Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative

Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a... Abstract In this study, we synthesized a novel photochromic inhibitor of the mitotic kinesin Eg5, which is composed of the photochromic compound spiropyran to photo-control the function of Eg5. The compound (S)-2, 3-dispiropyran propionic acid (DSPPA) exhibits reversible spiropyran–merocyanine photo-isomerization upon irradiation with visible or ultra-violet light. DSPPA induced reversible changes in the inhibitory effect on Eg5 ATPase and motor activities, which correlates with the spiropyran–merocyanine photo-isomerization. Microtubule-dependent ATPase activity was significantly more inhibited by the spiropyran isomer of DSPPA than by the merocyanine isomer. Additionally, an in vitro motility assay revealed that the microtubule gliding velocity was reduced more by the spiropyran isomer than by the merocyanine isomer. This indicates that the spiropyran derivative may be useful in regulating the function of the mitotic kinesin. Eg5, inhibitor, kinesin, photo-control, photochromic molecule The mitotic kinesin Eg5 is a member of the kinesin-5 family and a plus-end directed homotetrameric molecular motor. Eg5 plays an important role in regulating centrosome separation and formation of the bipolar spindle apparatus (1, 2). Inhibition of Eg5 induces monopolar spindles and eventually apoptosis. Therefore, Eg5 is an important drug target in anti-mitotic cancer therapy (3, 4). Eg5-specific inhibitors exhibit improved safety profiles compared with other traditional anti-mitotic drugs targeting the multi-functional relevant microtubule (MT) (5). It is known that there are several small-molecule inhibitors of Eg5. Monastrol was the first Eg5-specific inhibitor to be characterized (3). S-trityl-L-cysteine (STLC) is also a potent inhibitor that binds to Eg5 much more tightly than monastrol does (6, 7). The molecular mechanism of the inhibition of Eg5 by STLC has been well-studied (8). Other potent inhibitors, such as ispinesib, have also been reported (9). Interestingly, although the structures of the inhibitors are not similar, they bind to the same pocket, which is composed of the L5 loop, α2 helix and α3 helix near the ATPase site (10). For the inhibitors, the single bond linkage between aromatic rings and hydrophilic groups on the aromatic rings were observed as a common structure. Previously, we photo-reversibly controlled the ATPase activity of Eg5 using photochromic molecules that were incorporated into the entrance region of the Eg5 inhibitor binding site (11). Eg5 was modified with photochromic molecules that exhibited photo-reversible changes in the ATPase activity upon irradiation with ultra-violet (UV) and visible light (VIS). However, chemical modification to Eg5 with photochromic molecules to achieve photo-control has not been adapted for application in living cells. Consequently, we have synthesized a photochromic azobenzene derivative, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)- azobenzene (ACTAB), which is a novel photochromic inhibitor of Eg5 (12). The compound is composed of a trithyl group, which acts as the key moiety in STLC inhibition of Eg5. The ATPase and motor activities of Eg5 were effectively controlled using this photo-reversible photochromic inhibitor. We have demonstrated that photochromic compounds can be used as a photo-switching molecular control for the Eg5 inhibitor. Previously, we demonstrated that the azobenzene derivative inhibitor, ACTAB, photo-reversibly inhibited the ATPase activity of Eg5 (12). Consequently, other photochromic compounds with structures similar to potent Eg5 inhibitors previously reported may also be applicable as photochromic inhibitors for Eg5. We used spiropyran as a photochromic molecule to reversibly inhibit Eg5 because the molecular structure of spiropyran resembles that of the potent Eg5 inhibitor ispinesib (9). In this study, we used a spiropyran derivative to synthesize a novel photo-switching Eg5 inhibitor. The novel photochromic inhibitor photo-isomerizes upon UV and VIS irradiation. Furthermore, we successfully controlled the activity of the spiropyran derivative with light. It is expected that the novel photochromic Eg5 inhibitor composed of the spiropyran derivative has a potential use in achieving photo-control of cell division, and therefore a potential use in cancer therapy. Materials and Methods Synthesis of photochromic Eg5 inhibitor (S)-2, 3-dispiropyran-propionic acid (DSPPA) was synthesized by the established coupling reaction between 1-(β-carboxyethyl)-3′, 3′-dimethyl-6–nitrospiro (indoline-2′, 2 (2H-1) benzopyran) (spiro (SP)-COOH) (13) and (S)-(+)-2, 3-Diaminopropionic acid. Briefly, prior to the coupling reaction, the N-hydroxy succinimide ester (NHS-ester) of SP-COOH was prepared. SP-COOH (78.0 µmol) was activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (780 µmol) for 10 min in 3.0 ml N, N-dimethylformamide (DMF), and then N-hydroxy succinimide (780 µmol) was added. The mixture was stirred with a magnetic stirrer at room temperature for 3 h. The reactant was evaporated to dryness and the SP-COOH-NHS-ester was purified by flash column chromatography packed using a silica gel in a solvent of 20% methanol and 80% chloroform. Subsequently, the purified SP-COOH-NHS-ester was reacted with (S)-(+)-2, 3-Diaminopropionic acid. The SP-COOH-NHS-ester (20.0 µmol) in 3 ml DMF was added to a solution of (S)-(+)-2, 3-Diaminopropionic acid hydrochloride (40.0 µmol) in 1.5 ml of 50 mM trimethylamine-bicarbonate (TEA-HCO3) at pH 8.5 with vigorous stirring. After reacting for 3.0 h at room temperature, the reactant was evaporated to dryness. The product was purified by high performance liquid chromatography (HPLC) using C18 reverse-phase column chromatography with a linear gradient of 0–90% ethanol in 0.1 M TEA-HCO3 at pH 7.5. Further purification was performed using flash column chromatography packed with ultra-pure Wakogel C-200 in 20% methanol and 80% chloroform developing solvents. The purity of the products was analysed by silica gel TLC with 20% methanol and 80% chloroform developing solvent. The Rf value of DSPPA was 0.64. Fast atom bombardment mass spectrum of the product showed a molecular ion (M + H)+ at m/z 828 + 1, which corresponds to a molecular mass of 828,865 as calculated from the formula of C45H44N6O10 of DSPPA. Photo-isomerization of DSPPA Photo-isomerization of the Eg5 inhibitor DSPPA was performed by irradiation at 366 nm UV and VIS. The UV and VIS lights were used for conversion of the spiropyran-form (SP-form) to the merocyanine-form (MC-form) and the MC-form to the SP-form, respectively. The photo-isomerization of DSPPA was performed under a light source at distance of 1 cm from the surface of the solvents for the DSPPA dose-dependent ATPase inhibition assay. For the assay of reproducible reversibility of the photo-control of the MT-stimulated ATPase activity, the photo-isomerization of DSPPA was performed with a light source 10 cm from the surface of the solvents. Expression and purification of the kinesin Eg5 motor domain The cDNA of the motor domain of mouse wild-type (WT) Eg5 (WT, amino residue 1-367) was amplified by polymerase chain reaction and ligated into a pET21a vector. The Eg5 WT expression plasmids were used to transform Escherichia coli BL21 (DE3). Eg5 WT was purified by using a Co-nitrilotriacetic acid (NTA) column. The Co-NTA column was washed with lysis buffer containing 30 mM imidazole; bound Eg5 was eluted with lysis buffer containing 150 mM imidazole. The elutant fractions were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified Eg5 was dialysed in buffer (30 mM Tris-HCl at pH 7.5, 120 mM NaCl, 2.0 mM MgCl2, 0.1 mM adenosine triphosphate (ATP) and 0.5 mM dithiothreitol) and stored at −80°C. Expression and purification of the truncated conventional kinesin・kinesin-1 containing motor doamin Expression and purification of the N-terminal 560 amino acids of mouse brain kinesin (KIF5A) containing motor domain were performed according to the established methods we have previously reported (14). The plasmid pET21a carried a mouse conventional kinesin construct comprising residues 1-560 with a C-terminal 6 His-tag. This plasmid (pET21a: kinesin 1-560) was transformed into E.coli BL21 (DE3) cells for expression. Expressed kinesin-1 was purified by using a Co-NTA column. The purified kinesin-1 was dialysed and stored in the same