Highly efficient photocontrol of mitotic kinesin Eg5 ATPase activity using a novel photochromic compound composed of two azobenzene derivatives

Highly efficient photocontrol of mitotic kinesin Eg5 ATPase activity using a novel photochromic... Abstract Mitotic kinesin Eg5 plays an important physiological role in cell division. Several small-molecule inhibitors of Eg5 are the focus of cancer therapies. Azobenzene is a photochromic compound exhibiting cis–trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. Photochromic compounds of azobenzene derivatives, mimicking Eg5-specific inhibitors of STLC, indicated photoreversible inhibitory effects on Eg5 ATPase activity; however, the photoreversible switching efficiency was not significant. This study presents a novel synthesized photochromic Eg5 inhibitor 2, 3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB), which is composed of two azobenzenes. BDPSB exhibited cis–trans isomerization with UV and VIS light irradiation. The trans form of BDPSB significantly inhibited microtubule-dependent ATPase activity of Eg5, with an IC50 of 74 μM. Cis BDPSB showed weak effects on the microtubule-dependent ATPase activity. The results suggest that the novel photochromic Eg5 inhibitor BDPSB, which exhibits highly efficient photoswitching, shows a switch ‘ON’ and ‘OFF’ behaviour with VIS and UV light irradiation. azobenzene, inhibitor, mitotic kinesin, photochromic compound Kinesin, an ATP-driven motor protein, converts the hydrolysis energy of ATP and moves along microtubules. The structure and the function of kinesin has been well studied and shown that kinesin has a molecular mechanism of energy transduction similar to myosin (1, 2). The previous studies suggested that the chemical energy generated by ATP hydrolysis is transmitted mechanically for the physical motility energy to occur like a cam-shaft motion through subdomain steric interactions. Therefore, it is expected that incorporation of an external stimuli responsive molecular device will enable regulation of the function of the ATP-driven motor proteins artificially. Photochromic molecules are known to change structure and properties photoreversibly and are of great interest as nanodevices with optical switches (3–5). Azobenzene is a typical photochromic molecule, which is photoisomerized to a hydrophilic cis isomer by ultraviolet (UV) irradiation and to a hydrophobic trans isomer using visible (VIS) light (3, 6). Spiropyran photoisomerizes to merocyanine, showing a ring-opened zwitterion under UV irradiation and a closed ring hydrophobic spiro type under VIS light (7). Consequently, photochromic molecules might be one of the possible candidates for the external stimuli responsive molecular device to regulate the function of the ATP-driven motor proteins. In fact, we have attempted to control the function of motor proteins using photochromic compounds. Cis–trans isomerization of azobenzene derivatives incorporated into the functional site of skeletal muscle myosin induced conformational changes in the myosin head, reflecting energy transduction (8). We also succeeded in photoreversibly controlling the ATPase activity of kinesin by introducing azobenzene and spiropyran derivatives into the catalytic site and the microtubule-binding site of kinesin (9, 10). However, specific introduction of a photochromic molecule into a functional site of a motor protein by chemical modification presents challenges. Preparations of motor protein mutants with a high-reactive cysteine side chain at the target site allowed for the introduction of a photochromic molecule. However, amino acid substitution and chemical modification occasionally decrease the original function of kinesin and induce partial denaturation, causing inconsistencies. Thus, instead of the direct incorporation of photochromic molecules into the functional site of motor proteins, we focused on the utilization of the specific inhibitors of motor proteins conjugated with photochromic molecules to control the function of motor proteins (11–13). Kinesin Eg5 is essential for mitosis, has physiologically important roles related to various spindle dynamics (14), and is studied as a target for cancer therapy. Many inhibitors of Eg5 have been developed as anticancer drugs (15). S-Trityl-l-cysteine (STLC) is well known as one of the potent inhibitors of Eg5. The molecular mechanism of inhibition by STLC and its binding site on Eg5 are known (16, 17). We have previously synthesized the photochromic STLC analogue composed of azobenzene and trityl groups that acts as the key part in STLC inhibition of Eg5. The photochromic inhibitor, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′-(N-(2-(N-(triphenylmethyl)amino)acetyl) amino)-azobenzene (ACTAB), changed its inhibitory activity by cis–trans isomerization and ATPase inhibition was photoreversible (18). However, its efficiency as a photoswitching compound was not significant. This study shows a photochromic Eg5 inhibitor composed of two azobenzene derivatives with extremely efficient photoswitching. The trans isomer and cis isomer of the photoswitching nanodevice exhibited almost ‘ON’ and ‘OFF’ states, respectively. The method of controlling Eg5 using a specific photochromic inhibitor whose inhibitory activity changes dramatically is expected to be widely applied to biofunctional molecules having specific regulatory factors. Materials and Methods Synthesis of photochromic inhibitor composed of azobenzene derivative 2,3-Bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) was synthesized by coupling of meso-2,3-dimercaptosuccinic acid (27.44 µM) and phenylazo maleinanil (PAM) (82.32 µM) in 1 ml of THF for 4 h at room temperature. The product was purified with preparative TLC (PLC Silica gel 60, MERCK) using a developing solvent of 100% acetonitrile. The expected product migrated at Rf 0.03, it was extracted by methanol, and evaporated to dryness. Further purification was performed by HPLC using C18 reverse-phase column chromatography with a linear gradient of 0–90% acetonitrile in 0.1% trifluoroacetic acid. The product eluted as the main peak at 90% acetonitrile. The TLC (Silica gel 70 F254 Plate, Wako) analysis exhibited a single spot with an Rf of 0.53 in n-butanol:acetic acid:water (5:2:3). The yield of the product was 24.8%. Fast atom bombardment mass spectrum of the product showed a molecular ion (M+H)+ at m/z 736 + 1, which corresponds to a molecular mass of 736.14 as calculated from the formula C36H28N6O8S2 of BDPSB. Photoisomerization of the Eg5 inhibitor BDPSB Irradiations at 366 nm using a Black-Ray lamp (16 W; UVP, Upland, CA, USA) and VIS light using a fluorescent lamp (27 W) were used for the conversion of the trans-BDPSB to cis-BDPSB and the cis-BDPSB to trans-BDPSB, respectively. BDPSB dissolved in DMF or in a solution (20 mM HEPES–KOH pH 7.2, 50 mM KCl, 2.0 mM MgCl2, 0.1 mM EDTA and 0.1 mM EGTA) was irradiated with the light source located 1 cm from the surface of the solvents at 25°C. Expression and purification of the mitotic kinesin Eg5 motor domain and conventional kinesin The motor domain of Eg5 was prepared according to the following method, which is a modification of the previously reported method (18). The cDNA of the motor domain of wild-type (WT) mouse Eg5 (WT, amino residue 1–367) was amplified by polymerase chain reaction and ligated into the pET21a vector. The Eg5 WT expression plasmids were used to transform Escherichia coli BL21 (DE3). Eg5 WT was purified by using a Co-NTA column. The Co-NTA column was washed with lysis buffer containing 30 mM imidazole, and bound Eg5 