible over many cycles ( Movies S1 and S2 Figure 5 A; see also Supplemental Information ). We used the MATLAB software u-track () to identify, follow, and quantify the EB3 comets from several independent movies ( Movie S3 ), conservatively analyzing for the total number of EB3 comets, as well as their lifetime, speed, and distance traveled. The combined statistics show that trans↔cis photoisomerizations of PST-1 inside living cells achieve optical switching of these functional parameters of MT polymerization dynamics, thus allowing fully reversible switching between phases of ordinary MT dynamics and of MT catastrophe simply by applying light ( Figures 5 B–5E). Together with the preceding results, these experiments indicate that PSTs are a robust and powerful tool for precise, rapid, reversible, and noninvasive optical control over both MT dynamics and MT-dependent processes, suitable for both short- and long-term use in a range of systems from in vitro to in cellulo.

(B–E) Statistical analysis of the number of EB3 comets (B), and their lifetime (C), speed (D), and total distance traveled (E) show that PST-1 treatment allows for full, reversible optical control over microtubule dynamics in cellulo, with MT dynamics being blocked under phases of blue illumination but resuming normal behavior under phases of green illumination. Grey bars represent data for cells illuminated but without PST-1 treatment, while colored bars represent cells illuminated and treated with PST-1 at 10 μM (for which the color indicates the phase’s illumination wavelength). Data were assembled from u-track analysis of multiple independent movies acquired as in (A), and are given as mean ± SEM for each phase, with phases arranged in chronological order; each phase lasted 2 min. The bleaching of the mCherry tag over the course of the experiment reduces the automatic detection of EB3 comets by u-track, which progressively reduces the apparent comet number determined by conservative analysis (B); for unbiased analysis consult the original data in Movies S1 and S2

(A) Reversible trans↔cis photoisomerization of PST-1 in cellulo by alternating phases of blue and green light causes microtubule dynamics to stop and start again, with <1 s response time and with full reversibility (see full data in Movies S1 and S2 ). MDA-MB-231 cells transiently expressing EB3-mCherry were incubated for 10 min in the dark with 10 μM PST-1 or with cosolvent control, then imaged at 561 nm under alternating phases of illuminations at 405 nm and 514 nm (images from the time-lapse sequence are shown in chronological order; scale bars, 20 μm).

We next assayed PST-1 for inhibition of tubulin polymerization in a biochemical assay using purified tubulin. PST-1 strongly inhibited in vitro tubulin polymerization under 390 nm illumination in a dose-dependent manner (EC∼5 μM), but exerted no inhibitory effects in the dark (EC>> 40 μM; Figure 4 B). Lastly, we confirmed PST-1’s functional potency as photoswitchable MT inhibitor in live cells by immunofluorescence imaging of endogenous tubulin. In the dark, PST-1 had no effects on MT structure, but under the toxic regime PST-1 caused dose-dependent MT depolymerization, as well as nuclear fragmentation that is typical of apoptotic cells, as seen with the reference compound CA4 ( Figure 4 C, Figure S4 A). This indicates that cis-PST-1 is a powerful inhibitor of tubulin polymerization both in vitro and in cellulo, while trans-PST-1 is not.

(A) Representative confocal microscope image showing microtubule network following CA4P treatment (15 nM final concentration). This positive control accompanies immunofluorescence microscopy images from Figure 4 C (α-tubulin is shown in green, nuclei are stained blue, white bar corresponds to a scale of 20 μm). (B-E) Controls for lighting parameters in the C. elegans experiments. Images show representative C. elegans embryos expressing mCherry::H2B and mCherry::PH, permeabilized and bathed in M9 buffer with or without 40 μM PST-1; all scale bars, 10 μm. (B) Embryos, incubated for 50 min (left), 120 min (middle), and 150 min (right), with PST-1 but without photoisomerization illumination, show normal mitotic progression. (C and D) The encircled cell has been illuminated for 2 to 5 ms every 30 s, using the 405 nm laser only (blue circle, C) or with twin pulses of 405 nm then 514 nm lasers (green circle, D), but without PST-1, showing no disruption of mitosis due to lighting protocol only. (E) Embryos incubated in 40 μM PST-1; the encircled cell has been illuminated for 2 to 5 ms every 30 s using the 514 nm laser, without giving mitotic arrest. (F-G) Positive control timelapse sequences showing C. elegans embryos expressing histone and tubulin fused to GFP, under treatment with the known MT inhibitor, colchicine. Embryos were permeabilized and bathed in M9 buffer, and colchicine was added at t = 0 s. (F) Colchicine added during metaphase. (G) Colchicine added during anaphase. Stationary chromosomes and moving chromosomes are shown with red and yellow arrows, respectively. Scale bar, 10 μm.