manner as Eg5. Purification and polymerization of tubulin Tubulin was purified from porcine brains using the method described by Hackney (15). Tubulin was polymerized for 30 min at 37°C in a buffer containing 100 mM piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES) (pH 6.8), 1.0 mM ethyleneglycol-bis(β-aminoethyl)-N, N, N′, N′-tetraacetic acid (EGTA), 1.0 mM MgCl2 and 1.0 mM guanosine triphosphate (GTP). Taxol was then added to a final concentration of 10 µM. The polymerized MTs were collected by centrifugation at 280,000 × g for 15 min at 37°C. The supernatant was removed, and the pellet was collected in a buffer containing 100 mM PIPES at pH 6.8, 1.0 mM EGTA, 1.0 mM MgCl2, 1.0 mM GTP and 10 μM taxol. For the MT gliding assay, MTs were labelled with rhodamine. Rhodamine-labelled tubulin was mixed with unlabelled tubulin at a ratio of 1:5 and polymerized for 40 min at 37°C in the buffer (100 mM PIPES, pH 6.8, 2.0 mM EGTA and 1.0 mM MgSO4). Taxol was then added to a final concentration of 20 µM. Eg5 ATPase assay The ATPase activity of Eg5 was measured at 25°C in an assay solution containing 0.1 µM Eg5 (MT-stimulated ATPase) or 0.5 µM Eg5 (basal ATPase), 20 mM 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES) (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM (ethylenedinitrilo)tetraacetic acid (EDTA), 0.1 mM EGTA and 1.0 mM β-mercaptoethanol in the presence or absence of 3.0 µM MTs. To examine the effect of the photo-isomerization of the inhibitor, DSPPA dissolved in DMF was irradiated by UV or VIS light and added to the assay buffer containing Eg5 to a final concentration of 0.0–10.0 µM. The conditions for the photo-isomerization of DSPPA are as described in the earlier section, ‘Photo-isomerization of the DSPPA’. The final DMF concentration in the Eg5 ATPase buffer was 5.0%. The ATPase reaction was started by adding 2.0 mM ATP and terminated by adding 10% trichloroacetic acid. The released inorganic phosphate (Pi) in the supernatant was measured using the method of Youngburg (16). To determine KMT and Vmax for MT concentration in the presence of DSPPA, the MT concentration-dependent ATPase activities (0.0–5.0 µM) were measured in the presence of 10.0 µM DSPPA. Microtubule gliding assay Coverslips were coated with an anti-6× histidine monoclonal antibody (Wako) in the assay buffer (10 mM Tris-acetate at pH 7.5, 50 mM potassium-acetate, 2.5 mM EGTA and 4.0 mM MgSO4). Subsequently, 0.1 µM Eg5 in assay buffer A (10 mM Tris-acetate at pH 7.5, 50 mM potassium-acetate, 2.5 mM EGTA, 4.0 mM MgSO4, 0.5 mg/ml Casein and 0.2% β-mercaptoethanol) was added through the flow chambers, and the chambers were incubated for 5 min. After washing the chambers with assay buffer A, rhodamine-labelled MTs in assay buffer B (assay buffer A with 20.0 µM taxol) were infused through the flow chambers, and the chambers were incubated for 2 min. The chambers were then washed with assay buffer B. Next, 1.0 mM ATP and 10.0 µM photo-isomerized DSPPA in assay buffer C (assay buffer B with 1.5 mg/ml glucose, 0.01 mg/ml catalase and 0.05 mg/ml glucose oxidase) were infused through the flow chambers. The photo-isomerization of DSPPA was performed as described in the earlier section, ‘Photo-isomerization of DSPPA’, prior to adding to the motility assay buffer. The final DMF concentration in the assay buffer was 5.0%. Rhodamine-labelled MTs were visualized using an Olympus BX50 microscope equipped with a charge coupled device (CCD) camera (LK-TU53H: Toshiba, Japan). Statistical analysis Data are expressed as mean ± SE. Statically significant differences between groups was determined by using either the Welch’s t-test or Student’s t-test. P-values of < 0.05 were considered statistically significant. Results Photo-isomerization of the spiropyran derivative photochromic inhibitor of Eg5 To control photo-reversibly the ATPase and motor activities of Eg5, we designed and synthesized a novel photochromic Eg5 inhibitor, DSPPA (Fig. 1). The synthesis of DSPPA was performed by established methods as described in the Materials and Methods. The inhibitor is composed of two photochromic spiropyran moieties with a polar carboxylic acid. DSPPA contains two spiropyran moieties that undergo reversible SP and MC isomerization with significant conformational changes upon irradiation with VIS and UV as shown in Fig. 1. The isomerization of spiropyran has been used to photo-regulate other enzymes’ activity (17). Therefore, it is strongly expected to have a significant photo-reversible effect on the inhibition of Eg5. Fig. 1 View largeDownload slide Structures of DSPPA and its photo-isomers. The spiropyran derivative DSPPA undergoes reversible photo-isomerization between the spiro- and merocyanine-isomer after irradiation with visible or UV light, respectively. Fig. 1 View largeDownload slide Structures of DSPPA and its photo-isomers. The spiropyran derivative DSPPA undergoes reversible photo-isomerization between the spiro- and merocyanine-isomer after irradiation with visible or UV light, respectively. Figure 2 shows the absorption spectra changes that accompany the photo-isomerization of DSPPA. It is known that the conversion between the SP and MC states of spiropyran derivatives can be monitored by changes in the UV/VIS light spectrum around 500 nm (18). The SP isomer of DSPPA showed almost no absorption around 500 nm in the same manner as the other spiropyran. In total, 366 nm UV irradiation induced conversion of the SP to the MC state, and the absorption maximum at 560 nm increased in a time-dependent manner as shown in Fig. 2A. However, the VIS irradiation of the MC-form of DSPPA resulted in the conversion to the SP-form and a significant reduction in the absorbance peak at 560 nm (Fig. 2B). The time course of spectral changes upon UV and VIS light irradiations in an aqueous solution were almost similar to those in DMF (Supplementary Fig. S1). Interestingly, DSPPA dissolved in an aqueous solution also showed reverse photochromism (19). In the dark, the conversion of DSPPA from the SP-form to the MC-form occurred spontaneously, resulting in an increase in the absorption maximum at 520 nm (Fig. 2C). However, DSPPA in DMF converted from the MC to SP state as a normal photochromism and the absorption maximum at 520 nm was not increased in the dark (Supplementary Fig. S2). The reverse photochromism of DSPPA in the aqueous solution enabled us to convert between the SP and MC state without using UV light. Fig. 2 View largeDownload slide Absorption spectra changes of DSPPA accompanied by its photo-isomerization. (A) Time-dependent changes in the absorption spectra of SP-DSPPA to MC-DSPPA in DMF upon irradiation at 366 nm. In this experiment, 5.0 μM DSPPA was irradiated for 0, 30, 60, 120, 180, 240 and 300 s at 25°C. (B) Time-dependent changes in the absorption spectra of MC-DSPPA in DMF upon VIS irradiation for 0, 10, 20, 30, 60, 120 and 180 s at 25°C. (C) Time-dependent spectral changes of SP-DSPPA to the MC-form in an aqueous solution of 20 mM HEPES-potassium hydroxide (KOH) (pH 7.20), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA and 0.1 mM EDTA in the dark at 25°C. Fig. 2 View largeDownload slide Absorption spectra changes of DSPPA accompanied by its photo-isomerization. (A) Time-dependent changes in the absorption spectra of SP-DSPPA to MC-DSPPA in DMF upon irradiation at 366 nm. In this experiment, 5.0 μM DSPPA was irradiated for 0, 30, 60, 120, 180, 240 and 300 s at 25°C. (B) Time-dependent changes in the absorption spectra of MC-DSPPA in DMF upon VIS irradiation for 0, 10, 20, 30, 60, 120 and 180 s at 25°C. (C) Time-dependent spectral changes of SP-DSPPA to the MC-form in an aqueous solution of 20 mM HEPES-potassium hydroxide (KOH) (pH 7.20), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA and 0.1 mM EDTA in the dark at 25°C. Photo-control of Eg5 inhibitory activity The inhibitory activity of the SP and MC forms of DSPPA on the basal ATPase activity of Eg5 was examined. DSPPA potently inhibited the ATPase activity of Eg5. Furthermore, a difference in the inhibitory activity between the SP and MC isomers was observed. To determine the half-maximal inhibitory concentration (IC50), the rate of ATP hydrolysis of the Eg5 motor domain was measured in the absence of MTs and in the presence of increasing concentrations of the SP- or MC-form of DSPPA. Both DSPPA isomers inhibited the basal ATPase activity of the Eg5 motor domain in a concentration-dependent manner as shown in Fig. 