was eluted with lysis buffer containing 150 mM imidazole. Fractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purified Eg5 was dialyzed in buffer (30 mM Tris–HCl at pH 7.5, 120 mM NaCl, 2.0 mM MgCl2, 0.1 mM ATP and 0.5 mM DTT) and stored at −80°C. Truncated conventional kinesin·kinesin-1 (the N-terminal 560 amino acids of mouse brain kinesin containing motor domain) was prepared according to the established methods we have previously reported (19). The plasmid pET21a carried a mouse conventional kinesin (KIF5A) 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 dialyzed 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 (20). Tubulin was polymerized for 30 min at 37°C in 100 mM PIPES (pH 6.8), 1.0 mM EGTA, 1.0 mM MgCl2 and 1.0 mM GTP. Next, taxol was added to a final concentration of 10 µM. The polymerized microtubules were collected by centrifugation at 280,000 × g for 15 min at 37°C. The supernatant was removed, and the microtubule pellet was collected in a buffer containing 100 mM PIPES (pH 6.8), 1.0 mM EGTA, 1.0 mM MgCl2, 1.0 mM GTA and 10 μM taxol. Kinesin ATPase assay The ATPase assay was performed according to the following methods we have previously reported (18). The ATPase activity of kinesin was measured in 20 mM HEPES (pH 7.2), containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA and 1.0 mM β-mercaptoethanol. To examine the effect of the photoisomerization of the inhibitor, BDPSB dissolved in DMF was irradiated by UV or VIS light and added to the assay buffer to a final concentration 0.0–150.0 µM. The conditions for the photoisomerization of BDPSB are as described in the above section, ‘Photoisomerization of the Eg5 inhibitor BDPSB’. The final DMF concentration in the Eg5 ATPase buffer was 5.0%. For the basal ATPase activity, 0.5 µM Eg5 was added in the assay buffer without microtubules. For the microtubule-stimulated ATPase activity, 100 nM Eg5 motor domain or 100 nM conventional kinesin (kinesin-1) motor domain was added to the assay buffer in the presence of a 3.0 µM microtubule solution. The ATPase reaction was started by adding 2.0 mM ATP and terminated by adding 10% trichloroacetic acid. The reaction time of microtubule-stimulated ATPase or basal ATPase activity was 5 or 20 min at 25°C, respectively. The released inorganic phosphate (Pi) in the supernatant was measured according to the method of Youngburg (21). To determine KMT and Vmax for microtubule concentration in the presence of Eg5 and BDPSB, the microtubule concentration-dependent ATPase activities (0–12 µM) were measured in the presence of 100 µM BDPSB. Microtubule gliding assay The in vitro motility assay was performed according to the slightly changed method that we previously reported (18). Coverslips were coated with an anti-6× histidine monoclonal antibody (Wako) in assay buffer (10 mM Tris–acetate, pH 7.5, 50 mM potassium–acetate, 2.5 mM EGTA and 4.0 mM MgSO4). Subsequently, 0.5 µM Eg5 in assay buffer A (10 mM Tris–acetate, pH7.5, 50 mM potassium–acetate, 2.5 mM EGTA, 4.0 mM MgSO4, 0.2% β-mercaptoethanol) was perfused through the flow chambers and the chambers were incubated for 2 min at 25°C. After washing the chambers with assay buffer A, 1.0 mg/ml casein in assay buffer A was perfused through the flow chambers and the chambers were incubated for 5 min at 25°C. After washing the chambers with assay buffer A, 100 µM photo-isomerized BDPSB, which was irradiated with VIS light or UV (366 nm) for 10 min in assay buffer A, were perfused through the flow chambers and the anchored Eg5 were treated for 2 min at 25°C. And then, rhodamine-labelled microtubules in assay buffer B (assay buffer A with 100 µM photo-isomerized BDPSB and 20.0 µM taxol) were perfused through the flow chambers and the chambers were incubated for 2 min at 25°C. The chambers were then washed with assay buffer B. Finally, 1.0 mM ATP and 100 µM photo-isomerized BDPSB 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 perfused through the flow chambers. The final DMF concentration in the assay buffer was 5.0%. Rhodamine-labelled microtubules were visualized using an Olympus BX50 microscope equipped with a CCD camera (LK-TU53H: Toshiba, Japan). Statistical analysis Data are expressed as mean ±standard deviation (S.D.). Significant differences between groups were determined using Welch’s t-test or Student’s t-test. P-Values of <0.05 were considered statistically significant. Results and Discussion Design, synthesis and spectroscopic characterization of BDPSB Chemical structures of the Eg5 inhibitors previously reported were not highly conserved (22). The only similarity among the inhibitors is that the single bond linkage between aromatic rings and some hydrophilic groups on the aromatic rings were observed. The photochromic compounds are expected to exhibit inhibitory activity on Eg5. In fact, previously we showed that azobenzene derivatives ACTAB that mimic STLC, a potent inhibitor of Eg5, changes its inhibitory activity accompanied by cis–trans isomerization and regulates the ATPase activity of Eg5 photoreversibly (18). However, the photoswitching was not efficient. The difference in inhibitory activities between the cis and trans isomer was not significant. Our recent study revealed that the photochromic compounds composed of two spiropyrans inhibited Eg5 ATPase activity significantly, indicating that the two-headed structure of the aromatic group might be an effective inhibitor (23). In this study, we designed and synthesized the novel photochromic Eg5 inhibitor, BDPSB which is composed of two azobenzene moieties, and formed a two-headed structure (Fig. 1A). The synthesis of BDPSB was done by the established coupling reaction. Thiol reactive azobenzene derivative, 4-(N-Maleimido)azobenzene, was incorporated into the two thiol groups of 2,3-dimercapto succinic acid, according to the procedure described in the ‘Materials and Methods’ section. The two azobenzene moieties of BDPSB exhibit cis–trans isomerization. Consequently, BDPSB changes conformation and physical properties more drastically than compounds composed of a single azobenzene. Cis–trans reversible isomerization of BDPSB was monitored with absorption spectral changes during UV and VIS light irradiation. Azobenzene absorption spectra change at 300– 400 nm with cis–trans isomerization (3, 24). BDPSB also exhibited similar spectral changes during UV and VIS light irradiation. Figure 1B shows that irradiation of trans-BDPSB by UV light led to a reduction of the peak at 325 nm, and the spectral change was complete after 10 min. Irradiation of cis-BDPSB by VIS light led to an almost complete isomerization to the trans form, as shown in Fig. 1C, within 10 min. Fig. 1 View largeDownload slide Structural formula of 2,3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl] phenyl} pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) and its spectral changes accompanied by photoisomerization. (A) Structural formula of BDPSB. The azobenzene derivative BPSBA has reversible photoisomerization between the trans-form and cis-form with VIS or UV light irradiation. (B) Time-dependent change in the absorption spectra of trans-BDPSB to cis-BDPSB upon UV light irradiation (366 nm). Ten micromoles of BDPSB were irradiated in DMF for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. (C) Time-dependent change in the absorption spectra of cis-BDPSB to trans-BDPSB upon VIS light irradiation. Ten micromoles of BDPSB were irradiated for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. Fig. 1 View largeDownload slide Structural formula of 