https://orcid.org/0000-0003-4289-7891
Correction
17 May 2024: Saito R, Goto M, Katakura S, Ohba T, Kawata R, et al. (2024) Correction: Pterin-based small molecule inhibitor capable of binding to the secondary pocket in the active site of ricin-toxin A chain. PLOS ONE 19(5): e0304251. https://doi.org/10.1371/journal.pone.0304251 View correction
Figures
Abstract
The Ricin toxin A chain (RTA), which depurinates an adenine base at a specific region of the ribosome leading to death, has two adjacent specificity pockets in its active site. Based on this structural information, many attempts have been made to develop small-molecule RTA inhibitors that simultaneously block the two pockets. However, no attempt has been successful. In the present study, we synthesized pterin-7-carboxamides with tripeptide pendants and found that one of them interacts with both pockets simultaneously to exhibit good RTA inhibitory activity. X-ray crystallographic analysis of the RTA crystal with the new inhibitor revealed that the conformational change of Tyr80 is an important factor that allows the inhibitors to plug the two pockets simultaneously.
Citation: Saito R, Goto M, Katakura S, Ohba T, Kawata R, Nagatsu K, et al. (2022) Pterin-based small molecule inhibitor capable of binding to the secondary pocket in the active site of ricin-toxin A chain. PLoS ONE 17(12): e0277770. https://doi.org/10.1371/journal.pone.0277770
Editor: Israel Silman, Weizmann Institute of Science, ISRAEL
Received: July 25, 2022; Accepted: November 3, 2022; Published: December 12, 2022
Copyright: © 2022 Saito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All crystal structure data files are available from the Protein Data Bank at DOI: 10.2210/pdb7y4k/pdb (https://doi.org/10.2210/pdb7Y4K/pdb) and DOI: 10.2210/pdb7y4m/pdb (https://doi.org/10.2210/pdb7Y4M/pdb).
Funding: “RS received financial supports from JSPSKAKENHI (Grant Number 19K05699) and the Faculty of Science Special Grant for Promoting Scientific Research at Toho University. The founders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.”
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ricin is a highly toxic protein that can be easily isolated from the seeds of Ricinus communis; however, no effective antidote has been developed against it [1]. Ricin is an AB toxin consisting of an enzymatically active A subunit (Ricin Toxin A-chain, RTA) and a cell-binding B subunit (Ricin Toxin B-chain, RTB) linked by a disulfide bond [2, 3]. RTA is highly catalytically active in depurinating the adenine base at position 4324 (A4324) selectively in a specific region in the 28S rRNA to inhibit ribosomal protein biosynthesis, causing cell death [4]. The active site of RTA consists of two pockets, the primary pocket catalyzing the depurination of A4324 and the secondary pocket accommodating the guanine residue adjacent to A4324, with Tyr80 dividing these pockets. Based on the structural information of RTA, many inhibitor candidates based on nucleobases, pterins, and pyrimidines have been proposed, but all have been found to exhibit low RTA inhibitory activity [1]. Subsequently, Schramm et al. reported that the cyclic transition state model showed high RTA inhibitory activity, with an IC50 of 1 μM [5]. X-ray crystallographic analysis of this compound complexed with RTA showed that the model compound interacted with both the primary and secondary pockets. Since all the RTA inhibitors reported so far interacted only with the primary pocket, this result suggests that simultaneous interaction with the two pockets is a key factor for high RTA inhibitory activity. Despite its fascinating inhibitory activity, the cyclic model compound has not been put to practical use as an antidote for RTA because its high inhibitory activity was measured under non-physiological conditions and its synthesis is complex. Therefore, there is a requirement for development of more easily accessible and small-molecule RTA inhibitors. Our research group has also been interested in developing RTA inhibitors and has reported that several pterin-7-carboxamides (7PCAs) exhibited weak to good inhibition of RTA in vitro [6]. For instance, 7PCAs with (glycyl)phenylalanine pendants (1 and 2 in Fig 1) exhibited high RTA inhibitory activity (IC50 = 20 and 15 μM, respectively), while the corresponding glycylglycine analog (3) was found to be a weak RTA inhibitor (IC50 = 400 μM). X-ray crystallographic analysis of 2/RTA complex revealed that the C-terminal residue of the tripeptide pendant (P3) was oriented toward the secondary pocket, but was not long enough to reach it [6]. This indicates that a 7PCA with a tripeptide pendant with an appropriately modified the C-terminal residue can interact simultaneously with the primary and secondary pockets. Thus, in this study, we report the synthesis of 7PCAs with tripeptide pendants having elongated C-terminal residues, in which ornithine and lysine residues with an aromatic ring were employed as the C-terminal residues to direct the interaction with the secondary pocket. X-ray crystallographic analysis of the new ligands revealed that they complexed with RTA to be the first example of a small-molecule inhibitor that binds simultaneously with the primary and the secondary pockets of RTA.