3A. The IC50 value of the SP isomer was 2.09 ± 0.10 μM, whereas that of the MC isomer was 3.67 ± 0.12 μM. Fig. 3 View largeDownload slide Effects of DSPPA isomers on the basal and MT-stimulated ATPase activity of Eg5. (A) The rate of basal ATP hydrolysis in the presence of 0.5 μM Eg5 was measured in a solution of 20 mM HEPES-KOH at pH 7.20 containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP as a function of SP-DSPPA (○) or MC-DSPPA (•) concentration at 25°C. (B) The rate of MT-stimulated ATP hydrolysis at various concentrations of DSPPA was measured in the presence of 3.0 µM MTs, 2.0 mM ATP and SP-DSPPA (○) or MC-DSPPA (•) in a buffer of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Each data point is the average of three independent trials. (Data are expressed as mean ± SE, n = 3). Fig. 3 View largeDownload slide Effects of DSPPA isomers on the basal and MT-stimulated ATPase activity of Eg5. (A) The rate of basal ATP hydrolysis in the presence of 0.5 μM Eg5 was measured in a solution of 20 mM HEPES-KOH at pH 7.20 containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP as a function of SP-DSPPA (○) or MC-DSPPA (•) concentration at 25°C. (B) The rate of MT-stimulated ATP hydrolysis at various concentrations of DSPPA was measured in the presence of 3.0 µM MTs, 2.0 mM ATP and SP-DSPPA (○) or MC-DSPPA (•) in a buffer of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Each data point is the average of three independent trials. (Data are expressed as mean ± SE, n = 3). The MT-dependent ATPase activity of Eg5 was also significantly inhibited by DSPPA as shown in Fig. 3B. The IC50 value for the SP isomer of DSPPA for the ATPase activity of Eg5 in the presence of MTs was 3.77 ± 0.27 μM. However, the MC isomer of DSPPA had a lower inhibitory activity and an IC50 of 7.67 ± 0.31 μM. The changes in the inhibitory activity of the SP and MC isomers of DSPPA induced by VIS and UV irradiation was completely and reproducibly reversible as shown in Fig. 4. To avoid degradation of inhibitor by repeated UV irradiations, we employed the weaker UV light irradiation in the experiment of Fig. 4 than that in Fig. 3 as described in Materials and Methods. Therefore, the conversion from SP to MC isomer is not achieved completely and the ATPase activity under UV irradiation is lower than that at the same concentration of DSPPA in Fig. 3. Fig. 4 View largeDownload slide Reproducible reversibility of the photo-control of the MT-stimulated ATPase activity. DSPPA was alternately irradiated with 366 nm UV and VIS prior to the ATPase assay. The ATPase activity was measured in the presence of 3 μM MTs, 2.0 mM ATP and 7.5 μM SP or MC-DSPPA in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Alternation of UV and VIS was repeated three times. The MT-stimulated ATPase activity was measured in three independent experiments. Fig. 4 View largeDownload slide Reproducible reversibility of the photo-control of the MT-stimulated ATPase activity. DSPPA was alternately irradiated with 366 nm UV and VIS prior to the ATPase assay. The ATPase activity was measured in the presence of 3 μM MTs, 2.0 mM ATP and 7.5 μM SP or MC-DSPPA in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Alternation of UV and VIS was repeated three times. The MT-stimulated ATPase activity was measured in three independent experiments. We confirmed the specific inhibition of Eg5 by DSPPA using recombinant conventional kinesin・kinesin-1 (KIF5A). For truncated kinesin-1(1-560 amino acids), DSPPA did not exhibited a photo-reversible change in inhibitory activity accompanied by SP-MC isomerization (Supplementary Fig. S3). We also examined the influence of DSPPA on the interaction between Eg5 and MTs. Figure 5 shows the MT concentration-dependent ATPase activity of Eg5 in the presence of SP or MC-DSPPA. Both isomers decreased the Vmax to almost the same level as that of the ATPase activity in the absence of isomers. However, in the presence of the SP isomer of DSPPA, Eg5 showed a lower affinity for MTs than that in the presence of the MC isomer. Fig. 5 View largeDownload slide Effect of DSPPA isomers on the affinity of Eg5 for MTs. The inhibitory effects of DSPPA photo-isomers on Eg5 ATPase activity as a function of the MT concentration was measured in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol with 0.1 μM Eg5, 2.5 μM of SP-DSPPA (○) or MC-DSPPA (•) and 0–5 μM MTs. Fig. 5 View largeDownload slide Effect of DSPPA isomers on the affinity of Eg5 for MTs. The inhibitory effects of DSPPA photo-isomers on Eg5 ATPase activity as a function of the MT concentration was measured in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol with 0.1 μM Eg5, 2.5 μM of SP-DSPPA (○) or MC-DSPPA (•) and 0–5 μM MTs. From the data, we concluded that the optimal conditions for DSPPA to control the MT-dependent ATPase activity as a photo-switch is 5.0–6.0 μM DSPPA in the presence of 1.0–1.5 μM MTs (Figs 3B and 5). Photo-control of the Eg5-driven MT gliding velocity We examined the photo-control of the Eg5 motor activity using SP- and MC-DSPPA isomers. An in vitro motility assay using rhodamine-labelled MTs was performed according to established methods as described in the Materials and Methods. The velocity of the MTs gliding on Eg5-coated glass surfaces in flow cells was measured in the presence of SP or MC-DSPPA. The average MT gliding velocity (in the absence of DSPPA) was 23.22 ± 0.381 nm/s (Fig. 6A). The histogram of the velocity in the presence of DSPPA showed a slightly broader distribution. In the presence of SP-DSPPA, the average velocity significantly decreased to 15.70 ± 0.535 nm/s as shown in Fig. 6B. However, in the presence of MC-DSPPA, the average velocity decreased slightly (21.04 ± 0.436 nm/s) (Fig. 6C) compared with the control. Fig. 6 View largeDownload slide Photo-control of the motor activity of Eg5 with DSPPA. Histogram of the distribution of the MT gliding velocities on Eg5 in the in vitro motility assay (A) in the absence of DSPPA (mean velocity 23.22 ± 0.38 nm/s, n = 35), (B) in the presence of 5 μM SP-DSPPA (mean velocity 24.6 ± 0.54 nm/s, n = 77) and (C) in the presence of 5 mM MC-DSPPA (mean velocity 21.04 ± 0.44 nm/s, n = 77). The in vitro motility assay and the statistical analysis of the data were performed according to the methods described in the Materials and Methods section. Fig. 6 View largeDownload slide Photo-control of the motor activity of Eg5 with DSPPA. Histogram of the distribution of the MT gliding velocities on Eg5 in the in vitro motility assay (A) in the absence of DSPPA (mean velocity 23.22 ± 0.38 nm/s, n = 35), (B) in the presence of 5 μM SP-DSPPA (mean velocity 24.6 ± 0.54 nm/s, n = 77) and (C) in the presence of 5 mM MC-DSPPA (mean velocity 21.04 ± 0.44 nm/s, n = 77). The in vitro motility assay and the statistical analysis of the data were performed according to the methods described in the Materials and Methods section. During in vitro motility assays and in the presence of SP-DSPPA, the gliding MTs detached from the Eg5-coated glass surfaces significantly more often and time dependently as shown in Fig. 7 and the Supplementary Movies S1–3. For MC-DSPPA, the time-dependent detachment of MTs from the Eg5-coated glass surface was also observed but occurred slightly later than in the presence SP-DSPPA. This phenomena may be due to the conversion from MC- to SP-DSPPA upon the VIS illumination (530–550 nm) from the fluorescence microscope used during the in vitro motility assay. This is consistent with the KMT values in the presence of SP- and MC-DSPPA as observed in Fig. 5. Fig. 7 View largeDownload slide DSPPA induces dissociation of MTs from Eg5 during the in vitro gliding assay. Microtubule dissociation from the surface of flow cells during the in vitro motility assay performed in Fig. 6 were analysed in the presence of SP-DSPPA (○) or MC-DSPPA (•) and in the absence of DSPPA (Δ). DSPPA was added at time 0 s to start the assay. The relative population of MTs bound to Eg5 was calculated by the following equation: relative population of bound MTs (%) = (number of MTs bound to Eg5 and gliding on the glass surface/the number of MTs bound to Eg5 and gliding on the glass surface at time 0) × 100. Fig. 7 View largeDownload slide DSPPA induces dissociation of MTs from Eg5 during the in vitro gliding assay. Microtubule dissociation from the surface of flow cells during the in vitro motility assay performed in Fig. 6 were analysed in the presence of SP-DSPPA (○) or MC-DSPPA (•) and in the absence of DSPPA (Δ). DSPPA was added at time 0 s to start the assay. The relative population of MTs bound to Eg5 was calculated by the following equation: relative population of bound MTs (%) = (number of MTs bound to Eg5 and gliding on the glass surface/the number of MTs bound to Eg5 and gliding on the glass surface at time 0) × 100. Discussion The aim of this study was to develop a novel kinesin Eg5 inhibitor with a photo-switching mechanism to control the functions of the mitotic kinesin Eg5. It is well-known that there are several potent Eg5 inhibitors. Interestingly, the inhibitors share the same binding pocket composed of loop L5, helix α2 and helix α3 near the nucleotide binding site; and the structure of the inhibitors is not very well-conserved (9, 20). The only common structural property among the typical potent Eg5 inhibitors is the aromatic rings linked by rotatable single bonds with localized polar functional groups (Fig. 8). We expected that the photochromic compounds, which are similar to the inhibitors previously reported, may exhibit a photo-reversible inhibitory effect on Eg5. Previously, we synthesized an STLC analogue composed of a photochromic molecule of azobenzene, ACTAB. ACTAB has a structure where the azobenzene was incorporated into the position between the trithyl group and cysteine of STLC. ACTAB showed different inhibitory activities correlating to its cis–trans photo-isomerization (12). Consequently, it has been demonstrated that the photochromic compound derivative did function as an Eg5 inhibitor that has a photo-switching mechanism. Fig. 8 View largeDownload slide Comparison of the structures of DSPPA with potent Eg5 inhibitors. The structures of potent Eg5 inhibitors: STLC (A), monastrol (B) and ispinesib (C) were previously reported. The photochromic Eg5 inhibitors are ACTAB (D), DSPPA (E). The similarity between DSPPA and ispinesib are highlighted with a dotted line circle. Fig. 8 View largeDownload slide Comparison of the structures of DSPPA with potent Eg5 inhibitors. The structures of potent Eg5 inhibitors: STLC (A), monastrol (B) and ispinesib (C) were previously reported. The photochromic Eg5 inhibitors are ACTAB (D), DSPPA (E). The similarity between DSPPA and ispinesib are highlighted with a dotted line circle. In this study, we focused on spiropyran as a photochromic molecule and incorporated it into an Eg5 inhibitor. As shown in Fig. 8E, spiropyran exhibits isomerization between a hydrophobic closed ring SP-form and zwitterionized open ring MC-form upon UV and VIS irradiation, respectively. Moreover, the structure of spiropyran resembles the potent Eg5 inhibitor ispinesib (Fig. 8C). Consequently, it is strongly expected that the photochromic Eg5 inhibitor composed of spiropyran will have a significant change in its inhibitory activity correlating with its isomerization. In fact, the spiropyran derivative, DSPPA, inhibited Eg5. Additionally, the activity correlated with photo-isomerization. The inhibitory activity of DSPPA (IC50: SP isomer 2.09 ± 0.10 μM and MC isomer 3.67 ± 0.12 μM) for the basal ATPase activity of Eg5 was more potent than that of another photochromic inhibitor, ACTAB (IC50: trans isomer 8.1 μM and cis isomer 16.5 μM), which we previously reported (12). DSPPA showed a different inhibitory effect on Eg5 than ACTAB. Cis-ACTAB showed almost no influence on KMT but conversion to trans-ACTAB decreased the Vmax of the ATPase (12). However, as shown in Fig. 5, MT-dependent ATPase activities indicated that isomerization of MC- to SP-DSPPA decreased the affinity of Eg5 for MTs but did not influence the Vmax of the ATPase. Therefore, SP-DSPPA may have an allosteric site on Eg5, where it influences MT binding. We observed more effective inhibition with higher concentration of DSPPA at the higher range of MTs concentration. Consequently, the photo-switch of this compound is useful by taking the proper concentration of the compound. The sigmoidal shape of the MT-dependent ATPase activity in the presence of SP-DSPPA (Fig. 5) may reflect the existence of an allosteric binding site. Furthermore, in the presence of SP-DSPPA, the gliding MTs in the in vitro motility assay dissociated significantly from Eg5 adhered to the glass surface of the flow cells as shown in Fig. 7. The results also suggested that SP-DSPPA binds to the allosteric site and influences the affinity of Eg5 for MTs. It is known that the conversion of the SP to MC isomer of spiropyran is dependent on the polarity of the solvent in the dark. The concentration of the MC isomer in the thermal equilibrium state increases as the water content of the binary solvent mixtures increases (21). In the case of neat organic solvents, spiropyran with the C8 position was substituted with a carboxyl or alcohol group has shown the reverse photochromism. DSPPA also exhibited reverse photochromism in an aqueous solution. The closed ring, colourless SP-form of DSPPA spontaneously converted to the open ring, coloured MC-form in the dark. However, the reverse photochromism was not observed in a neat organic solvent (data not shown). Although the spontaneous conversion of SP- to MC-DSPPA in the dark was much slower than the conversion under UV irradiation (Fig. 2A and C), SP-MC isomerization can be regulated with VIS and darkness instead of with UV light. The reverse photochromism of DSPPA enables us to photo-regulate Eg5 in vivo without harmful UV light irradiation. We have demonstrated that the photochromic molecules are applicable as a photo-switching device to control the inhibitory activity of the mitotic kinesin Eg5 inhibitor. In conclusion, we have demonstrated that the photochromic compound spiropyran can be used as a photo-switching device for kinesin Eg5 inhibitors to regulate their inhibitory activities. Supplementary Data Supplementary Data are available at JB Online. Conflict of Interest None declared. References 1 Cole D.G., Saxton W.M., Sheehan K.B., Scholey J.M. ( 1994) A ‘slow’ homotetrameric kinesin-related motor protein purified from Drosophila embryos. J. Biol. Chem . 269, 22913– 22916 Google Scholar PubMed  2 Slangy A., Lane H.A., d’Hérin P., Harper M., Kress M., Niggt E.A. ( 1995) Phosphorylation by p34cdc2 regulated spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell  83, 1159– 1169 Google Scholar CrossRef Search ADS PubMed  3 Mayer T.U., Kapoor T.M., Haggarty S.J., King R.W., Schreiber S.L., Mitchison T.J. ( 1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science  286, 971– 974 Google Scholar CrossRef Search ADS PubMed  4 Sakowicz R., Finer J.T., Beraud C., Crompton A., Lewis E., Fritsch A., Lee Y., Mak J., Moody R., Turincio R., Chabala J.C., Gonzales P., Roth S., Weitman S., Wood K.W. ( 2004) Antitumor activity of a kinesin inhibitor. Cancer Res.  64, 3276– 3280 Google Scholar CrossRef Search ADS PubMed  5 Rath O., Kozielski F. ( 2012) Kinesins and cancer. Nat. Rev. Cancer  12, 527– 539 Google Scholar CrossRef Search ADS PubMed  6 DeBonis S., Skoufias D.A., Lebeau L., Lopez R., Robin G., Margolis R.L., Wade R.H., Kozielski F. ( 2004) In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol. Cancer Ther . 3, 1079– 1090 Google Scholar PubMed  7 Ogo N., Oishi S., Matsuno K., Sawada J., Fujii N., Asai A. ( 2007) Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorg. Med. Chem. 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Med . 16, 158– 166 17 Zhang X., Heng S., Abell A.D. ( 2015) Photoregulation of α-chymotrypsin activity by spiropyran-based inhibitors in solution and attached to an optical fiber. Chem. Eur. J.  21, 10915– 10713 Google Scholar CrossRef Search ADS   18 Kalisky Y., Orlowski T.E., Williams D.J. ( 1983) Dynamics of the spiropyran-merocyanine conversion in solution. J. Phys. Chem.  87, 5333– 5338 Google Scholar CrossRef Search ADS   19 Wu Y., Sasaki T., Kazushi K., Seo T., Sakurai K. ( 2008) Interactions between spiropyrans and room-temperature ionic liquids: photochromism and solvatochromism. J. Phys. Chem. B  112, 7530– 7536 Google Scholar CrossRef Search ADS PubMed  20 Yan Y., Sardana V., Xu B., Homnick C., Halczenko W., Buser C.A., Schaber M., Hartman G.D., Huber H.E., Kuo L.C. ( 2004) Inhibition of a mitotic motor protein: where, how, and conformational consequences. J. Mol. 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Soc.  104, 4904– 4907 Google Scholar CrossRef Search ADS   Abbreviations Abbreviations ACTAB 4-(N-(2-(N-acetylcysteine-S-yl)acetyl)amino)-4′- (N-(2-(N-(triphenylmethyl)amino)acetyl) amino)- azobenzene ATP adenosine triphosphate CCD charge coupled device DMF N, N-dimethylformamide DSPPA (S)-2, 3-dispiropyran propionic acids EDTA (ethylenedinitrilo)tetraacetic acid EGTA ethyleneglycol-bis(β-aminoethyl)-N, N, N′, N′-tetraacetic acid GTP guanosine triphosphate HEPES 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid HPLC high performance liquid chromatography IC inhibitory concentration KOH potassium hydroxide MC merocyanine NHS N-hydroxy succinimide NTA nitrilotriacetic acid Pi inorganic phosphate PIPES piperazine-1, 4-bis (2-ethanesulfonic acid) SP spiro SP-COOH 1-(β-carboxyethyl)-3′, 3′-dimethyl-6–nitrospiro (indoline-2′, 2 (2H-1) benzopyran) STLC S-trityl-L-cysteine TEA-HCO3 trimethylamine-bicarbonate UV ultra-violet VIS visible light © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy046