2,3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl] phenyl} pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) and its spectral changes accompanied by photoisomerization. (A) Structural formula of BDPSB. The azobenzene derivative BPSBA has reversible photoisomerization between the trans-form and cis-form with VIS or UV light irradiation. (B) Time-dependent change in the absorption spectra of trans-BDPSB to cis-BDPSB upon UV light irradiation (366 nm). Ten micromoles of BDPSB were irradiated in DMF for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. (C) Time-dependent change in the absorption spectra of cis-BDPSB to trans-BDPSB upon VIS light irradiation. Ten micromoles of BDPSB were irradiated for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. Photo-control of the BDPSB inhibitory activity on the basal Eg5 ATPase The inhibitory activity of the cis and trans isomers of BDPSB on the basal ATPase activity of Eg5 was determined. Trans-BDPSB dose-dependent ATPase activity of Eg5 showed biphase alteration of ATPase activity (Fig. 2A). The first phase of ATPase activity modulation with trans-BDPSB showed an increase of 400% at 25 μM. Subsequently, in the second phase, the ATPase activity decreased dose-dependently, and reached almost complete inhibition at 150 μM trans-BDPSB. This indicates some cooperative interactions between the compound and binding sites on Eg5. Cis-BDPSB increased ATPase activity by 400% at 125 μM in a single phase. The ATPase activation corresponds to that in the first phase of trans-BDPSB. The decrease in ATPase activity in the second phase of trans-BDPSB reflects a binding site different from that in the first phase. Fig. 2 View largeDownload slide Photocontrol of the ATPase activity of Eg5 by BDPSB. (A) Dose-dependent inhibition of basal Eg5 ATPase activities by cis- and trans-BDPSB in 0.5 µM Eg5, 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. (B) Dose-dependent inhibition of microtubule stimulated Eg5 ATPase activities by cis- and trans-BDPSB in 0.1 µM Eg5, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 3.0 µM microtubule and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. The dashed line indicates the inhibition of true cis-BDPSB obtained by subtracting the inhibition of co-existing 25% trans-BDPAB. (C) Inhibitory effect of BDPSB photoisomers on the interaction of Eg5 and microtubules. The microtubule dose-dependent ATPase activities in 0.1 µM Eg5, 0–12 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 5% DMF in the presence of 80 µM trans-BDPSB (filled circle), cis-BDPSB (open circle) and in the absence of BDPSB (filled circle) at 25°C. Fig. 2 View largeDownload slide Photocontrol of the ATPase activity of Eg5 by BDPSB. (A) Dose-dependent inhibition of basal Eg5 ATPase activities by cis- and trans-BDPSB in 0.5 µM Eg5, 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. (B) Dose-dependent inhibition of microtubule stimulated Eg5 ATPase activities by cis- and trans-BDPSB in 0.1 µM Eg5, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 3.0 µM microtubule and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. The dashed line indicates the inhibition of true cis-BDPSB obtained by subtracting the inhibition of co-existing 25% trans-BDPAB. (C) Inhibitory effect of BDPSB photoisomers on the interaction of Eg5 and microtubules. The microtubule dose-dependent ATPase activities in 0.1 µM Eg5, 0–12 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 5% DMF in the presence of 80 µM trans-BDPSB (filled circle), cis-BDPSB (open circle) and in the absence of BDPSB (filled circle) at 25°C. Photo-control of the BDPSB inhibitory activity on the microtubule-stimulated Eg5 ATPase The microtubule-stimulated ATPase activity of Eg5 was significantly inhibited by trans-BDPSB as shown in Fig. 2B. The IC50 value of trans-BDPSB for ATPase activity of Eg5 in the presence of microtubules was 74.2 ± 2.5 μM. The phase of trans-BDPSB dose-dependent inhibition exhibited a sigmoidal response, which indicates the existence of a subsite of binding and corresponds to the biphase alteration of basal ATPase activity of trans-BDPSB (Fig. 2A). The effect of the trans-BDPSB binding to a subsite on the microtubule-stimulated ATPase activity may be reduced comparing the 400% increase in the first phase in the basal ATPase activity. Eg5 ATPase activity is highly stimulated by microtubules. Then, the effect on the trans-BDPSB binding to the subsite may be buried by the microtubules stimulation of ATPase activity. Cis-BDPSB showed extremely low inhibitory activity, as shown in Fig. 2B. Isomerization of azobenzene from trans to cis under UV irradiation (25) is not complete and for BDPSB, 25% trans isomer remains at the equilibrium point under UV irradiation as shown in Fig. 1B. The inhibitory effect of the trans isomer coexisting with cis-BDPSB was controlled for by subtracting trans inhibition from the apparent results of cis-BDPSB inhibition (open circle in Fig. 2B). The estimated true inhibitory activity of only cis-BDPSB was extremely low, as shown in Fig. 2B (dashed line). The highly efficient photocontrol of the ATPase activity of Eg5 was achieved by cis–trans isomerization of BDPSB. ACTAB, composed of a single azobenzene, exhibited little inhibitory activity between cis and trans isomers (18). The highly efficient photocontrol of BDPSB on ATPase activity is proposed to be due to the double azobenzene head structure, which induced the conformational change. The changes in inhibitory activity of trans and cis isomers of BDPSB, as induced by VIS and UV light irradiation, were completely and reproducibly reversible (Fig. 3). BDPSB exhibited almost ‘ON’ and ‘OFF’ photoswitching by trans–cis isomerization under VIS and UV light irradiations. Fig. 3 View largeDownload slide Reproducible reversibility of the photocontrol of the microtubule stimulated ATPase activity by BDPSB. BDPSB was alternately irradiated with 366 nm UV light and VIS light prior to the ATPase assay. Alternating irradiations of UV and VIS lights were repeated three times. ATPase activities in 0.1 µM Eg5, 3 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP and 120 µM trans-BDPSB (grey bar) or cis-BDPSB (white bar) at 25°C. Results of three independent experiments and expressed as mean±standard deviation (error bar). Fig. 3 View largeDownload slide Reproducible reversibility of the photocontrol of the microtubule stimulated ATPase activity by BDPSB. BDPSB was alternately irradiated with 366 nm UV light and VIS light prior to the ATPase assay. Alternating irradiations of UV and VIS lights were repeated three times. ATPase activities in 0.1 µM Eg5, 3 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP and 120 µM trans-BDPSB (grey bar) or cis-BDPSB (white bar) at 25°C. Results of three independent experiments and expressed as mean±standard deviation (error bar). Figure 2C shows the microtubule concentration-dependent ATPase activity of Eg5 in the presence of 80 μM cis- or trans-BDPSB. The trans isomer significantly affected the Vmax and KMT. The results suggested that the trans isomer decreases the affinity of Eg5 for microtubules and affects the ATPase catalytic activity of Eg5. Cis-BDPSB had minimal effect on KMT and Vmax. The photochromic inhibitor ACTAB showed almost similar values of KMT in the cis and trans isomers, but Vmax in the presence of trans-ACTAB was lower than that in the presence of cis-ACTAB (18). These results show BDPSB has an inhibitory mechanism different from that of ACTAB. BDPSB also regulated the microtubule-dependent ATPase activity of conventional kinesin (kinesin-1) photoreversibly. However, as