To validate tubulin as the molecular target of cis-PSTs, we first assayed the ability of PST-1 to bind to the colchicine domain on tubulin. We used purified tubulin in an in vitro radioligand scintillation proximity assay (SPA) () to examine the competitive displacement ofH-colchicine from its tubulin binding site by PST-1 under light and dark conditions. This assay confirmed that cis-PST-1 competes dose dependently with colchicine for tubulin binding, as does its isosteric parent drug CA4 ( Figure 4 A). The SPA returned an ECvalue of 30 μM for PST-1 under 390 nm illumination, with the reference compound CA4 showing higher affinity (EC= 0.16 μM), which mirrors the activity difference shown previously in the cytotoxicity assays. Importantly, trans-PST-1 showed no significant competitive binding to tubulin, further supporting the off↔on design conjecture of our study.

(C) Under the toxic regime, PST-1 induces MT breakdown and nuclear fragmentation dose-dependently, while under the dark regime, MT structure is unaffected (MDA-MB-231 cells treated for 20 hr with PST-1, then stained for α-tubulin (green) and DNA (blue); scale bars, 20 μm; see also Figure S4 A).

(A) PST-1 exposed to 390 nm competes with colchicine for tubulin binding, mirroring the effect of the CA4 positive control, while no such interaction can be detected in the dark (radioligand binding assay). Results are given as mean ± SD.

Taken together, these results support the interpretation that the cis-PSTs generated in situ upon blue light exposure potently induce mitotic arrest, presumably linked to the activation of the spindle assembly checkpoint, and result in cell death which displays many characteristics of apoptosis. By contrast, the trans-PSTs that are established under dark conditions (or which predominate under longer wavelength illumination) are all but inactive due to their ∼100-fold weaker cytotoxicity, which additionally does not involve mitotic arrest. Both these results are general across a range of cell lines, supporting the conjecture that PSTs should be appropriate for use in any eukaryotic system. These experiments thereby supported our overall design, showing that PSTs can be used as antimitotic cytotoxins that can be robustly and reversibly photoswitched in living cells between the essentially “off” trans form and the potent “on” cis form. We now turned our focus to testing the specific molecular premise of the PSTs’ design: by evaluating their capacity to effect MT disruption in situ in a reversibly photoswitchable manner.

Finally, cell-cycle analysis of MDA-MB-231 cells treated with PST-1 showed G/M phase arrest beginning sharply around 500 nM under the toxic regime ( Figures 3 D and 3E), matching the ECvalues seen in the cytotoxicity assays. By contrast, in the dark even 100 μM of PST-1 had no effect on cell-cycle repartition ( Figures S3 F and S3G). As G/M arrest is typical of MT-disrupting agents (), this supports the design conjecture that only cis-PSTs inhibit MT dynamics. Similar results were obtained in HEK293T (human embryonic kidney) and HeLa cells ( Figures S3 I and S3J). We also explored a dual wavelength “rescue protocol,” to illustrate repeated, dynamic photocontrol over PST cytotoxicity. In this protocol, each pulse contains a component at 390 nm immediately followed by another at 515 nm (so that the transiently formed cis is reisomerized back to trans during each pulse). The rescue substantially reduced G/M arrest compared to the toxic regime ( Figure S3 H). This highlights that PSTs can be reversibly and efficiently photoisomerized in cellulo in the long term (>5,000 trans→cis→trans photoswitching cycles over 2 days), without degradation of the drug and without phototoxicity to the cell.