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Abstract

Abstract In this study, we synthesized a novel photochromic inhibitor of the mitotic kinesin Eg5, which is composed of the photochromic compound spiropyran to photo-control the function of Eg5. The compound (S)-2, 3-dispiropyran propionic acid (DSPPA) exhibits reversible spiropyran–merocyanine photo-isomerization upon irradiation with visible or ultra-violet light. DSPPA induced reversible changes in the inhibitory effect on Eg5 ATPase and motor activities, which correlates with the spiropyran–merocyanine photo-isomerization. Microtubule-dependent ATPase activity was significantly more inhibited by the spiropyran isomer of DSPPA than by the merocyanine isomer. Additionally, an in vitro motility assay revealed that the microtubule gliding velocity was reduced more by the spiropyran isomer than by the merocyanine isomer. This indicates that the spiropyran derivative may be useful in regulating the function of the mitotic kinesin. Eg5, inhibitor, kinesin, photo-control, photochromic molecule The mitotic kinesin Eg5 is a member of the kinesin-5 family and a plus-end directed homotetrameric molecular motor. Eg5 plays an important role in regulating centrosome separation and formation of the bipolar spindle apparatus (1, 2). Inhibition of Eg5 induces monopolar spindles and eventually apoptosis. Therefore, Eg5 is an important drug target in anti-mitotic cancer therapy (3, 4). Eg5-specific inhibitors exhibit improved safety profiles compared with other traditional anti-mitotic drugs targeting the multi-functional relevant microtubule (MT) (5). It is known that there are several small-molecule inhibitors of Eg5. Monastrol was the first Eg5-specific inhibitor to be characterized (3). S-trityl-L-cysteine (STLC) is also a potent inhibitor that binds to Eg5 much more tightly than monastrol does (6, 7). The molecular mechanism of the inhibition of Eg5 by STLC has been well-studied (8). Other potent inhibitors, such as ispinesib, have also been reported (9). Interestingly, although the structures of the inhibitors are not similar, they bind to the same pocket, which is composed of the L5 loop, α2 helix and α3 helix near the ATPase site (10). For the inhibitors, the single bond linkage between aromatic rings and hydrophilic groups on the aromatic rings were observed as a common structure. Previously, we photo-reversibly controlled the ATPase activity of Eg5 using photochromic molecules that were incorporated into the entrance region of the Eg5 inhibitor binding site (11). Eg5 was modified with photochromic molecules that exhibited photo-reversible changes in the ATPase activity upon irradiation with ultra-violet (UV) and visible light (VIS). However, chemical modification to Eg5 with photochromic molecules to achieve photo-control has not been adapted for application in living cells. Consequently, we have synthesized a photochromic azobenzene derivative, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)- azobenzene (ACTAB), which is a novel photochromic inhibitor of Eg5 (12). The compound is composed of a trithyl group, which acts as the key moiety in STLC inhibition of Eg5. The ATPase and motor activities of Eg5 were effectively controlled using this photo-reversible photochromic inhibitor. We have demonstrated that photochromic compounds can be used as a photo-switching molecular control for the Eg5 inhibitor. Previously, we demonstrated that the azobenzene derivative inhibitor, ACTAB, photo-reversibly inhibited the ATPase activity of Eg5 (12). Consequently, other photochromic compounds with structures similar to potent Eg5 inhibitors previously reported may also be applicable as photochromic inhibitors for Eg5. We used spiropyran as a photochromic molecule to reversibly inhibit Eg5 because the molecular structure of spiropyran resembles that of the potent Eg5 inhibitor ispinesib (9). In this study, we used a spiropyran derivative to synthesize a novel photo-switching Eg5 inhibitor. The novel photochromic inhibitor photo-isomerizes upon UV and VIS irradiation. Furthermore, we successfully controlled the activity of the spiropyran derivative with light. It is expected that the novel photochromic Eg5 inhibitor composed of the spiropyran derivative has a potential use in achieving photo-control of cell division, and therefore a potential use in cancer therapy. Materials and Methods Synthesis of photochromic Eg5 inhibitor (S)-2, 3-dispiropyran-propionic acid (DSPPA) was synthesized by the established coupling reaction between 1-(β-carboxyethyl)-3′, 3′-dimethyl-6–nitrospiro (indoline-2′, 2 (2H-1) benzopyran) (spiro (SP)-COOH) (13) and (S)-(+)-2, 3-Diaminopropionic acid. Briefly, prior to the coupling reaction, the N-hydroxy succinimide ester (NHS-ester) of SP-COOH was prepared. SP-COOH (78.0 µmol) was activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (780 µmol) for 10 min in 3.0 ml N, N-dimethylformamide (DMF), and then N-hydroxy succinimide (780 µmol) was added. The mixture was stirred with a magnetic stirrer at room temperature for 3 h. The reactant was evaporated to dryness and the SP-COOH-NHS-ester was purified by flash column chromatography packed using a silica gel in a solvent of 20% methanol and 80% chloroform. Subsequently, the purified SP-COOH-NHS-ester was reacted with (S)-(+)-2, 3-Diaminopropionic acid. The SP-COOH-NHS-ester (20.0 µmol) in 3 ml DMF was added to a solution of (S)-(+)-2, 3-Diaminopropionic acid hydrochloride (40.0 µmol) in 1.5 ml of 50 mM trimethylamine-bicarbonate (TEA-HCO3) at pH 8.5 with vigorous stirring. After reacting for 3.0 h at room temperature, the reactant was evaporated to dryness. The product was purified by high performance liquid chromatography (HPLC) using C18 reverse-phase column chromatography with a linear gradient of 0–90% ethanol in 0.1 M TEA-HCO3 at pH 7.5. Further purification was performed using flash column chromatography packed with ultra-pure Wakogel C-200 in 20% methanol and 80% chloroform developing solvents. The purity of the products was analysed by silica gel TLC with 20% methanol and 80% chloroform developing solvent. The Rf value of DSPPA was 0.64. Fast atom bombardment mass spectrum of the product showed a molecular ion (M + H)+ at m/z 828 + 1, which corresponds to a molecular mass of 828,865 as calculated from the formula of C45H44N6O10 of DSPPA. Photo-isomerization of DSPPA Photo-isomerization of the Eg5 inhibitor DSPPA was performed by irradiation at 366 nm UV and VIS. The UV and VIS lights were used for conversion of the spiropyran-form (SP-form) to the merocyanine-form (MC-form) and the MC-form to the SP-form, respectively. The photo-isomerization of DSPPA was performed under a light source at distance of 1 cm from the surface of the solvents for the DSPPA dose-dependent ATPase inhibition assay. For the assay of reproducible reversibility of the photo-control of the MT-stimulated ATPase activity, the photo-isomerization of DSPPA was performed with a light source 10 cm from the surface of the solvents. Expression and purification of the kinesin Eg5 motor domain The cDNA of the motor domain of mouse wild-type (WT) Eg5 (WT, amino residue 1-367) was amplified by polymerase chain reaction and ligated into a pET21a vector. The Eg5 WT expression plasmids were used to transform Escherichia coli BL21 (DE3). Eg5 WT was purified by using a Co-nitrilotriacetic acid (NTA) column. The Co-NTA column was washed with lysis buffer containing 30 mM imidazole; bound Eg5 was eluted with lysis buffer containing 150 mM imidazole. The elutant fractions were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified Eg5 was dialysed