shown in Fig. 4, trans-BDPSB showed lower inhibitory activity for kinesin-1 (IC50 of 100 μM) than for Eg5 (74 μM for Eg5). The inhibition may be induced by the binding of BDPSB to a similar site on both Eg5 and conventional kinesin but not to the site where the Eg5-specific inhibitors bind. Previous crystallographic studies of Eg5 specific inhibitors, STLC, Monastrol and Ispinesib, revealed these inhibitors bind to the same pocket composed of loop L5, α2 and α3 (17, 26, 27). Binding of these inhibitors significantly stabilizes the bound nucleotide, and induces formation of a weak-binding state, resulting in inhibition of basal and microtubule stimulated ATPase activity. On the other hand, trans-BDPSB dose-dependent basal ATPase activity of Eg5 showed biphase alteration as shown in Fig. 2A. In the first phase, ATPase activity was four times greater and in the subsequent second phase, ATPase was significantly inhibited. The second phase may be due to the binding of BDPSB to the pocket composed of loop L5, α2 and α3. The first phase may reflect the BDPSB binding to a new site which has not been shown so far. The second binding site might be the conserved region between Eg5 and conventional kinesin, which is related to the inhibition of microtubule stimulated ATPase activity. At this stage, we have no evidence of the exact BDPSB binding site. Therefore, it is required to elucidate the structure of BDPSB bound Eg5. Fig. 4 View largeDownload slide Inhibitory effect of BDPSB on the ATPase activity of conventional kinesin. The microtubule-stimulated ATPase activity of conventional kinesin (kinesin-1) was measured in the presence of BDPSB and compared with Eg5. ATPase activities of 0.1 µM motor domain truncated kinesin-1 (1–650 amino acids) or Eg5 were measured in a solution of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 3.0 µM microtubule and 0–150 µM trans-BDPSB or cis-BDPSB at 25°C. Results are of three independent experiments and are expressed as mean±standard deviation (error bars). Fig. 4 View largeDownload slide Inhibitory effect of BDPSB on the ATPase activity of conventional kinesin. The microtubule-stimulated ATPase activity of conventional kinesin (kinesin-1) was measured in the presence of BDPSB and compared with Eg5. ATPase activities of 0.1 µM motor domain truncated kinesin-1 (1–650 amino acids) or Eg5 were measured in a solution of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 3.0 µM microtubule and 0–150 µM trans-BDPSB or cis-BDPSB at 25°C. Results are of three independent experiments and are expressed as mean±standard deviation (error bars). Photo-control of Eg5 motor activity with BDPSB We also examined the effect of BDPSB on Eg5 motor activity. In the presence of cis- or trans-BDPSB, fluorescently labelled microtubules gliding on the Eg5 adsorbed glass surface in flow cells was monitored according to the established methods of the in vitro motility assay described in the Materials and Methods. In the absence of BDPSB (as control), the microtubules showed normal gliding at the average velocity of 11.54 ± 1.65 nm/s (Fig. 5D). Cis-BDPSB (100 µM) exhibited almost no effect on the microtubule gliding as shown in Fig. 5B and E. The average velocity (9.67 ± 2.46 nm/s) of microtubules was slightly reduced compared with control. On the other hand, in the presence of 100 µM trans-BDPSB, fluorescently labelled microtubules significantly dissociated from the Eg5-coated glass surface in flow cell (Fig. 5C). Moreover, the average velocity (4.00 ± 1.54 nm/s) of gliding of the microtubules remaining on the Eg5-coated glass surface was significantly reduced as shown in Fig. 5F. The results are consistent with the effect of trans-BDPSB on the microtubule stimulated ATPase activity (Fig. 2B and C) and suggest that trans-BDPSB predominantly interferes with the interaction of microtubules with Eg5 resulting in inhibition of microtubule stimulated ATPase activity. Fig. 5 View largeDownload slide Effect of cis- and trans-BDPSB on the Eg5 motor activity. Fluorescence microscopy images at the initial time of the in vitro motility assay. (A) In the absence of BDPSB; (B) in the presence of 100 µM cis-BDPSB; (C) in the presence 100 µM trans-BDPSB. Scale bar indicates 50 µm. A histogram of the distribution of microtubule gliding velocities on Eg5 (D-F). (D) In the absence of BDPSB (mean velocity 11.54 ± 1.65 nm/s, n = 184); (E) in the presence of 100 µM cis-BDPSB (mean velocity 9.67 ± 2.46 nm/s, n = 161); (F) in the presence of 100 µM trans-BDPSB (mean velocity 4.00 ± 1.54 nm/s, n = 19). 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. 5 View largeDownload slide Effect of cis- and trans-BDPSB on the Eg5 motor activity. Fluorescence microscopy images at the initial time of the in vitro motility assay. (A) In the absence of BDPSB; (B) in the presence of 100 µM cis-BDPSB; (C) in the presence 100 µM trans-BDPSB. Scale bar indicates 50 µm. A histogram of the distribution of microtubule gliding velocities on Eg5 (D-F). (D) In the absence of BDPSB (mean velocity 11.54 ± 1.65 nm/s, n = 184); (E) in the presence of 100 µM cis-BDPSB (mean velocity 9.67 ± 2.46 nm/s, n = 161); (F) in the presence of 100 µM trans-BDPSB (mean velocity 4.00 ± 1.54 nm/s, n = 19). 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. Conclusion This study demonstrates that a photochromic compound composed of two azobenzene derivatives photoreversibly inhibits the ATPase activity and motor activity of mitotic kinesin Eg5. About 150 μM of trans-BDPSB completely inhibited ATPase activity of Eg5, whereas cis-BDPSB showed extremely low inhibition. The highly efficient ‘ON’ and ‘OFF’ photoswitching may be applicable to other motor proteins. Conflict of Interest None declared. 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( 2016 ) Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy . Future Med. Chem. 8 , 463 – 489 Google Scholar CrossRef Search ADS PubMed 23 Sadakane K. , Takaichi M. , Maruta S. , ( 2018 ) Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative . J. Biochem in press. doi: 10.1093/jb/mvy046. 24 Rau H. ( 1990 ) Photoisomerization of azobenzenes in Photochemistry and Photophysics ( Rabeck J.F. , ed.) Vol. II , pp. 119 – 141 , CRC , Boca Raton, FL 25 Behrendt R. , Renner C. , Schenk M. , Wang F. , Wachtveitl J. , Oesterhelt D. , Moroder L. ( 1999 ) Photomodulation of the conformation of cyclic peptides with azobenzene moieties in the peptide backbone . Angew. Chem. Int. Ed. Engl. 38 , 2771 – 2774 Google Scholar CrossRef Search ADS PubMed 26 Maliga Z. , Mitchison T.J. ( 2006 ) Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol . BMC Chem. Biol. 6 , 2 Google Scholar CrossRef Search ADS PubMed 27 Talapatra S.K. , Schuttelkopf A.W. , Kozielski F. ( 2012 ) The structure of the ternary Eg5-ADP-ispinesib complex . Acta Crystallogr. D Biol. Crystallogr. 68 , 1311 – 1319 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ACTAB 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′-(N-(2-(N-(triphenylmethyl) amino) acetyl) amino)-azobenzene BDPSB 2, 3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid DMF N,N-dimethylformamide EDTA (ethylenedinitrilo)tetraacetic acid EGTA ethylene glycol tetraacetic acid; HEPES, 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic acid PIPES piperazine-1,4-bis(2-ethanesulfonic acid) TCA trichloroacetic acid; Pi, inorganic phosphate UV ultraviolet VIS visible © 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

Highly efficient photocontrol of mitotic kinesin Eg5 ATPase activity using a novel photochromic compound composed of two azobenzene derivatives