Results and discussion
The synthesis of new 7PCAs, 4a and 4b, were started from appropriate commercially available amino acid derivatives, which were converted into the corresponding methyl esters 5a and 5b. 5a and 5b were then processed using the conventional peptide synthetic procedures to produce crude tripeptides 8a and 8b, respectively. Finally, the tripeptides were respectively reacted with 7-methoxycarbonylpterin in the presence of 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) as an accererator [6–9] to synthesize the corresponding 7PCAs, 4a and 4b (Scheme 1).
Reagents and conditions: (i) CH3I, K2CO3, dimethylformamide (DMF), 0°C to r.t.; (ii) 4-M HCl in 1,4-dioxane, r.t.; (iii) Boc-Phe-OH, PyBOP, iPr2NEt, DMF, r.t.; (iv) Fmoc-Gly-OH, PyBOP, iPr2NEt, DMF, r.t.; (v) piperidine, DMF, r.t.; (vi) 7-methoxycarbonylpterin, DBU, MeOH, r.t.
Co-crystals of RTA with 4a and 4b were prepared using the hanging drop vapor diffusion crystallization method and their crystal structures were analyzed. The resulting 3D structures of the complexes are shown in Fig 2. In each case, the pterin ring interacted with Gly121, Asn122, Tyr123, Ile172, and Arg180 in the primary pocket of RTA through the same interactions as those previously observed with other pterin derivatives [6–8, 10]. This demonstrates that the binding mode of the pterin moiety of 7PCAs to the primary pocket of RTA is constant, regardless of the structure of the pendants. For 4a, the tripeptide moiety was unsuccessfully assigned, probably because only the pterin moiety was accommodated in the primary pocket and the pendant moiety was free to move. In stark contrast, the entire structure of 4b was assigned, and, notably, the Cbz group on the C-terminal residue was accommodated in the secondary pocket. This is the first example of a single and small organic molecule plugging the two pockets in the active site of RTA simultaneously. Closer examination of the crystal structure of 4b/RTA complex revealed that the phenylalanine residue in 4b interacts with Trp211 of RTA in a T-shaped CH–π interaction, and Tyr80, the partition between the two pockets in the active site of RTA, was moved to the opposite side of Trp211 owing to repulsion with the phenyl group. Besides, the carbonyl group of the Cbz in 4b made hydrogen bonds with Asn48 and Arg78 in the “entrance” of the secondary pocket at 3.15 Å and 2.67 Å, respectively, and accordingly the benzene ring of Cbz group was placed in the secondary pocket (Fig 3).
X-ray crystal structures of (A) 4a-RTA and (B) 4b-RTA complexes in surface representation styles. The ligands are colored in green, and the amino acid residues around the ligands are in blue except Tyr80 in magenta.
All interactions were detected and visualized by Discovery Studio Visualizer [11] in both 2D (right) and 3D (left) styles.
The same movements of Tyr80 were observed in the crystal structures of RTA with the potent inhibitors 1, 2 (Fig 4B) [6], while no such conformational change was observed in RTA crystals with 4a and other low-activity inhibitors such as 3 (Fig 4C), for which the positions of Tyr80 was the same as in the inhibitor-free crystal structure [6]. Furthermore, the same rearrangement of Tyr80 residue was observed in the crystal structure of RTA with the Schramm’s cyclic nucleotide as a ligand (Fig 4D). These results strongly suggest that the conformational change in Tyr80 is essential for inhibitors to interact with the secondary pocket and to increase the inhibitory activity. For 4A, the displacement of Tyr80, as well as the T-shaped interactions, were not observed, even though it had the same P1-P2 moiety, Gly-Phe. It is likely that the alkyl chain of the C-terminal residue in 4A is not long enough to form the hydrogen bonds with the residues at the entrance of the secondary pocket; consequently, the unfixed large side chain is free to move, preventing the phenyl ring in P2 from forming a T-shape interaction.
(A) crystal structure of 4b-RTA complex, (B) crystal structure of 2-RTA complex (PDB ID: 4HUP), (C) crystal structure of 3-RTA complex (PDB ID: 4HV7), and (D) crystal structure of RTAS complexed with the cyclic transition-model inhibitor (PDB ID: 3HIO). Images are drawn by Discovery Studio Visualizer [11].
The activity of RTA in the absence and presence of 4a and 4b was measured using an in vitro translation reaction of firefly luciferase utilizing rabbit reticulocyte lysate and a subsequent luciferase quantification by measuring the bioluminescence intensity. The inhibitory activity of 1, as a positive standard, was also evaluated. The results are presented in Table 1.