in buffer (30 mM Tris-HCl at pH 7.5, 120 mM NaCl, 2.0 mM MgCl2, 0.1 mM adenosine triphosphate (ATP) and 0.5 mM dithiothreitol) and stored at −80°C. Expression and purification of the truncated conventional kinesin・kinesin-1 containing motor doamin Expression and purification of the N-terminal 560 amino acids of mouse brain kinesin (KIF5A) containing motor domain were performed according to the established methods we have previously reported (14). The plasmid pET21a carried a mouse conventional kinesin construct comprising residues 1-560 with a C-terminal 6 His-tag. This plasmid (pET21a: kinesin 1-560) was transformed into E.coli BL21 (DE3) cells for expression. Expressed kinesin-1 was purified by using a Co-NTA column. The purified kinesin-1 was dialysed and stored in the same manner as Eg5. Purification and polymerization of tubulin Tubulin was purified from porcine brains using the method described by Hackney (15). Tubulin was polymerized for 30 min at 37°C in a buffer containing 100 mM piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES) (pH 6.8), 1.0 mM ethyleneglycol-bis(β-aminoethyl)-N, N, N′, N′-tetraacetic acid (EGTA), 1.0 mM MgCl2 and 1.0 mM guanosine triphosphate (GTP). Taxol was then added to a final concentration of 10 µM. The polymerized MTs were collected by centrifugation at 280,000 × g for 15 min at 37°C. The supernatant was removed, and the pellet was collected in a buffer containing 100 mM PIPES at pH 6.8, 1.0 mM EGTA, 1.0 mM MgCl2, 1.0 mM GTP and 10 μM taxol. For the MT gliding assay, MTs were labelled with rhodamine. Rhodamine-labelled tubulin was mixed with unlabelled tubulin at a ratio of 1:5 and polymerized for 40 min at 37°C in the buffer (100 mM PIPES, pH 6.8, 2.0 mM EGTA and 1.0 mM MgSO4). Taxol was then added to a final concentration of 20 µM. Eg5 ATPase assay The ATPase activity of Eg5 was measured at 25°C in an assay solution containing 0.1 µM Eg5 (MT-stimulated ATPase) or 0.5 µM Eg5 (basal ATPase), 20 mM 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES) (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM (ethylenedinitrilo)tetraacetic acid (EDTA), 0.1 mM EGTA and 1.0 mM β-mercaptoethanol in the presence or absence of 3.0 µM MTs. To examine the effect of the photo-isomerization of the inhibitor, DSPPA dissolved in DMF was irradiated by UV or VIS light and added to the assay buffer containing Eg5 to a final concentration of 0.0–10.0 µM. The conditions for the photo-isomerization of DSPPA are as described in the earlier section, ‘Photo-isomerization of the DSPPA’. The final DMF concentration in the Eg5 ATPase buffer was 5.0%. The ATPase reaction was started by adding 2.0 mM ATP and terminated by adding 10% trichloroacetic acid. The released inorganic phosphate (Pi) in the supernatant was measured using the method of Youngburg (16). To determine KMT and Vmax for MT concentration in the presence of DSPPA, the MT concentration-dependent ATPase activities (0.0–5.0 µM) were measured in the presence of 10.0 µM DSPPA. Microtubule gliding assay Coverslips were coated with an anti-6× histidine monoclonal antibody (Wako) in the assay buffer (10 mM Tris-acetate at pH 7.5, 50 mM potassium-acetate, 2.5 mM EGTA and 4.0 mM MgSO4). Subsequently, 0.1 µM Eg5 in assay buffer A (10 mM Tris-acetate at pH 7.5, 50 mM potassium-acetate, 2.5 mM EGTA, 4.0 mM MgSO4, 0.5 mg/ml Casein and 0.2% β-mercaptoethanol) was added through the flow chambers, and the chambers were incubated for 5 min. After washing the chambers with assay buffer A, rhodamine-labelled MTs in assay buffer B (assay buffer A with 20.0 µM taxol) were infused through the flow chambers, and the chambers were incubated for 2 min. The chambers were then washed with assay buffer B. Next, 1.0 mM ATP and 10.0 µM photo-isomerized DSPPA in assay buffer C (assay buffer B with 1.5 mg/ml glucose, 0.01 mg/ml catalase and 0.05 mg/ml glucose oxidase) were infused through the flow chambers. The photo-isomerization of DSPPA was performed as described in the earlier section, ‘Photo-isomerization of DSPPA’, prior to adding to the motility assay buffer. The final DMF concentration in the assay buffer was 5.0%. Rhodamine-labelled MTs were visualized using an Olympus BX50 microscope equipped with a charge coupled device (CCD) camera (LK-TU53H: Toshiba, Japan). Statistical analysis Data are expressed as mean ± SE. Statically significant differences between groups was determined by using either the Welch’s t-test or Student’s t-test. P-values of < 0.05 were considered statistically significant. Results Photo-isomerization of the spiropyran derivative photochromic inhibitor of Eg5 To control photo-reversibly the ATPase and motor activities of Eg5, we designed and synthesized a novel photochromic Eg5 inhibitor, DSPPA (Fig. 1). The synthesis of DSPPA was performed by established methods as described in the Materials and Methods. The inhibitor is composed of two photochromic spiropyran moieties with a polar carboxylic acid. DSPPA contains two spiropyran moieties that undergo reversible SP and MC isomerization with significant conformational changes upon irradiation with VIS and UV as shown in Fig. 1. The isomerization of spiropyran has been used to photo-regulate other enzymes’ activity (17). Therefore, it is strongly expected to have a significant photo-reversible effect on the inhibition of Eg5. Fig. 1 View largeDownload slide Structures of DSPPA and its photo-isomers. The spiropyran derivative DSPPA undergoes reversible photo-isomerization between the spiro- and merocyanine-isomer after irradiation with visible or UV light, respectively. Fig. 1 View largeDownload slide Structures of DSPPA and its photo-isomers. The spiropyran derivative DSPPA undergoes reversible photo-isomerization between the spiro- and merocyanine-isomer after irradiation with visible or UV light, respectively. Figure 2 shows the absorption spectra changes that accompany the photo-isomerization of DSPPA. It is known that the conversion between the SP and MC states of spiropyran derivatives can be monitored by changes in the UV/VIS light spectrum around 500 nm (18). The SP isomer of DSPPA showed almost no absorption around 500 nm in the same manner as the other spiropyran. In total, 366 nm UV irradiation induced conversion of the SP to the MC state, and the absorption maximum at 560 nm increased in a time-dependent manner as shown in Fig. 2A. However, the VIS irradiation of the MC-form of DSPPA resulted in the conversion to the SP-form and a significant reduction in the absorbance peak at 560 nm (Fig. 2B). The time course of spectral changes upon UV and VIS light irradiations in an aqueous solution were almost similar to those in DMF (Supplementary Fig. S1). Interestingly, DSPPA dissolved in an aqueous solution also showed reverse photochromism (19). In the dark, the conversion of DSPPA from the SP-form to the MC-form occurred spontaneously, resulting in an increase in the absorption maximum at 520 nm (Fig. 2C). However, DSPPA in DMF converted from the MC to SP state as a normal photochromism and the absorption maximum at 520 nm was not increased in the dark (Supplementary Fig. S2). The reverse photochromism of DSPPA in the aqueous solution enabled us to convert between the SP and MC state without using UV light. Fig. 2 View largeDownload slide Absorption spectra changes of DSPPA accompanied by its photo-isomerization. (A) Time-dependent changes in the absorption spectra of SP-DSPPA to MC-DSPPA in DMF upon irradiation at 366 nm. In this experiment, 5.0 μM DSPPA was irradiated for 0, 30, 60, 120, 180, 240 and 300 s at 25°C. (B) Time-dependent changes in the absorption spectra of MC-DSPPA in DMF upon VIS irradiation for 0, 10, 20, 30, 60, 120 and 180 s at 25°C. (C) Time-dependent spectral changes of SP-DSPPA to the MC-form in an aqueous solution of 20 mM HEPES-potassium hydroxide (KOH) (pH 7.20), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA and 0.1 mM EDTA in the dark at 25°C. Fig. 2 View largeDownload slide Absorption spectra changes of DSPPA accompanied by its photo-isomerization. (A) Time-dependent changes in the absorption spectra of SP-DSPPA to MC-DSPPA in DMF upon irradiation at 366 nm. In this experiment, 5.0 μM DSPPA was irradiated for 0, 30, 60, 120, 180, 240 and 300 s at 25°C. (B) Time-dependent changes in the absorption spectra of MC-DSPPA in DMF upon VIS irradiation for 0, 10, 20, 30, 60, 120 and 180 s at 25°C. (C) Time-dependent spectral changes of SP-DSPPA to the MC-form in an aqueous solution of 20 mM