We pursued further studies of the mechanism and light-dependency of PST-induced cytotoxicity focusing on PST-1 and its prodrug PST-1P. We first assayed cell membrane permeability (propidium iodide exclusion assay) and nuclear fragmentation (quantification of DNA content) in MDA-MB-231 cells. Neither assay showed a response to PST-1 below 50 μM in the dark regime. However, under the toxic regime, even submicromolar PST-1 induced loss of membrane integrity, and depletion of nuclear DNA content resulting in the emergence of hypodiploid (sub-G) cells ( Figures 3 A and 3B and Figure S3 E). Similar results were obtained in Jurkat (T cell lymphoma) and HeLa cells ( Figures S3 A–S3D). We subsequently examined cleavage of poly(ADP-ribose) polymerase (PARP). Full-length PARP (116 kDa) is involved in the repair of DNA damage caused by a variety of cellular stresses. During apoptosis, PARP is cleaved by caspase-3, caspase-7, and possibly by other suicidal proteases, into an 89-kDa catalytic fragment and a 24-kDa DNA-binding domain (). HeLa cells treated with PST-1P were analyzed by western blot and showed the PARP proteolytic signature typical of apoptosis only under the toxic lighting regime ( Figure 3 C).

(A) PST-1 induces cell membrane permeability in HeLa cells in a light-dependent manner; cells were treated for 70 hr with PST-1 under the dark and toxic regimes, and the percentage of PI positive cells in the total amount of cells determined. (B–E) PST-1 induces dose-dependent nuclear fragmentation in HeLa (B), Jurkat (C and D), and MDA-MB-231 cells (E) under the toxic regime only. Dose response curve for Jurkat cells (C) was fitted based on the raw data partially presented in (D), for which an EC= 79 nM was determined for PST-1 upon 390 nm illumination. Graph (E) represents data corresponding to dose response curves shown in Figure 3 B. (F–J) Light-dependent effects of PSTs on cell cycle. (F) When kept in the dark, PST-1 shows no influence on cell-cycle repartition even up to 100 μM (MDA-MB-231 cells, 48 hr of dark PST-1 treatment). (G) Prodrug PST-1P reproduces the cell-cycle arrest seen for PST-1 when illuminated with blue light; compare to Figure 3 E. (H) The rescue lighting regime reduces the effect on cell-cycle arrest exerted by PST-1 relative to what is experienced when only the 390 nm pulse component is used. Cells were pulsed with 390 nm light followed by 515 nm pulse that greatly decreased the effective cis-PST-1 concentration and contributed to reduction in G/M arrest. (I–J) PST-1 provokes light-dependent G/M phase arrest in HEK293T (I) and HeLa (J) cells.

We then investigated the dependency of the PSTs’ bioactivity on the irradiating wavelength in detail. We examined the proliferation of HeLa cells exposed to PST-1P, after pulsed illuminations at a range of wavelengths from 370–535 nm, by MTT assay ( Figures 2 C and 2D). Illumination at 380–390 nm gave the most potent cytotoxicity, while the dose-response curves for wavelengths up to 525 nm were translated progressively higher concentrations by factors that match the relative trans/cis ratios at those wavelengths ( Figure S1 C). This supports the conclusions that the choice of illuminating wavelength determines the concentration of the cis form generated, and that this cis concentration is the primary determinant of the PSTs’ bioactivity. For comparison, the trans isomer (assayed under dark conditions) was confirmed to be > 250 times less toxic than the cis form, and also showed a markedly shallower dose-dependency that could argue for a different biochemical mechanism of toxicity ( Figure 2 D, Figure S1 C, and Supplemental Information , Part C1). These experiments indicate how the PSTs’ biological effects can be not only sharply controlled (illuminated or dark), but also finely tuned (wavelength dependence) by lighting conditions.

(D) Dose-response curves for PST-1P are horizontally translated according to the irradiating wavelength used (same data as in C; see also Figure S1 C).

(B) PSTs are between 20- to 100-fold more cytotoxic under 390 nm illuminations than in the dark (same data as in A; see also Figures S2 A–S2I).