Compound 4b exhibited an IC50 value of 33.4±2.7 μM, whereas compound 4a exhibited an IC50 value of 485±33 μM. An approximately 10-fold difference was observed between the two inhibitory activities. The weak inhibitory activity of 4a was comparable to that of pterin-7-carboxylic acid, which is consistent with the fact that only the pterin-7-carboxylic acid portion was assigned in the X-ray crystallography of 4a/RTA and no conformational change in Tyr80 was observed. The more potent 4b, which changed the orientation of Tyr80, exhibited the same level of inhibitory activity as 1 and 2, bringing about the same movement of Tyr80. From this result, it appears that the inhibitory activity was not enhanced by the inhibitor plugging the secondary pocket. This apparent ineffectiveness may be due to the fact that the Cbz group is located at a shallow position near the entrance of the secondary pocket. Therefore, replacement of the Cbz group with larger aromatic ring systems having substituents of forming extra interactions with residues in the secondary pocket could lead to the enhancement of the RTA inhibitory activity. More detailed structure-activity relationship studies to determine the residues in the secondary pocket to be interactes with will be conducted in the near future.
Conclusion
In the present study, new pterin compounds 4a and 4b were synthesized; among them, 4b, having a Gly-Phe-Lys(Cbz)-OH pendant, exhibited good RTA inhibitory activity comparable to 1 and 2 previously reported as potent RTA inhibitors. X-ray crystallographic analysis of 4b/RTA co-crystal revealed that the potent 4b not only bound tightly to the primary pocket but also plugged the secondary pocket in the active site of RTA. This is the first example of a small organic inhibitor that is capable of simultaneously plugging the two pockets of the RTA sensitive site. A more detailed analysis of 4b/RTA co-crystal revealed that the conformational change in Tyr80, induced by the T-shaped interaction between the phenyl ring in the pendant moiety of 4b and Trp211, is an important factor in determining the RTA inhibitory activity and achieving the interaction between inhibitors and the secondary pocket of the RTA active site. These findings should guide the development of more potent RTA inhibitors.
Materials and methods
General
1H-NMR spectra were recorded using an ECP-400 spectrometer (JEOL Ltd., Japan). Chemical shifts (δ) are reported in ppm using tetramethylsilane or an undeuterated solvent as the internal standard in the deuterated solvent used. The coupling constants (J) are given in hertz. 13C-NMR spectra were recorded using an ECP-100 spectrometer (JEOL Ltd., Japan) or an Avance II 400 spectrometer (Bruker Biospin, Billerica, MA, USA). Chemical shift multiplicities are reported as s = singlet, d = doublet, t = triplet, and m = multiplet. Electrospray ionization (ESI) mass spectra were recorded on a JEOL JMS-T100CS mass spectrometer. Column chromatography was performed on silica gel (particle size: 46–50 μm; Fuji Silysia Chemical Ltd.). SephadexTM LH-20, purchased from Cytiva (Tokyo, Japan), was used for the gel filtration. All chemicals were purchased from Sigma-Aldrich Co. LLC. or Tokyo Chemical Industry Co., Ltd. (TCI) and used as received, unless otherwise mentioned below.
Chemistry
Synthesis of Boc-Orn(Cbz)-OMe (5a) [12].
CH3I (500 μL, 8 mmol) was added dropwise to a suspension of Boc-Orn(Cbz)-OH (1.84 g, 5 mmol) and K2CO3 (1.43 g, 10 mmol) in DMF (10 mL) at 0°C, and the mixture was stirred at 0°C for 30 min, then room temperature for overnight. The mixture was then diluted with ethyl acetate, washed with water followed by brine, and dried over anhydrous Na2SO4. Evaporation of the solvent resulted in the formation of 5a as a colorless oil (1.86 g, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.55 (9H, s), 1.57–1.67 (3H, m), 1.81–1.87 (1H, m), 3.22 (2H, dt, J = 12.8, 6.6 Hz), 3.73 (3H, s), 4.30 (1H, dt, J = 12.8, 6.6 Hz), 4.83 (1H, br s), 5.05 (1H, br s), 5.09 (2H, s), 7.36 (5H, s). 13C NMR (100 MHz, CDCl3): δ/ppm 26.02, 28.37, 30.09, 40.56, 50.77, 52.44, 53.14, 66.72, 80.08, 128.17, 128.58, 136.60, 155.51, 156.54, 173.16. HR-ESIMS Calcd for C19H28N2NaO6 [M+Na]+ 403.1845. Found 403.1853.
Synthesis of Boc-Lys(Cbz)-OMe (5b).
CH3I (600 μL, 9.6 mmol) was added dropwise to a suspension of Boc-Lys(Cbz)-OH (2.28 g, 6 mmol) and K2CO3 (1.67 g, 12 mmol) in DMF (10 mL) at 0°C, and the mixture was stirred at 0°C for 30 min, and then at room temperature overnight. The mixture was then diluted with ethyl acetate, washed with water followed by brine, and dried over anhydrous Na2SO4. Evaporation of the solvent resulted in the formation of 5b as a colorless oil (2.28 g, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.34–1.40 (2H, m), 1.43 (9H, s), 1.43–1.54 (2H, m), 1.61–1.65 (1H, m), 1.74–1.90 (1H, m), 3.19 (2H, dt, J = 13.0, 6.0 Hz), 3.73 (3H, s), 4.29 (1H, dd, J = 8.4, 12.3 Hz), 4.79 (1H, br s), 5.05 (1H, br s), 5.09 (2H, s), 7.35 (5H, s). 13C NMR (100 MHz, CDCl3): δ/ppm 22.51, 28.44, 29.49, 32.51, 40.75, 52.43, 53.27, 66.78, 80.07, 128.23, 128.64, 136.70, 155.60, 156.58, 173.39. These spectral data are congruent with those previously reported [13, 14].