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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/mvy051
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Abstract

Abstract Mitotic kinesin Eg5 plays an important physiological role in cell division. Several small-molecule inhibitors of Eg5 are the focus of cancer therapies. Azobenzene is a photochromic compound exhibiting cis–trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. Photochromic compounds of azobenzene derivatives, mimicking Eg5-specific inhibitors of STLC, indicated photoreversible inhibitory effects on Eg5 ATPase activity; however, the photoreversible switching efficiency was not significant. This study presents a novel synthesized photochromic Eg5 inhibitor 2, 3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB), which is composed of two azobenzenes. BDPSB exhibited cis–trans isomerization with UV and VIS light irradiation. The trans form of BDPSB significantly inhibited microtubule-dependent ATPase activity of Eg5, with an IC50 of 74 μM. Cis BDPSB showed weak effects on the microtubule-dependent ATPase activity. The results suggest that the novel photochromic Eg5 inhibitor BDPSB, which exhibits highly efficient photoswitching, shows a switch ‘ON’ and ‘OFF’ behaviour with VIS and UV light irradiation. azobenzene, inhibitor, mitotic kinesin, photochromic compound Kinesin, an ATP-driven motor protein, converts the hydrolysis energy of ATP and moves along microtubules. The structure and the function of kinesin has been well studied and shown that kinesin has a molecular mechanism of energy transduction similar to myosin (1, 2). The previous studies suggested that the chemical energy generated by ATP hydrolysis is transmitted mechanically for the physical motility energy to occur like a cam-shaft motion through subdomain steric interactions. Therefore, it is expected that incorporation of an external stimuli responsive molecular device will enable regulation of the function of the ATP-driven motor proteins artificially. Photochromic molecules are known to change structure and properties photoreversibly and are of great interest as nanodevices with optical switches (3–5). Azobenzene is a typical photochromic molecule, which is photoisomerized to a hydrophilic cis isomer by ultraviolet (UV) irradiation and to a hydrophobic trans isomer using visible (VIS) light (3, 6). Spiropyran photoisomerizes to merocyanine, showing a ring-opened zwitterion under UV irradiation and a closed ring hydrophobic spiro type under VIS light (7). Consequently, photochromic molecules might be one of the possible candidates for the external stimuli responsive molecular device to regulate the function of the ATP-driven motor proteins. In fact, we have attempted to control the function of motor proteins using photochromic compounds. Cis–trans isomerization of azobenzene derivatives incorporated into the functional site of skeletal muscle myosin induced conformational changes in the myosin head, reflecting energy transduction (8). We also succeeded in photoreversibly controlling the ATPase activity of kinesin by introducing azobenzene and spiropyran derivatives into the catalytic site and the microtubule-binding site of kinesin (9, 10). However, specific introduction of a photochromic molecule into a functional site of a motor protein by chemical modification presents challenges. Preparations of motor protein mutants with a high-reactive cysteine side chain at the target site allowed for the introduction of a photochromic molecule. However, amino acid substitution and chemical modification occasionally decrease the original function of kinesin and induce partial denaturation, causing inconsistencies. Thus, instead of the direct incorporation of photochromic molecules into the functional site of motor proteins, we focused on the utilization of the specific inhibitors of motor proteins conjugated with photochromic molecules to control the function of motor proteins (11–13). Kinesin Eg5 is essential for mitosis, has physiologically important roles related to various spindle dynamics (14), and is studied as a target for cancer therapy. Many inhibitors of Eg5 have been developed as anticancer drugs (15). S-Trityl-l-cysteine (STLC) is well known as one of the potent inhibitors of Eg5. The molecular mechanism of inhibition by STLC and its binding site on Eg5 are known (16, 17). We have previously synthesized the photochromic STLC analogue composed of azobenzene and trityl groups that acts as the key part in STLC inhibition of Eg5. The photochromic inhibitor, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′-(N-(2-(N-(triphenylmethyl)amino)acetyl) amino)-azobenzene (ACTAB), changed its inhibitory activity by cis–trans isomerization and ATPase inhibition was photoreversible (18). However, its efficiency as a photoswitching compound was not significant. This study shows a photochromic Eg5 inhibitor composed of two azobenzene derivatives with extremely efficient photoswitching. The trans isomer and cis isomer of the photoswitching nanodevice exhibited almost ‘ON’ and ‘OFF’ states, respectively. The method of controlling Eg5 using a specific photochromic inhibitor whose inhibitory activity changes dramatically is expected to be widely applied to biofunctional molecules having specific regulatory factors. Materials and Methods Synthesis of photochromic inhibitor composed of azobenzene derivative 2,3-Bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) was synthesized by coupling of meso-2,3-dimercaptosuccinic acid (27.44 µM) and phenylazo maleinanil (PAM) (82.32 µM) in 1 ml of THF for 4 h at room temperature. The product was purified with preparative TLC (PLC Silica gel 60, MERCK) using a developing solvent of 100% acetonitrile. The expected product migrated at Rf 0.03, it was extracted by methanol, and evaporated to dryness. Further purification was performed by HPLC using C18 reverse-phase column chromatography with a linear gradient of 0–90% acetonitrile in 0.1% trifluoroacetic acid. The product eluted as the main peak at 90% acetonitrile. The TLC (Silica gel 70 F254 Plate, Wako) analysis exhibited a single spot with an Rf of 0.53 in n-butanol:acetic acid:water (5:2:3). The yield of the product was 24.8%. Fast atom bombardment mass spectrum of the product showed a molecular ion (M+H)+ at m/z 736 + 1, which corresponds to a molecular mass of 736.14 as calculated from the formula C36H28N6O8S2 of BDPSB. Photoisomerization of the Eg5 inhibitor BDPSB Irradiations at 366 nm using a Black-Ray lamp (16 W; UVP, Upland, CA, USA) and VIS light using a fluorescent lamp (27 W) were used for the conversion of the trans-BDPSB to cis-BDPSB and the cis-BDPSB to trans-BDPSB, respectively. BDPSB dissolved in DMF or in a solution (20 mM HEPES–KOH pH 7.2, 50 mM KCl, 2.0 mM MgCl2, 0.1 mM EDTA and 0.1 mM EGTA) was irradiated with the light source located 1 cm from the surface of the solvents at 25°C. Expression and purification of the mitotic kinesin Eg5 motor domain and conventional kinesin The motor domain of Eg5 was prepared according to the following method, which is a modification of the previously reported method (18). The cDNA of the motor domain of wild-type (WT) mouse Eg5 (WT, amino residue 1–367) was amplified by polymerase chain reaction and ligated into the pET21a vector. The Eg5 WT expression plasmids were used to transform Escherichia coli BL21 (DE3). Eg5 WT was purified by using a Co-NTA column. The Co-NTA column was washed with lysis buffer containing 30 mM imidazole, and bound Eg5 was eluted with lysis buffer containing 150 mM