The potency, robustness, and light-specificity of the PSTs’ biological effects were directly assessed in cellulo. Trans-PSTs were assayed by shielding experiments from light (100% trans; “dark regime”), while cis-PSTs were assayed by applying a “toxic regime” of frequently pulsed, short illuminations (e.g., 75 ms pulses of 390 nm light every 15 s). To apply these illuminations in cell culture, we hand-built a cheap, computerized LED lighting system to illuminate > 30 well plates separately with independent wavelengths and timings ( Figure S6 and Supplemental Information ). Crystal violet assays in the MDA-MB-231 human breast cancer cell line showed that PSTs are powerfully cytotoxic under the toxic lighting regime, but not in the dark. Their ECvalues of 0.5–5.4 μM under the toxic regime represent potencies up to 100 times greater than under the dark regime ( Figures 2 A and 2B and Figures S2 A–S2I), and closely similar results were obtained for the PSTs’ light-dependent cytotoxicity in HeLa (cervical cancer) cells ( Figures S2 J–S2N).

(A-I) Cytotoxicity profiles of all PSTs in the dark and upon illumination with 390 nm light under the toxic regime, assessed in MDA-MB-231 cells by crystal violet staining after 48 hr. Profiles of PST prodrugs PST-1P, PST-1CL and PST-2S are presented in comparison to the toxic regime profiles of their active cores. The profile of peptidase-triggered stilbene prodrug CA4CL is compared to that of CA4P as a control of the cyclisation prodrug strategy. Data were summarized in Figure 2 B. (J-N) The light-dependent cytotoxicity of PSTs as shown by MTT assay in HeLa cells after 45 hr incubation with PSTs under dark or toxic regimes (vertical axis is the formazan absorption readout); the assay with PST-1 used 1% MeCN as cosolvent, the other experiments used 1% DMSO. (O) MTT assay in HEK293T cells after 72 hr incubation with PST-1 under different lighting pattern timings at 390 nm. Results illustrate the dependency of observed cytotoxicity upon the specific pulse program as opposed to the time-averaged photon flux (e.g., compare programs 3-5).

The switching of the PSTs was demonstrated by UV-Vis spectroscopy ( Figure 1 D and Supplemental Information , Part B). All compounds were rapidly and fully reversibly photoswitched in phosphate-buffered saline (PBS) buffer, without degradation over hundreds of cycles, by low-power illumination. Their para-methoxy substituents enable PSTs to be trans↔cis photoswitched using visible light (), benefiting in vivo compatibility: 380–420 nm light gives approximately 90% cis isomer, which bears the CDI pharmacophore; longer wavelengths give decreasing cis percentages until 500–530 nm gives approximately 85% trans isomer, which does not bear the pharmacophore ( Figure S1 A). In the dark, spontaneous (unidirectional) cis→trans isomerization leads to 100% trans isomer, with half-lives τ, ranging from 0.8–120 min ( Figure S1 B), being modulated by the substitution pattern (). This process acts as a safety mechanism, whereby the concentration of bioactive cis isomer will drop to zero unless blue light pulses are reapplied on the timescale of this reversion half-life. Therefore, even without using green light to actively switch off the PSTs, any cis-PST diffusing into a non-illuminated area, or remaining after a period of illumination, will spontaneously and rapidly be isomerized to trans, thus spatiotemporally restricting the PSTs’ inhibitory bioactivity to illuminated zones only.

(A) Absorption spectra of trans (solid lines) and cis (dotted lines) isomers of the non-prodrug PSTs (PST-1 – PST-5) in water-acetonitrile solution (upper), and their φ(λ) (dotted lines) and E(λ) (solid lines) (lower), determined as per supplementary sections B.1-B.2. (B) τ, λand ε(λstrong) determined as per supplementary sections B.1 and B.4. (C) Summary results from MTT assay in HeLa cells after 44 hr incubation with PST-1P under lighting with different wavelengths but constant pulse timings. Results illustrate the predictable dependency of observed cytotoxicity upon wavelength. That the observed cytotoxicity is governed by the underlying [cis-PST-1] with an ECof about 0.68 μM in this assay, is supported since the back-calculated PSS(λ) values [PSS (analysis)] at different wavelengths are reliably correlated with the calculated φ(λ) values from Figure S1 A [PSS (model)]. (D) Model comparing an on-target dose profile OTD(t) which can be established for a cis-PST to the typical PK pattern of an always-on drug. (E) Two-wavelength active photoisomerization concept for in vivo spatial restriction of photoswitchable cytotoxins to a specific zone, illustrating the importance of assaying the toxic regime (left zone), the rescue protocol (overlap zone), green-only illumination (central zone) and the dark regime (right zone) as the limiting cases for the cytotoxicity experienced in each area.