Synthesis of H-Orn(Cbz)-OMe hydrochloride.
To a solution of 5a (1.22 g, 3 mmol) in 1,4-dioxane (10 mL), 4 M HCl in 1,4-dioxane (10 mL) was added at room temperature, and the mixture was stirred at room temperature for 8 h. After the completion of the reaction, HCl and 1,4-dioxane were removed by evaporation. The residual solvents were further removed by azeotropy with benzene three times to give H-Orn(Cbz)-OMe hydrochloride as a colorless solid (1.02 g, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.47–1.81 (2H, m), 1.89–2.17 (2H, m), 3.01–3.22 (2H, m), 3.71 (3H, s), 4.00–4.17 (1H, m), 5.04 (1H, s), 5.68 (1H, br s), 7.27–7.32 (5H, m), 8.63 (3H, br s); 13C NMR (100 MHz, CDCl3): δ/ppm 25.39, 27.58, 40.24, 53.05, 53.58, 66.61, 128.09, 128.56, 128.74, 136.75, 156.86, 188.43. These spectral data are congruent with those previously reported [12].
Synthesis of Boc-Phe-Orn(Cbz)-OMe (6a).
H-Orn(Cbz)-OMe hydrocloride (1.60 g, 5.1 mmol) was dissolved in DMF (20 mL), and Boc-Phe-OH (1.52 g, 5.7 mmol), PyBOP (3.33 g, 6.4 mmol) and diisopropylethylamine (1.90 mL, 11.0 mmol) were successively added to the DMF solution. The mixture was stirred for 2 days at room temperature. The reaction mixture was poured into H2O (50 mL), and the organic materials were extracted with ethyl acetate (50 mL). The organic layer was successively washed with 1M KHSO4 (150 mL), saturated Na2CO3 aq. (150 mL), and brine (150 mL), and then dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel (140 g) and eluted with hexane/ethyl acetate = 1/2 to give 6a as a colorless solid (2.04 g, 76%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.40 (9H, s), 1.44–1.67 (3H, m), 1.80–1.86 (1H, m), 3.06 (2H, d, J = 6.7 Hz), 3.17 (2H, dt, J = 13.5, 6.7 Hz), 3.69 (3H, s), 4.35 (1H, d, J = 6.7 Hz), 4.54 (1H, dd, J = 7.8, 13.5 Hz), 4.91 (1H, br s), 5.03 (1H, br s), 5.09 (2H, s), 6.53 (1H, d, J = 7.8 Hz), 7.18–7.24 (5H, m), 7.28–7.36 (5H, m). 13C NMR (100 MHz, CDCl3): δ/ppm 25.87, 28.39, 29.67, 38.24, 40.51, 52.00, 52.60, 56.03, 66.84, 80.45, 127.11, 128.25(×2), 128.66, 128.80, 129.49, 136.65, 136.71, 156.60(×2), 171.22, 172.13. HR-ESIMS Calcd for C28H37N3NaO7 [M+Na]+ 550.2529. Found 550.2507.
Synthesis of H-Lys(Cbz)-OMe hydrochloride.
To a solution of 5b (1.57 g, 4.7 mmol) in 1,4-dioxane (10 mL) 4 M HCl in 1,4-dioxane (10mL) was added at room temperature, and the mixture was stirred at room temperature for 3 h. After the completion of the reaction, HCl and 1,4-dioxane were removed by evaporation. Residual solvents were further removed by azeotropy with benzene three times to give H-Lys(Cbz)-OMe hydrochloride [14, 15] as colorless solid (1.30 g, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.36–1.71 (4H, m), 1.90–2.11 (2H, m), 3.07–3.22 (2H, m), 3.75 (3H, s), 4.00–4.23 (1H, m), 5.06 (2H, s), 5.50 (1H, br s), 7.27–7.37 (5H, m), 8.73 (3H, br s); 13C NMR (100 MHz, CDCl3): δ/ppm 22.08, 28.93, 29.73, 40.36, 53.12, 53.32, 66.55, 128.05, 128.13, 128.55, 136.84, 156.72, 170.00. These spectral data are congruent with those previously reported [14, 15].
Synthesis of Boc-Phe-Lys(Cbz)-OMe (6b).