imidazole. Fractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purified Eg5 was dialyzed in buffer (30 mM Tris–HCl at pH 7.5, 120 mM NaCl, 2.0 mM MgCl2, 0.1 mM ATP and 0.5 mM DTT) and stored at −80°C. Truncated conventional kinesin·kinesin-1 (the N-terminal 560 amino acids of mouse brain kinesin containing motor domain) was prepared according to the established methods we have previously reported (19). The plasmid pET21a carried a mouse conventional kinesin (KIF5A) 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 dialyzed 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 (20). Tubulin was polymerized for 30 min at 37°C in 100 mM PIPES (pH 6.8), 1.0 mM EGTA, 1.0 mM MgCl2 and 1.0 mM GTP. Next, taxol was added to a final concentration of 10 µM. The polymerized microtubules were collected by centrifugation at 280,000 × g for 15 min at 37°C. The supernatant was removed, and the microtubule pellet was collected in a buffer containing 100 mM PIPES (pH 6.8), 1.0 mM EGTA, 1.0 mM MgCl2, 1.0 mM GTA and 10 μM taxol. Kinesin ATPase assay The ATPase assay was performed according to the following methods we have previously reported (18). The ATPase activity of kinesin was measured in 20 mM HEPES (pH 7.2), containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA and 1.0 mM β-mercaptoethanol. To examine the effect of the photoisomerization of the inhibitor, BDPSB dissolved in DMF was irradiated by UV or VIS light and added to the assay buffer to a final concentration 0.0–150.0 µM. The conditions for the photoisomerization of BDPSB are as described in the above section, ‘Photoisomerization of the Eg5 inhibitor BDPSB’. The final DMF concentration in the Eg5 ATPase buffer was 5.0%. For the basal ATPase activity, 0.5 µM Eg5 was added in the assay buffer without microtubules. For the microtubule-stimulated ATPase activity, 100 nM Eg5 motor domain or 100 nM conventional kinesin (kinesin-1) motor domain was added to the assay buffer in the presence of a 3.0 µM microtubule solution. The ATPase reaction was started by adding 2.0 mM ATP and terminated by adding 10% trichloroacetic acid. The reaction time of microtubule-stimulated ATPase or basal ATPase activity was 5 or 20 min at 25°C, respectively. The released inorganic phosphate (Pi) in the supernatant was measured according to the method of Youngburg (21). To determine KMT and Vmax for microtubule concentration in the presence of Eg5 and BDPSB, the microtubule concentration-dependent ATPase activities (0–12 µM) were measured in the presence of 100 µM BDPSB. Microtubule gliding assay The in vitro motility assay was performed according to the slightly changed method that we previously reported (18). Coverslips were coated with an anti-6× histidine monoclonal antibody (Wako) in assay buffer (10 mM Tris–acetate, pH 7.5, 50 mM potassium–acetate, 2.5 mM EGTA and 4.0 mM MgSO4). Subsequently, 0.5 µM Eg5 in assay buffer A (10 mM Tris–acetate, pH7.5, 50 mM potassium–acetate, 2.5 mM EGTA, 4.0 mM MgSO4, 0.2% β-mercaptoethanol) was perfused through the flow chambers and the chambers were incubated for 2 min at 25°C. After washing the chambers with assay buffer A, 1.0 mg/ml casein in assay buffer A was perfused through the flow chambers and the chambers were incubated for 5 min at 25°C. After washing the chambers with assay buffer A, 100 µM photo-isomerized BDPSB, which was irradiated with VIS light or UV (366 nm) for 10 min in assay buffer A, were perfused through the flow chambers and the anchored Eg5 were treated for 2 min at 25°C. And then, rhodamine-labelled microtubules in assay buffer B (assay buffer A with 100 µM photo-isomerized BDPSB and 20.0 µM taxol) were perfused through the flow chambers and the chambers were incubated for 2 min at 25°C. The chambers were then washed with assay buffer B. Finally, 1.0 mM ATP and 100 µM photo-isomerized BDPSB 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 perfused through the flow chambers. The final DMF concentration in the assay buffer was 5.0%. Rhodamine-labelled microtubules were visualized using an Olympus BX50 microscope equipped with a CCD camera (LK-TU53H: Toshiba, Japan). Statistical analysis Data are expressed as mean ±standard deviation (S.D.). Significant differences between groups were determined using Welch’s t-test or Student’s t-test. P-Values of <0.05 were considered statistically significant. Results and Discussion Design, synthesis and spectroscopic characterization of BDPSB Chemical structures of the Eg5 inhibitors previously reported were not highly conserved (22). The only similarity among the inhibitors is that the single bond linkage between aromatic rings and some hydrophilic groups on the aromatic rings were observed. The photochromic compounds are expected to exhibit inhibitory activity on Eg5. In fact, previously we showed that azobenzene derivatives ACTAB that mimic STLC, a potent inhibitor of Eg5, changes its inhibitory activity accompanied by cis–trans isomerization and regulates the ATPase activity of Eg5 photoreversibly (18). However, the photoswitching was not efficient. The difference in inhibitory activities between the cis and trans isomer was not significant. Our recent study revealed that the photochromic compounds composed of two spiropyrans inhibited Eg5 ATPase activity significantly, indicating that the two-headed structure of the aromatic group might be an effective inhibitor (23). In this study, we designed and synthesized the novel photochromic Eg5 inhibitor, BDPSB which is composed of two azobenzene moieties, and formed a two-headed structure (Fig. 1A). The synthesis of BDPSB was done by the established coupling reaction. Thiol reactive azobenzene derivative, 4-(N-Maleimido)azobenzene, was incorporated into the two thiol groups of 2,3-dimercapto succinic acid, according to the procedure described in the ‘Materials and Methods’ section. The two azobenzene moieties of BDPSB exhibit cis–trans isomerization. Consequently, BDPSB changes conformation and physical properties more drastically than compounds composed of a single azobenzene. Cis–trans reversible isomerization of BDPSB was monitored with absorption spectral changes during UV and VIS light irradiation. Azobenzene absorption spectra change at 300– 400 nm with cis–trans isomerization (3, 24). BDPSB also exhibited similar spectral changes during UV and VIS light irradiation. Figure 1B shows that irradiation of trans-BDPSB by UV light led to a reduction of the peak at 325 nm, and the spectral change was complete after 10 min. Irradiation of cis-BDPSB by VIS light led to an almost complete isomerization to the trans form, as shown in Fig. 1C, within 10 min. Fig. 1 View largeDownload slide Structural formula of 2,3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl] phenyl} pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) and its spectral changes accompanied by photoisomerization. (A) Structural formula of BDPSB. The azobenzene derivative BPSBA has reversible photoisomerization between the trans-form and cis-form with VIS or UV light irradiation. (B) Time-dependent change in the absorption spectra of trans-BDPSB to cis-BDPSB upon UV light irradiation (366 nm). Ten micromoles of BDPSB were irradiated in DMF for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. (C) Time-dependent change in the absorption spectra of cis-BDPSB to trans-BDPSB upon VIS light irradiation. Ten micromoles of BDPSB were irradiated for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. Fig. 1 View largeDownload slide Structural formula of 2,3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl] phenyl} pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB) and