HEPES-potassium hydroxide (KOH) (pH 7.20), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA and 0.1 mM EDTA in the dark at 25°C. Photo-control of Eg5 inhibitory activity The inhibitory activity of the SP and MC forms of DSPPA on the basal ATPase activity of Eg5 was examined. DSPPA potently inhibited the ATPase activity of Eg5. Furthermore, a difference in the inhibitory activity between the SP and MC isomers was observed. To determine the half-maximal inhibitory concentration (IC50), the rate of ATP hydrolysis of the Eg5 motor domain was measured in the absence of MTs and in the presence of increasing concentrations of the SP- or MC-form of DSPPA. Both DSPPA isomers inhibited the basal ATPase activity of the Eg5 motor domain in a concentration-dependent manner as shown in Fig. 3A. The IC50 value of the SP isomer was 2.09 ± 0.10 μM, whereas that of the MC isomer was 3.67 ± 0.12 μM. Fig. 3 View largeDownload slide Effects of DSPPA isomers on the basal and MT-stimulated ATPase activity of Eg5. (A) The rate of basal ATP hydrolysis in the presence of 0.5 μM Eg5 was measured in a solution of 20 mM HEPES-KOH at pH 7.20 containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP as a function of SP-DSPPA (○) or MC-DSPPA (•) concentration at 25°C. (B) The rate of MT-stimulated ATP hydrolysis at various concentrations of DSPPA was measured in the presence of 3.0 µM MTs, 2.0 mM ATP and SP-DSPPA (○) or MC-DSPPA (•) in a buffer of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Each data point is the average of three independent trials. (Data are expressed as mean ± SE, n = 3). Fig. 3 View largeDownload slide Effects of DSPPA isomers on the basal and MT-stimulated ATPase activity of Eg5. (A) The rate of basal ATP hydrolysis in the presence of 0.5 μM Eg5 was measured in a solution of 20 mM HEPES-KOH at pH 7.20 containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP as a function of SP-DSPPA (○) or MC-DSPPA (•) concentration at 25°C. (B) The rate of MT-stimulated ATP hydrolysis at various concentrations of DSPPA was measured in the presence of 3.0 µM MTs, 2.0 mM ATP and SP-DSPPA (○) or MC-DSPPA (•) in a buffer of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Each data point is the average of three independent trials. (Data are expressed as mean ± SE, n = 3). The MT-dependent ATPase activity of Eg5 was also significantly inhibited by DSPPA as shown in Fig. 3B. The IC50 value for the SP isomer of DSPPA for the ATPase activity of Eg5 in the presence of MTs was 3.77 ± 0.27 μM. However, the MC isomer of DSPPA had a lower inhibitory activity and an IC50 of 7.67 ± 0.31 μM. The changes in the inhibitory activity of the SP and MC isomers of DSPPA induced by VIS and UV irradiation was completely and reproducibly reversible as shown in Fig. 4. To avoid degradation of inhibitor by repeated UV irradiations, we employed the weaker UV light irradiation in the experiment of Fig. 4 than that in Fig. 3 as described in Materials and Methods. Therefore, the conversion from SP to MC isomer is not achieved completely and the ATPase activity under UV irradiation is lower than that at the same concentration of DSPPA in Fig. 3. Fig. 4 View largeDownload slide Reproducible reversibility of the photo-control of the MT-stimulated ATPase activity. DSPPA was alternately irradiated with 366 nm UV and VIS prior to the ATPase assay. The ATPase activity was measured in the presence of 3 μM MTs, 2.0 mM ATP and 7.5 μM SP or MC-DSPPA in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Alternation of UV and VIS was repeated three times. The MT-stimulated ATPase activity was measured in three independent experiments. Fig. 4 View largeDownload slide Reproducible reversibility of the photo-control of the MT-stimulated ATPase activity. DSPPA was alternately irradiated with 366 nm UV and VIS prior to the ATPase assay. The ATPase activity was measured in the presence of 3 μM MTs, 2.0 mM ATP and 7.5 μM SP or MC-DSPPA in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol at 25°C. Alternation of UV and VIS was repeated three times. The MT-stimulated ATPase activity was measured in three independent experiments. We confirmed the specific inhibition of Eg5 by DSPPA using recombinant conventional kinesin・kinesin-1 (KIF5A). For truncated kinesin-1(1-560 amino acids), DSPPA did not exhibited a photo-reversible change in inhibitory activity accompanied by SP-MC isomerization (Supplementary Fig. S3). We also examined the influence of DSPPA on the interaction between Eg5 and MTs. Figure 5 shows the MT concentration-dependent ATPase activity of Eg5 in the presence of SP or MC-DSPPA. Both isomers decreased the Vmax to almost the same level as that of the ATPase activity in the absence of isomers. However, in the presence of the SP isomer of DSPPA, Eg5 showed a lower affinity for MTs than that in the presence of the MC isomer. Fig. 5 View largeDownload slide Effect of DSPPA isomers on the affinity of Eg5 for MTs. The inhibitory effects of DSPPA photo-isomers on Eg5 ATPase activity as a function of the MT concentration was measured in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol with 0.1 μM Eg5, 2.5 μM of SP-DSPPA (○) or MC-DSPPA (•) and 0–5 μM MTs. Fig. 5 View largeDownload slide Effect of DSPPA isomers on the affinity of Eg5 for MTs. The inhibitory effects of DSPPA photo-isomers on Eg5 ATPase activity as a function of the MT concentration was measured in an assay buffer consisting of 20 mM HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA and 1.0 mM β-mercaptoethanol with 0.1 μM Eg5, 2.5 μM of SP-DSPPA (○) or MC-DSPPA (•) and 0–5 μM MTs. From the data, we concluded that the optimal conditions for DSPPA to control the MT-dependent ATPase activity as a photo-switch is 5.0–6.0 μM DSPPA in the presence of 1.0–1.5 μM MTs (Figs 3B and 5). Photo-control of the Eg5-driven MT gliding velocity We examined the photo-control of the Eg5 motor activity using SP- and MC-DSPPA isomers. An in vitro motility assay using rhodamine-labelled MTs was performed according to established methods as described in the Materials and Methods. The velocity of the MTs gliding on Eg5-coated glass surfaces in flow cells was measured in the presence of SP or MC-DSPPA. The average MT gliding velocity (in the absence of DSPPA) was 23.22 ± 0.381 nm/s (Fig. 6A). The histogram of the velocity in the presence of DSPPA showed a slightly broader distribution. In the presence of SP-DSPPA, the average velocity significantly decreased to 15.70 ± 0.535 nm/s as shown in Fig. 6B. However, in the presence of MC-DSPPA, the average velocity decreased slightly (21.04 ± 0.436 nm/s) (Fig. 6C) compared with the control. Fig. 6 View largeDownload slide Photo-control of the motor activity of Eg5 with DSPPA. Histogram of the distribution of the MT gliding velocities on Eg5 in the in vitro motility assay (A) in the absence of DSPPA (mean velocity 23.22 ± 0.38 nm/s, n = 35), (B) in the presence of 5 μM SP-DSPPA (mean velocity 24.6 ± 0.54 nm/s, n = 77) and (C) in the presence of 5 mM MC-DSPPA (mean velocity 21.04 ± 0.44 nm/s, n = 77). The in vitro motility assay and the statistical analysis of the data were performed according to the methods described in the Materials and Methods section. Fig. 6 View largeDownload slide Photo-control of the motor activity of Eg5 with DSPPA. Histogram of the distribution of the MT gliding velocities on Eg5 in the in vitro motility assay (A) in the absence of DSPPA (mean velocity 23.22 ± 0.38 nm/s, n = 35), (B) in the presence of 5 μM SP-DSPPA (mean velocity 24.6 ± 0.54 nm/s, n = 77) and (C) in the presence of 5 mM MC-DSPPA (mean velocity 21.04 ± 0.44 nm/s, n = 77). The in vitro motility assay and the statistical analysis of the data were performed according to the methods described in the Materials and Methods section. During in vitro motility assays and in the presence of SP-DSPPA, the gliding MTs detached from the Eg5-coated glass surfaces significantly more often and time dependently as shown in Fig. 7 and the Supplementary Movies S1–3. For MC-DSPPA, the time-dependent detachment of MTs from the Eg5-coated glass surface was also observed but occurred slightly later than in the presence SP-DSPPA. This phenomena may be due to the conversion from MC- to SP-DSPPA upon the VIS illumination (530–550 nm) from the fluorescence microscope used during the in vitro motility assay. This is consistent with the KMT values in the presence of SP- and MC-DSPPA as observed in Fig. 5. Fig. 7 View largeDownload slide DSPPA