Boc-Phe (1.69 g, 6.4 mmol), PyBOP (3.76 g, 7.2 mmol) and diisopropylethylamine (2.00 mL, 12 mmol) were successively added to a solution of H-Lys(Cbz)-OMe hydrochloride (1.67 g, 5.1 mmol) in DMF (25 mL). The reaction mixture was then stirred for 24 h at room temperature. The mixture was poured into H2O (50 mL), and the organic materials were extracted with ethyl acetate (50 mL). The organic layer was successively washed with 1M KHSO4 (150 mL), saturated Na2CO3 aq. (150 mL), and brine (150 mL), and dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel (140 g) with hexane/ethyl acetate = 1/2 as the eluent to give 6b as a colorless solid (2.19 g, 80%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.40 (9H, s), 1.48–1.56 (2H, m), 1.61–1.67 (3H, m), 1.78–1.83 (1H, m), 3.04 (2H, t, J = 6.9 Hz), 3.16 (2H, dd, J = 6.0, 13.6 Hz), 3.70 (3H, s), 4.35 (1H, dd, J = 7.5, 13.3 Hz), 4.54 (1H, dt, J = 5.2, 6.9 Hz), 4.94 (1H, br s), 5.09 (1H, t, 13.6 Hz), 5.10 (2H, s), 6.47 (1H, d, J = 7.5 Hz), 7.17–7.24 (5H, m), 7.27–7.36 (5H, m). 13C NMR (100 MHz, CDCl3): δ/ppm 22.17, 28.40, 29.29, 32.09, 38.19, 40.65, 52.01, 52.53, 55.90, 66.81, 77.37, 127.06, 128.24, 128.27, 128.66, 128.77, 129.47, 136.73(×2), 156.64(×2), 171.32, 172.30. These spectral data are congruent with those previously reported [16].
Synthesis of H-Phe-Orn(Cbz)-OMe hydrochloride.
To a solution of 6a (966 mg, 1.8 mmol) in 1,4-dioxane (20 mL), 4 M HCl in 1,4-dioxane (10mL) was added at room temperature, and the mixture was stirred overnight at room temperature. After the completion of the reaction, HCl and 1,4-dioxane were removed by evaporation. Then, the residual solvents were further removed by azeotropy with benzene three times to give H-Phe-Orn(Cbz)-OMe hydrochloride as a colorless solid (875 mg, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.25–1.63 (2H, m), 1.68–1.81 (2H, m), 3.09 (2H, br s), 3.26–3.34 (2H, m), 3.60 (3H, s), 4.37–4.45 (1H, m), 4.58–4.60 (1H, m), 5.00 (2H, s), 5.62 (1H, br s), 6.47 (1H, br s), 7.15–7.42 (10H, m), 8.32 (3H, br s); 13C NMR (100 MHz, DMSO-d6): δ/ppm 25.75, 28.17, 36.80, 51.91, 52.03, 53.17, 64.93, 65.15, 127.11, 127.73, 127.78, 128.37, 128.46, 129.60, 134.87, 137.24, 156.14, 168.18, 171.72. HR-ESIMS Calcd for C23H30N3O5 [M+H]+ 428.2180. Found 428.2134. This material was used directly in the next step without further purification.
Synthesis of Fmoc-Gly-Phe-Orn(Cbz)-OMe (7a).
Fmoc-Gly-OH (614 mg, 2.1 mmol), PyBOP (1.21 g, 2.3 mmol) in diisopropylethylamine (700 μL, 4 mmol) were added successively to a solution of H-Phe-Orn(Cbz)-OMe hydrochloride (888 mg, 1.9 mmol), and DMF (20 mL). The reaction mixture was stirred for 24h at room temperature. The mixture was poured into H2O (30 mL), and the organic materials were extracted with ethyl acetate (30 mL). The organic layer was successively washed with 1M KHSO4 (90 mL), saturated Na2CO3 aq. (90 mL), and brine (90 mL), and dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel (130 g) and eluted with hexane/ethyl acetate = 1/19 to give 7a as a colorless solid (903 mg, 67%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.41–1.49 (1H, m), 1.60–1.66 (2H, m), 1.80–1.89 (1H, m), 3.01–3.13 (4H, m), 3.69 (3H, s), 3.73–3.84 (2H, m), 4.16 (1H, t, J = 7.2 Hz), 4.39 (2H, d, J = 7.2 Hz), 4.51–4.56 (1H, m), 4.66–4.72 (1H, m), 5.04 (2H, s), 5.16 (1H, br s), 5.59 (1H, br s), 6.64–6.77 (2H, m), 7.15–7.32 (14H, m), 7.57 (2H, d, J = 7.0 Hz), 7.76 (2H, d, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): δ/ppm 25.83, 29.00, 37.56, 40.53, 44.62, 47.20, 52.13, 52.62, 54.44, 66.88, 67.39, 120.16, 125.15, 127.19, 127.25, 127.92, 128.22, 128.28, 128.67, 128.81, 129.38, 136.35, 136.60, 141.44, 143.84, 156.81(×2), 169.46, 170.50, 172.03. HR-ESIMS Calcd for C40H42N4NaO8 [M+Na]+ 729.2895. Found 729.2877.