its spectral changes accompanied by photoisomerization. (A) Structural formula of BDPSB. The azobenzene derivative BPSBA has reversible photoisomerization between the trans-form and cis-form with VIS or UV light irradiation. (B) Time-dependent change in the absorption spectra of trans-BDPSB to cis-BDPSB upon UV light irradiation (366 nm). Ten micromoles of BDPSB were irradiated in DMF for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. (C) Time-dependent change in the absorption spectra of cis-BDPSB to trans-BDPSB upon VIS light irradiation. Ten micromoles of BDPSB were irradiated for 0, 60, 120, 180, 240, 300, 360, 480 and 600 s at 25°C. Photo-control of the BDPSB inhibitory activity on the basal Eg5 ATPase The inhibitory activity of the cis and trans isomers of BDPSB on the basal ATPase activity of Eg5 was determined. Trans-BDPSB dose-dependent ATPase activity of Eg5 showed biphase alteration of ATPase activity (Fig. 2A). The first phase of ATPase activity modulation with trans-BDPSB showed an increase of 400% at 25 μM. Subsequently, in the second phase, the ATPase activity decreased dose-dependently, and reached almost complete inhibition at 150 μM trans-BDPSB. This indicates some cooperative interactions between the compound and binding sites on Eg5. Cis-BDPSB increased ATPase activity by 400% at 125 μM in a single phase. The ATPase activation corresponds to that in the first phase of trans-BDPSB. The decrease in ATPase activity in the second phase of trans-BDPSB reflects a binding site different from that in the first phase. Fig. 2 View largeDownload slide Photocontrol of the ATPase activity of Eg5 by BDPSB. (A) Dose-dependent inhibition of basal Eg5 ATPase activities by cis- and trans-BDPSB in 0.5 µM Eg5, 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. (B) Dose-dependent inhibition of microtubule stimulated Eg5 ATPase activities by cis- and trans-BDPSB in 0.1 µM Eg5, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 3.0 µM microtubule and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. The dashed line indicates the inhibition of true cis-BDPSB obtained by subtracting the inhibition of co-existing 25% trans-BDPAB. (C) Inhibitory effect of BDPSB photoisomers on the interaction of Eg5 and microtubules. The microtubule dose-dependent ATPase activities in 0.1 µM Eg5, 0–12 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 5% DMF in the presence of 80 µM trans-BDPSB (filled circle), cis-BDPSB (open circle) and in the absence of BDPSB (filled circle) at 25°C. Fig. 2 View largeDownload slide Photocontrol of the ATPase activity of Eg5 by BDPSB. (A) Dose-dependent inhibition of basal Eg5 ATPase activities by cis- and trans-BDPSB in 0.5 µM Eg5, 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. (B) Dose-dependent inhibition of microtubule stimulated Eg5 ATPase activities by cis- and trans-BDPSB in 0.1 µM Eg5, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 3.0 µM microtubule and 2.0 mM ATP in the presence of 0–150 µM trans-BDPSB (filled circle) or cis-BDPSB (open circle) at 25°C. The dashed line indicates the inhibition of true cis-BDPSB obtained by subtracting the inhibition of co-existing 25% trans-BDPAB. (C) Inhibitory effect of BDPSB photoisomers on the interaction of Eg5 and microtubules. The microtubule dose-dependent ATPase activities in 0.1 µM Eg5, 0–12 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 5% DMF in the presence of 80 µM trans-BDPSB (filled circle), cis-BDPSB (open circle) and in the absence of BDPSB (filled circle) at 25°C. Photo-control of the BDPSB inhibitory activity on the microtubule-stimulated Eg5 ATPase The microtubule-stimulated ATPase activity of Eg5 was significantly inhibited by trans-BDPSB as shown in Fig. 2B. The IC50 value of trans-BDPSB for ATPase activity of Eg5 in the presence of microtubules was 74.2 ± 2.5 μM. The phase of trans-BDPSB dose-dependent inhibition exhibited a sigmoidal response, which indicates the existence of a subsite of binding and corresponds to the biphase alteration of basal ATPase activity of trans-BDPSB (Fig. 2A). The effect of the trans-BDPSB binding to a subsite on the microtubule-stimulated ATPase activity may be reduced comparing the 400% increase in the first phase in the basal ATPase activity. Eg5 ATPase activity is highly stimulated by microtubules. Then, the effect on the trans-BDPSB binding to the subsite may be buried by the microtubules stimulation of ATPase activity. Cis-BDPSB showed extremely low inhibitory activity, as shown in Fig. 2B. Isomerization of azobenzene from trans to cis under UV irradiation (25) is not complete and for BDPSB, 25% trans isomer remains at the equilibrium point under UV irradiation as shown in Fig. 1B. The inhibitory effect of the trans isomer coexisting with cis-BDPSB was controlled for by subtracting trans inhibition from the apparent results of cis-BDPSB inhibition (open circle in Fig. 2B). The estimated true inhibitory activity of only cis-BDPSB was extremely low, as shown in Fig. 2B (dashed line). The highly efficient photocontrol of the ATPase activity of Eg5 was achieved by cis–trans isomerization of BDPSB. ACTAB, composed of a single azobenzene, exhibited little inhibitory activity between cis and trans isomers (18). The highly efficient photocontrol of BDPSB on ATPase activity is proposed to be due to the double azobenzene head structure, which induced the conformational change. The changes in inhibitory activity of trans and cis isomers of BDPSB, as induced by VIS and UV light irradiation, were completely and reproducibly reversible (Fig. 3). BDPSB exhibited almost ‘ON’ and ‘OFF’ photoswitching by trans–cis isomerization under VIS and UV light irradiations. Fig. 3 View largeDownload slide Reproducible reversibility of the photocontrol of the microtubule stimulated ATPase activity by BDPSB. BDPSB was alternately irradiated with 366 nm UV light and VIS light prior to the ATPase assay. Alternating irradiations of UV and VIS lights were repeated three times. ATPase activities in 0.1 µM Eg5, 3 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP and 120 µM trans-BDPSB (grey bar) or cis-BDPSB (white bar) at 25°C. Results of three independent experiments and expressed as mean±standard deviation (error bar). Fig. 3 View largeDownload slide Reproducible reversibility of the photocontrol of the microtubule stimulated ATPase activity by BDPSB. BDPSB was alternately irradiated with 366 nm UV light and VIS light prior to the ATPase assay. Alternating irradiations of UV and VIS lights were repeated three times. ATPase activities in 0.1 µM Eg5, 3 µM microtubule, 20 mM HEPES-KOH (pH 7.2), 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP and 120 µM trans-BDPSB (grey bar) or cis-BDPSB (white bar) at 25°C. Results of three independent experiments and expressed as mean±standard deviation (error bar). Figure 2C shows the microtubule concentration-dependent ATPase activity of Eg5 in the presence of 80 μM cis- or trans-BDPSB. The trans isomer significantly affected the Vmax and KMT. The results suggested that the trans isomer decreases the affinity of Eg5 for microtubules and affects the ATPase catalytic activity of Eg5. Cis-BDPSB had minimal effect on KMT and Vmax. The photochromic inhibitor ACTAB showed almost similar values of KMT in the cis and trans isomers, but Vmax in the presence of trans-ACTAB was lower than that in the presence of cis-ACTAB (18). These results show BDPSB has an inhibitory mechanism different from that of ACTAB. BDPSB also regulated the microtubule-dependent ATPase activity of conventional kinesin (kinesin-1) photoreversibly. However, as