induces dissociation of MTs from Eg5 during the in vitro gliding assay. Microtubule dissociation from the surface of flow cells during the in vitro motility assay performed in Fig. 6 were analysed in the presence of SP-DSPPA (○) or MC-DSPPA (•) and in the absence of DSPPA (Δ). DSPPA was added at time 0 s to start the assay. The relative population of MTs bound to Eg5 was calculated by the following equation: relative population of bound MTs (%) = (number of MTs bound to Eg5 and gliding on the glass surface/the number of MTs bound to Eg5 and gliding on the glass surface at time 0) × 100. Fig. 7 View largeDownload slide DSPPA induces dissociation of MTs from Eg5 during the in vitro gliding assay. Microtubule dissociation from the surface of flow cells during the in vitro motility assay performed in Fig. 6 were analysed in the presence of SP-DSPPA (○) or MC-DSPPA (•) and in the absence of DSPPA (Δ). DSPPA was added at time 0 s to start the assay. The relative population of MTs bound to Eg5 was calculated by the following equation: relative population of bound MTs (%) = (number of MTs bound to Eg5 and gliding on the glass surface/the number of MTs bound to Eg5 and gliding on the glass surface at time 0) × 100. Discussion The aim of this study was to develop a novel kinesin Eg5 inhibitor with a photo-switching mechanism to control the functions of the mitotic kinesin Eg5. It is well-known that there are several potent Eg5 inhibitors. Interestingly, the inhibitors share the same binding pocket composed of loop L5, helix α2 and helix α3 near the nucleotide binding site; and the structure of the inhibitors is not very well-conserved (9, 20). The only common structural property among the typical potent Eg5 inhibitors is the aromatic rings linked by rotatable single bonds with localized polar functional groups (Fig. 8). We expected that the photochromic compounds, which are similar to the inhibitors previously reported, may exhibit a photo-reversible inhibitory effect on Eg5. Previously, we synthesized an STLC analogue composed of a photochromic molecule of azobenzene, ACTAB. ACTAB has a structure where the azobenzene was incorporated into the position between the trithyl group and cysteine of STLC. ACTAB showed different inhibitory activities correlating to its cis–trans photo-isomerization (12). Consequently, it has been demonstrated that the photochromic compound derivative did function as an Eg5 inhibitor that has a photo-switching mechanism. Fig. 8 View largeDownload slide Comparison of the structures of DSPPA with potent Eg5 inhibitors. The structures of potent Eg5 inhibitors: STLC (A), monastrol (B) and ispinesib (C) were previously reported. The photochromic Eg5 inhibitors are ACTAB (D), DSPPA (E). The similarity between DSPPA and ispinesib are highlighted with a dotted line circle. Fig. 8 View largeDownload slide Comparison of the structures of DSPPA with potent Eg5 inhibitors. The structures of potent Eg5 inhibitors: STLC (A), monastrol (B) and ispinesib (C) were previously reported. The photochromic Eg5 inhibitors are ACTAB (D), DSPPA (E). The similarity between DSPPA and ispinesib are highlighted with a dotted line circle. In this study, we focused on spiropyran as a photochromic molecule and incorporated it into an Eg5 inhibitor. As shown in Fig. 8E, spiropyran exhibits isomerization between a hydrophobic closed ring SP-form and zwitterionized open ring MC-form upon UV and VIS irradiation, respectively. Moreover, the structure of spiropyran resembles the potent Eg5 inhibitor ispinesib (Fig. 8C). Consequently, it is strongly expected that the photochromic Eg5 inhibitor composed of spiropyran will have a significant change in its inhibitory activity correlating with its isomerization. In fact, the spiropyran derivative, DSPPA, inhibited Eg5. Additionally, the activity correlated with photo-isomerization. The inhibitory activity of DSPPA (IC50: SP isomer 2.09 ± 0.10 μM and MC isomer 3.67 ± 0.12 μM) for the basal ATPase activity of Eg5 was more potent than that of another photochromic inhibitor, ACTAB (IC50: trans isomer 8.1 μM and cis isomer 16.5 μM), which we previously reported (12). DSPPA showed a different inhibitory effect on Eg5 than ACTAB. Cis-ACTAB showed almost no influence on KMT but conversion to trans-ACTAB decreased the Vmax of the ATPase (12). However, as shown in Fig. 5, MT-dependent ATPase activities indicated that isomerization of MC- to SP-DSPPA decreased the affinity of Eg5 for MTs but did not influence the Vmax of the ATPase. Therefore, SP-DSPPA may have an allosteric site on Eg5, where it influences MT binding. We observed more effective inhibition with higher concentration of DSPPA at the higher range of MTs concentration. Consequently, the photo-switch of this compound is useful by taking the proper concentration of the compound. The sigmoidal shape of the MT-dependent ATPase activity in the presence of SP-DSPPA (Fig. 5) may reflect the existence of an allosteric binding site. Furthermore, in the presence of SP-DSPPA, the gliding MTs in the in vitro motility assay dissociated significantly from Eg5 adhered to the glass surface of the flow cells as shown in Fig. 7. The results also suggested that SP-DSPPA binds to the allosteric site and influences the affinity of Eg5 for MTs. It is known that the conversion of the SP to MC isomer of spiropyran is dependent on the polarity of the solvent in the dark. The concentration of the MC isomer in the thermal equilibrium state increases as the water content of the binary solvent mixtures increases (21). In the case of neat organic solvents, spiropyran with the C8 position was substituted with a carboxyl or alcohol group has shown the reverse photochromism. DSPPA also exhibited reverse photochromism in an aqueous solution. The closed ring, colourless SP-form of DSPPA spontaneously converted to the open ring, coloured MC-form in the dark. However, the reverse photochromism was not observed in a neat organic solvent (data not shown). Although the spontaneous conversion of SP- to MC-DSPPA in the dark was much slower than the conversion under UV irradiation (Fig. 2A and C), SP-MC isomerization can be regulated with VIS and darkness instead of with UV light. The reverse photochromism of DSPPA enables us to photo-regulate Eg5 in vivo without harmful UV light irradiation. We have demonstrated that the photochromic molecules are applicable as a photo-switching device to control the inhibitory activity of the mitotic kinesin Eg5 inhibitor. In conclusion, we have demonstrated that the photochromic compound spiropyran can be used as a photo-switching device for kinesin Eg5 inhibitors to regulate their inhibitory activities. Supplementary Data Supplementary Data are available at JB Online. Conflict of Interest None declared. References 1 Cole D.G., Saxton W.M., Sheehan K.B., Scholey J.M. ( 1994) A ‘slow’ homotetrameric kinesin-related motor protein purified from Drosophila embryos. J. Biol. Chem . 269, 22913– 22916 Google Scholar PubMed  2 Slangy A., Lane H.A., d’Hérin P., Harper M., Kress M., Niggt E.A. ( 1995) Phosphorylation by p34cdc2 regulated spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell  83, 1159– 1169 Google Scholar CrossRef Search ADS PubMed  3 Mayer T.U., Kapoor T.M., Haggarty S.J., King R.W., Schreiber S.L., Mitchison T.J. ( 1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. 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Soc.  104, 4904– 4907 Google Scholar CrossRef Search ADS   Abbreviations Abbreviations ACTAB 4-(N-(2-(N-acetylcysteine-S-yl)acetyl)amino)-4′- (N-(2-(N-(triphenylmethyl)amino)acetyl) amino)- azobenzene ATP adenosine triphosphate CCD charge coupled device DMF N, N-dimethylformamide DSPPA (S)-2, 3-dispiropyran propionic acids EDTA (ethylenedinitrilo)tetraacetic acid EGTA ethyleneglycol-bis(β-aminoethyl)-N, N, N′, N′-tetraacetic acid GTP guanosine triphosphate HEPES 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid HPLC high performance liquid chromatography IC inhibitory concentration KOH potassium hydroxide MC merocyanine NHS N-hydroxy succinimide NTA nitrilotriacetic acid Pi inorganic phosphate PIPES piperazine-1, 4-bis (2-ethanesulfonic acid) SP spiro SP-COOH 1-(β-carboxyethyl)-3′, 3′-dimethyl-6–nitrospiro (indoline-2′, 2 (2H-1) benzopyran) STLC S-trityl-L-cysteine TEA-HCO3 trimethylamine-bicarbonate UV ultra-violet VIS visible light © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Apr 26, 2018

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