Synthesis of H-Phe-Lys(Cbz)-OMe hydrochloride.
To a solution of 6b (1.65 g, 3 mmol) in 1,4-dioxane (10 mL), 4 M HCl in 1,4-dioxane (10mL) was added at room temperature, and the mixture was stirred at room temperature overnight. After the completion of the reaction, HCl and 1,4-dioxane were removed by evaporation. The residual solvents were further removed by azeotropy with benzene three times to give H-Phe-Lys(Cbz)-OMe hydrochloride as a colorless solid (1.51 g, quant.). 1H NMR (400 MHz, CDCl3): δ/ppm 1.22–1.27 (2H, m), 1.38–1.45 (2H, m), 1.61–1.64 (1H, m), 1.74–1.79 (1H, m), 3.09 (2H, br. s), 3.15 (2H, d, J = 7.1 Hz), 3.66 (3H, s), 4.31–4.42 (2H, m), 5.02 (2H, s), 6.03 (1H, br s), 7.15–7.22 (5H, m), 7.28–7.35 (5H, m); 13C NMR (100 MHz, CDCl3): δ/ppm 22.49, 28.93, 30.46, 36.67, 51.96, 52.18, 53.12, 64.93, 65.10, 127.02, 127.69, 127.73, 128.35, 128.38, 129.66, 134.94, 137.29, 156.10, 168.15, 171.80. HR-ESIMS Calcd for C24H32N3O5 [M+H]+ 442.2336. Found 442.2364. This material was used directly in the next step without further purification.
Synthesis of Fmoc-Gly-Phe-Lys(Cbz)-OMe (7b).
Fmoc-Gly-OH (633 mg, 2.1 mmol), PyBOP (1.23 g, 2.4 mmol) and diisopropylethylamine (1.1 mL, 6.3 mmol) were successively added to a solution of H-Phe-Lys(Cbz)-OMe hydrochloride (848 mg, 1.8 mmol) in DMF (8 mL). The reaction mixture was stirred for 24h at room temperature. The mixture was poured into H2O (20 mL), and the organic materials were extracted with ethyl acetate (20 mL). The organic layer was successively washed with 1M KHSO4 (60 mL), saturated Na2CO3 aq. (60 mL), and brine (60 mL), and dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, the crude product was purified by column chromatography on silica gel (70 g) and eluted with CHCl3/MeOH = 49/1 to give 7b as a colorless solid (720 mg, 56%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.24–1.30 (2H, m), 1.42–1.48 (2H, m), 1.55–1.58 (1H, m), 1.79–1.82 (1H, m), 3.05–3.13 (4H, m), 3.68 (3H, s), 3.71–3.78 (2H, m), 4.18 (1H, t, J = 6.0 Hz), 4.39 (2H, d, J = 6.0 Hz) 4.47–4.52 (1H, m), 4.60–4.66 (1H, m), 5.06 (2H, s), 5.10 (1H, s), 5.47 (1H, br s), 6.48 (1H, br s), 7.165–7.32 (14H, m), 7.58 (2H, d, J = 7.5 Hz), 7.76 (2H, d, J = 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ/ppm 22.27, 29.25, 31.57, 37.84, 40.55, 44.50, 47.14, 52.16, 52.45, 54.53, 66.76, 67.34, 120.08, 125.12, 127.08, 127.18, 127.84, 128.19(×2), 128.60, 128.70, 129.32, 136.63, 141.36, 143.82, 143.83, 156.72, 169.39, 170.63, 172.22. HR-ESIMS Calcd for C41H44N4NaO8 [M+Na]+ 743.3057. Found 743.3038.
Synthesis of H-Gly-Phe-Orn(Cbz)-OMe (8a).
Piperidine (400 μL, 4.1 mmol) was added to a solution of compound 7a (802 mg, 1.1 mmol) in DMF (4.0 mL), and then the reaction mixture was stirred for 2 h at room temperature. After removal of the solvent under reduced pressure, the residue was purified by column chromatography on silica gel (60 g) and eluted with CHCl3/MeOH (9/1) to give 8a as a colorless solid (506 mg, 92%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.44–1.48 (2H, m), 1.63–1.66 (1H, m), 1.83–1.86 (1H, m), 3.01–3.30 (6H, m), 3.70 (3H, s), 4.51 (1H, dd, J = 2.2, 7.5 Hz), 4.66 (1H, dt, J = 14.6, 7.5 Hz), 5.07 (2H, s), 5.14–5.16 (1H, m), 6.88 (1H, d, J = 7.5 Hz), 7.19–7.35 (10H, m), 7.75 (1H, d, J = 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ/ppm 25.75, 29.29, 37.75, 40.48, 44.66, 52.08, 52.58, 54.35, 66.77, 127.06, 128.23, 128.72, 129.38(×2), 136.72(×2), 156.68, 170.98, 172.16, 173.51. HR-ESIMS Calcd for C25H32N4NaO6 [M+Na]+ 507.2214. Found 507.2179.