We synthesized a series of PSTs (PST-1–PST-5) by diazonium coupling, in two to four synthetic steps ( Figure 1 and Supplemental Information ) (). Considering the solubility problems that have hampered the combretastatins, we also prepared PST-1P and PST-2S, as azobenzene analogs (“azologues”) of two nonspecific combretastatin prodrugs that have entered Phase III clinical trials: PST-1P is an azologue of CA4 phosphate (known as CA4P or “fosbretabulin”) and PST-2S is an azologue of the serinyl anilide “ombrabulin” (). Lastly, PST-1CL was synthesized to explore dual optical-and-biochemical targeting via exopeptidases ().

We designed the photostatins (PSTs) as reversibly photoswitchable analogs of combretastatin A-4 (CA4) (). CA4 is one of the most prominent colchicine domain MT inhibitors (CDIs). Its potency, its vascular disrupting and antiangiogenic properties, and its avoidance of multidrug-resistance make it a promising candidate for tumor chemotherapy, and three CA4 derivatives have progressed to clinical trials (). The CA4 pharmacophore is a trimethoxybenzene ring held cis to a low-steric-demand, methoxy-bearing arene (). Crucially, trans-CA4 is several orders of magnitude less potent than the cis isomer (). We replaced the bridging C=C double bond of the stilbenoid combretastatins with an isosteric N=N double bond to give the azobenzene PSTs, which can be trans↔cis photoisomerized with full reversibility and excellent photostability over many cycles by low intensity visible light ( Figure 1 ). We anticipated that the cis-PSTs would reproduce the valuable pharmacology of CA4, while their trans isomers would be biologically inactive. We aimed to reversibly photoswitch PSTs between the cis and trans isomeric forms in situ, to turn their bioactivity on and off, with high spatiotemporal precision, inside living cells and organisms.

Discussion

Fehrentz et al., 2011 Fehrentz T.

Schönberger M.

Trauner D. Optochemical genetics. Dumontet and Jordan, 2010 Dumontet C.

Jordan M.A. Microtubule-binding agents: a dynamic field of cancer therapeutics. Bhattacharyya et al., 2008 Bhattacharyya B.

Panda D.

Gupta S.

Banerjee M. Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin. Biological implementations of photoswitchable small molecules have traditionally focused on transmembrane proteins, usually expressed in neurons, whose inherently nonlinear response has contributed to many successful applications (). Here, we show that photopharmacology also offers valuable applications to intracellular targets such as the microtubule cytoskeleton, which is highly conserved and fundamental to all eukaryotes (). The PSTs embed a photoswitch inside the CDI pharmacophore, generating minimal-complexity cell permeant compounds which are straightforward to synthesize and can be applied as water-soluble prodrugs. The PSTs have reproduced the MT disrupting, cytostatic, and cytotoxic effects of their CDI isosteres () through in vitro, in cellulo, and in vivo assays, with the novel advantage that PSTs can spatially and temporally target these effects with full reversibility.

PSTs can exploit visible light trans↔cis photoswitching, combined with spontaneous cis→trans relaxation, to create steep cis/trans spatiotemporal gradients even in vivo. Since the cis-PSTs are two orders of magnitude more potent than the trans-PSTs, this allows them to apply their strong bioactivity in a highly localized fashion. The PSTs’ temporal specificity on the order of seconds, and spatial specificity down to at least the single-cell level, enable a variety of precise and fully reversible studies which are inaccessible to the current MT inhibitors. We envision that finely localized light delivery, possibly combined with substitution patterns that give faster spontaneous cis→trans relaxation, may also allow PSTs to achieve subcellularly localized MT inhibition. The reversibility of the PSTs’ bioactivity allows quick recovery of normal MT function after PST treatment, so studies may establish not only the short-term consequences of inhibiting MT-dependent processes, but also their long-term effects on the whole organism. We therefore expect that PSTs will prove useful tools for spatiotemporally precise research into a range of MT-dependent processes, including intracellular transport, synaptic plasticity, cell division, motility, invasion, and angiogenesis, revealing new insights in cellular and developmental biology as well as in vivo physiology.