shown in Fig. 4, trans-BDPSB showed lower inhibitory activity for kinesin-1 (IC50 of 100 μM) than for Eg5 (74 μM for Eg5). The inhibition may be induced by the binding of BDPSB to a similar site on both Eg5 and conventional kinesin but not to the site where the Eg5-specific inhibitors bind. Previous crystallographic studies of Eg5 specific inhibitors, STLC, Monastrol and Ispinesib, revealed these inhibitors bind to the same pocket composed of loop L5, α2 and α3 (17, 26, 27). Binding of these inhibitors significantly stabilizes the bound nucleotide, and induces formation of a weak-binding state, resulting in inhibition of basal and microtubule stimulated ATPase activity. On the other hand, trans-BDPSB dose-dependent basal ATPase activity of Eg5 showed biphase alteration as shown in Fig. 2A. In the first phase, ATPase activity was four times greater and in the subsequent second phase, ATPase was significantly inhibited. The second phase may be due to the binding of BDPSB to the pocket composed of loop L5, α2 and α3. The first phase may reflect the BDPSB binding to a new site which has not been shown so far. The second binding site might be the conserved region between Eg5 and conventional kinesin, which is related to the inhibition of microtubule stimulated ATPase activity. At this stage, we have no evidence of the exact BDPSB binding site. Therefore, it is required to elucidate the structure of BDPSB bound Eg5. Fig. 4 View largeDownload slide Inhibitory effect of BDPSB on the ATPase activity of conventional kinesin. The microtubule-stimulated ATPase activity of conventional kinesin (kinesin-1) was measured in the presence of BDPSB and compared with Eg5. ATPase activities of 0.1 µM motor domain truncated kinesin-1 (1–650 amino acids) or Eg5 were measured in a solution of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 3.0 µM microtubule and 0–150 µM trans-BDPSB or cis-BDPSB at 25°C. Results are of three independent experiments and are expressed as mean±standard deviation (error bars). Fig. 4 View largeDownload slide Inhibitory effect of BDPSB on the ATPase activity of conventional kinesin. The microtubule-stimulated ATPase activity of conventional kinesin (kinesin-1) was measured in the presence of BDPSB and compared with Eg5. ATPase activities of 0.1 µM motor domain truncated kinesin-1 (1–650 amino acids) or Eg5 were measured in a solution of 20 mM HEPES-KOH (pH 7.2) containing 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM β-mercaptoethanol, 2.0 mM ATP, 3.0 µM microtubule and 0–150 µM trans-BDPSB or cis-BDPSB at 25°C. Results are of three independent experiments and are expressed as mean±standard deviation (error bars). Photo-control of Eg5 motor activity with BDPSB We also examined the effect of BDPSB on Eg5 motor activity. In the presence of cis- or trans-BDPSB, fluorescently labelled microtubules gliding on the Eg5 adsorbed glass surface in flow cells was monitored according to the established methods of the in vitro motility assay described in the Materials and Methods. In the absence of BDPSB (as control), the microtubules showed normal gliding at the average velocity of 11.54 ± 1.65 nm/s (Fig. 5D). Cis-BDPSB (100 µM) exhibited almost no effect on the microtubule gliding as shown in Fig. 5B and E. The average velocity (9.67 ± 2.46 nm/s) of microtubules was slightly reduced compared with control. On the other hand, in the presence of 100 µM trans-BDPSB, fluorescently labelled microtubules significantly dissociated from the Eg5-coated glass surface in flow cell (Fig. 5C). Moreover, the average velocity (4.00 ± 1.54 nm/s) of gliding of the microtubules remaining on the Eg5-coated glass surface was significantly reduced as shown in Fig. 5F. The results are consistent with the effect of trans-BDPSB on the microtubule stimulated ATPase activity (Fig. 2B and C) and suggest that trans-BDPSB predominantly interferes with the interaction of microtubules with Eg5 resulting in inhibition of microtubule stimulated ATPase activity. Fig. 5 View largeDownload slide Effect of cis- and trans-BDPSB on the Eg5 motor activity. Fluorescence microscopy images at the initial time of the in vitro motility assay. (A) In the absence of BDPSB; (B) in the presence of 100 µM cis-BDPSB; (C) in the presence 100 µM trans-BDPSB. Scale bar indicates 50 µm. A histogram of the distribution of microtubule gliding velocities on Eg5 (D-F). (D) In the absence of BDPSB (mean velocity 11.54 ± 1.65 nm/s, n = 184); (E) in the presence of 100 µM cis-BDPSB (mean velocity 9.67 ± 2.46 nm/s, n = 161); (F) in the presence of 100 µM trans-BDPSB (mean velocity 4.00 ± 1.54 nm/s, n = 19). 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. 5 View largeDownload slide Effect of cis- and trans-BDPSB on the Eg5 motor activity. Fluorescence microscopy images at the initial time of the in vitro motility assay. (A) In the absence of BDPSB; (B) in the presence of 100 µM cis-BDPSB; (C) in the presence 100 µM trans-BDPSB. Scale bar indicates 50 µm. A histogram of the distribution of microtubule gliding velocities on Eg5 (D-F). (D) In the absence of BDPSB (mean velocity 11.54 ± 1.65 nm/s, n = 184); (E) in the presence of 100 µM cis-BDPSB (mean velocity 9.67 ± 2.46 nm/s, n = 161); (F) in the presence of 100 µM trans-BDPSB (mean velocity 4.00 ± 1.54 nm/s, n = 19). 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. Conclusion This study demonstrates that a photochromic compound composed of two azobenzene derivatives photoreversibly inhibits the ATPase activity and motor activity of mitotic kinesin Eg5. About 150 μM of trans-BDPSB completely inhibited ATPase activity of Eg5, whereas cis-BDPSB showed extremely low inhibition. The highly efficient ‘ON’ and ‘OFF’ photoswitching may be applicable to other motor proteins. Conflict of Interest None declared. 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( 2016 ) Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy . Future Med. Chem. 8 , 463 – 489 Google Scholar CrossRef Search ADS PubMed 23 Sadakane K. , Takaichi M. , Maruta S. , ( 2018 ) Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative . J. Biochem in press. doi: 10.1093/jb/mvy046. 24 Rau H. ( 1990 ) Photoisomerization of azobenzenes in Photochemistry and Photophysics ( Rabeck J.F. , ed.) Vol. II , pp. 119 – 141 , CRC , Boca Raton, FL 25 Behrendt R. , Renner C. , Schenk M. , Wang F. , Wachtveitl J. , Oesterhelt D. , Moroder L. ( 1999 ) Photomodulation of the conformation of cyclic peptides with azobenzene moieties in the peptide backbone . Angew. Chem. Int. Ed. Engl. 38 , 2771 – 2774 Google Scholar CrossRef Search ADS PubMed 26 Maliga Z. , Mitchison T.J. ( 2006 ) Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol . BMC Chem. Biol. 6 , 2 Google Scholar CrossRef Search ADS PubMed 27 Talapatra S.K. , Schuttelkopf A.W. , Kozielski F. ( 2012 ) The structure of the ternary Eg5-ADP-ispinesib complex . Acta Crystallogr. D Biol. Crystallogr. 68 , 1311 – 1319 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ACTAB 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4′-(N-(2-(N-(triphenylmethyl) amino) acetyl) amino)-azobenzene BDPSB 2, 3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid DMF N,N-dimethylformamide EDTA (ethylenedinitrilo)tetraacetic acid EGTA ethylene glycol tetraacetic acid; HEPES, 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic acid PIPES piperazine-1,4-bis(2-ethanesulfonic acid) TCA trichloroacetic acid; Pi, inorganic phosphate UV ultraviolet VIS visible © 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: May 31, 2018

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