Synthesis of H-Gly-Phe-Lys(Cbz)-OMe (8b).
Piperidine (2 mL, 20.3 mmol) was added to a solution of compound 7b (1.78 g, 2.5 mmol) in DMF (8.0 mL), and the reaction mixture was stirred for 2 h at room temperature. After removing the solvent under reduced pressure, the residue was purified by column chromatography on silica gel (100 g) and eluted with dichloromethane/MeOH = 99/1 to give 8b as a colorless solid (874 mg, 71%). 1H NMR (400 MHz, CDCl3): δ/ppm 1.25–1.31 (2H, m), 1.44–1.50 (2H, m), 1.59–1.74 (1H, m), 1.76–1.85 (1H, m), 3.03 (1H, dd, J = 13.9, 7.7 Hz), 3.08–3.19 (3H, m), 3.19–3.30 (1H, m), 3.69 (3H, s), 4.50 (1H, dt, J = 12.7, 7.7 Hz), 4.64 (1H, q, J = 7.4 Hz), 5.07–5.12 (2H, m), 5.27–5.30 (1H, m), 6.84 (1H, d, J = 8.0 Hz), 7.12–7.35 (10H, m), 7.71 (1H, d, J = 7.8 Hz). 13C NMR (100 MHz, DMSO-d6): δ/ppm 22.70, 29.02, 30.51, 37.97, 40.05, 43.50, 51.86, 52.07, 53.20, 65.17, 126.34, 127.74, 127.76, 128.05, 128.37, 129.32, 137.31, 137.50, 156.16, 171.01, 171.26, 172.41. HR-ESIMS Calcd for C26H35N4O6 [M+H]+ 499.2551. Found 499.2525.
Synthesis of 7PC-Gly-Phe-Orn(Cbz)-OH (4a).
DBU (300 μL, 2 mol) was added to the slurry of 7MCP (100 mg, 450 μmol) in MeOH (1.5 mL). Compound 8a (506 mg, 1 mmol) was added to the mixture, which was then stirred at room temperature for 3 h. After the completion of the reaction, 0.01 M nBu4NCl aq. was added dropwise to the reaction mixture and the crude mixture was purified by gel filtration (LH-20, 80 g) and eluted with 0.01 M nBu4NCl. The pure fractions were collected and acidified with 6M HCl to pH 2, followed by filtration to yield compound 4a (75 mg, 25%). 1H NMR (400 MHz, DMSO-d6): δ/ppm 1.20–1.78 (4H, m), 2.55–2.84 (2H, m), 2.88–3.05 (4H, m), 3.79–4.00 (2H, m), 4.12–4.24 (1H, m), 4.54–4.67 (1H, m), 5.00 (2H, s), 7.19–7.35 (12H, m), 8.17–8.39 (2H, m), 8.80–8.88 (2H, m), 11.75 (1H, br s), 12.63 (1H, br s). 13C NMR (100 MHz, DMSO-d6): δ/ppm 26.10, 28.34, 37,63, 42.22, 51.84, 53.56, 65.14, 126.24, 127.70, 127.99, 128.33, 129.22, 129.27, 131.86, 136.42, 137.24, 137.47, 137.66, 147.16, 154.51, 156.08, 156.12, 160.26, 162.77, 167.92, 171.03, 173.31. HR-ESIMS Calcd for C31H32N9O8 [M–H]– 658.2379. Found 658.2356.
Synthesis of 7PC-Gly-Phe-Lys(Cbz)-OH (4b).
DBU (300 μL, 2 mol) was added to the slurry of 7MCP (109 mg, 492 μmol) in MeOH (2.0 mL). Compound 8b (515 mg, 1 mmol) was added to the mixture, which was then stirred at room temperature for 3 h. After the completion of the reaction, 0.01 M nBu4NCl aq. was added dropwise to the reaction mixture and the crude mixture was purified by gel filtration (LH-20, 80 g) eluted with 0.01 M nBu4NCl. The pure fractions were collected and acidified with 6M HCl to pH 2, followed by filtration to yield compound 4b (45 mg, 13%). 1H NMR (400 MHz, DMSO-d6): δ/ppm 1.24–1.36 (2H, m), 1.36–1.45 (2H, m), 1.56–1.76 (2H, m), 2.77 (1H, dd, J = 4.1, 11.8 Hz), 2.95–3.06 (3H, m), 3.83 (1H, dd, J = 5.9, 16.7 Hz), 3.95 (1H, dd, J = 5.9, 16.7 Hz), 4.12–4.17 (1H, m), 4.58–4.64 (1H, m), 4.90–5.07 (3H, m), 7.22–7.33 (12H, m), 8.23 (1H, d, J = 8.5 Hz), 8.30 (1H, d, J = 7.6 Hz), 8.79–8.89 (2H, m), 11.64 (1H, br. s), 12.58 (1H, br s). 13C NMR (100 MHz, DMSO-d6): δ/ppm 22.81, 29.08, 30.67, 37.64, 42.23, 52.03, 53.59, 65.13, 126.25, 127.72, 127.99, 128.33, 129.23, 129.28, 131.86, 136.46, 137.26, 137.67, 147.16, 154.56, 156.08(×2), 162.78, 167.95, 171.04, 173.45. HR-ESIMS Calcd for C32H34N9O8 [M–H]– 672.2536. Found 672.2553.
Mass expression of ricin toxin A-chain (RTA)
The pET28a plasmid containing the His-RTA expression sequence was purchased from GenScript and transformed into E. coli BL21(DE3) cells using the calcium treatment method. The transformed strains were incubated overnight at 32°C on LB agar culture supplemented with kanamycin. The generated colonies were picked up and inoculated into 5 litres of LB medium, and then incubated at 32°C with shaking. After approximately 6 h, the growth of the bacteria was visually confirmed by turbidity, IPTG was added to the medium to induce expression, and the cultivation was continued at 32°C overnight. Bacteria were collected by centrifugation and ultrasonically crushed to obtain a crude extract. His-tagged proteins were collected using a TALON column, dialyzed against 10 mM sodium acetate buffer (pH 4.0), and concentrated using an ultrafiltration filter to obtain the target proteins. Crystallization of RTA was then performed by the hanging drop vapor diffusion method using the obtained RTA protein solution.
X-ray crystallography
RTA crystals were grown at 23°C using the hanging drop method from 6.4–7.8 mg/mL protein and 11–12% PEG2000, 0.2 M Lithium sulfate, 0.1 M sodium acetate, pH 4.5. Crystals of RTA complex with synthetic ligands were obtained by soaking RTA crystals in a crystallization reservoir containing 3.0 mM of the compound.
Diffraction data of RTA-ligand complex crystals were collected at 95 K using PILATUS3 S 2M detector on the beamline BL-5A Photon Factory (PF, Tsukuba, Japan). The data set was processed and scaled using the XDS [17] and SCALA [18] from the CCP4 suite [19]. Processed data were phased by the molecular replacement method using Molrep [20] with the coordinates of RTA bound with N-(N-(pterin-7-yl)carbonylglycyl)-L-phenylalanine (PDBID: 4HUO) [6]. Model building and electron density map inspection were done using Coot [21]. Following model building, refinement was performed using Refmac5 [22]. Crystallographic data for the complexes are presented in Table 2. Coordinates of the refined model of 4a/RTA and 4b/RTA complexes were in the Protein data Bank with accession codes 7Y4K and 7Y4M, respectively.
RTA inhibitory activity measurements
The RTA inhibitory activity of the synthesized inhibitors was evaluated by measuring the amount of firefly luciferase produced by the in vitro translation reaction using rabbit reticulocyte lysate with luciferase quantification reagents.
A 1.5 mL eppendorf tube was filled with 0.2 μL of Amino Acid Mixtures (1 mM, Promega), 0.2 μL of RNasin® ribonuclease inhibitor solution (1 U/μL, Promega), 0.2 μL of Luciferase Control RNA (1 mg/mL, Promega), 1.0 μL of RTA solution (0.5 nM, 20 mM HEPES buffer, pH 7.5), 1.0 μL of inhibitor solution (prepared with a minimum volume of dimethylsulfoxide and 20 mM HEPES buffer, pH 7.5), 7.0 μL of Reticulocyte Lysate (Promega) and made up to 10.0 μL with purified water. After incubation in a warm bath at 30°C for 90 min, the reaction was stopped by freezing at –20°C for 15 min. To the resulting translation product, 70 μL of purified water was added to obtaine a total volume of 80 μL, and 20 μL of each product was dispensed into a white 96-well plate. Luminescence was analyzed using the Luciferase Quantification Reagent brightliteTM Plus (Perkin Elmer) and a microplate reader (ARVO X4, Perkin Elmer). The mean bioluminescence of each sample was determined in triplicates. The inhibition ratio was calculated using the following equation;
where RLURTA&inhibitor is the luminescence amount in the presence of both RTA and an inhibitor, RLUinhibitor is the luminescence amount in the presence of an inhibitor but the absence of RTA, RLURTA is the luminescence amount in the presence of RTA but the absence of an inhibitor, and RLUcontrol is the luminescence amount in the absence of RTA nor an inhibitor.
Supporting information
S1 File. Proton (1H) and carbon (13C) NMR spectra of new compounds.
https://doi.org/10.1371/journal.pone.0277770.s001
(PDF)
Acknowledgments
The authors would like to thank Editage (www.editage.jp) for English language editing.
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