Optimizing linker rigidity to improve intracellular behavior of PROTACs targeting hematopoietic prostaglandin D synthase

Hinata Osawa ab, Kosuke Saito c and Yosuke Demizu *abd
aGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
bDivision of Organic Chemistry, National Institute of Health Sciences, Kanagawa, Japan. E-mail: demizu@nihs.go.jp
cDivision of Medicinal Safety Science, National Institute of Health Sciences, Kanagawa, Japan
dGraduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan

Received 7th May 2025 , Accepted 2nd September 2025

First published on 2nd September 2025


Abstract

Proteolysis-targeting chimeras (PROTACs) are emerging as powerful tools for targeted protein degradation. Among the key factors influencing their efficacy, linker design plays a critical role by affecting membrane permeability, ternary complex formation, and degradation potency. In this study, we conducted a comparative analysis of three novel PROTACs targeting hematopoietic prostaglandin D synthase (H-PGDS), each incorporating linkers with distinct degrees of rigidity—including methylene modifications and spirocyclic structures. Although all compounds exhibited similar binding affinities and degradation activities, the most rigid derivative (PROTAC-3) showed markedly higher intracellular accumulation but formed the least stable ternary complex. These results reveal a trade-off between cell permeability and complex stability, emphasizing the importance of comprehensive linker optimization. Our findings highlight the value of integrating conformational rigidity and spatial design in the rational development of next-generation PROTACs.


Introduction

Proteolysis-targeting chimeras (PROTACs),1,2 which utilize ubiquitin-proteasome system (UPS)-mediated protein degradation, have attracted attention as a novel modality for selectively degrading proteins of interest (POIs).3,4 PROTAC molecules consist of three components: a ligand for the target protein (POI ligand), an E3 ligase ligand, and a linker connecting these two ligands, promoting ternary complex formation between the POI and the E3 ligase. This subsequently induces polyubiquitination and proteasome-dependent degradation of the POI. Among these components, the linker has garnered considerable attention due to its multifaceted role in governing ternary complex formation, protein–protein proximity, membrane permeability, and overall degradation efficiency.

A growing body of literature has demonstrated that linker attributes—such as length, flexibility, and spatial orientation—can dramatically impact PROTAC efficacy. For example, shortening the linker length or rigidifying its structure has been shown to enhance degradation activity by improving geometrical complementarity between the POI and the E3 ligase. Conversely, excessive rigidity can compromise the adaptability required for productive ternary complex formation. For instance, Shibata et al. reported that incorporation of rigid phenyl rings into the linker of androgen receptor-targeting SNIPERs led to a loss of activity, whereas flexible PEG chains retained degradation potency.5 These findings underscore the need for achieving an optimal balance between rigidity and flexibility.

In recent years, medicinal chemists have explored the incorporation of moderately rigid motifs—such as piperazine rings, alkyne spacers, and spirocyclic frameworks—to impose conformational constraints without sacrificing the adaptability needed for ternary complex assembly. Among these, spirocycles have attracted particular interest for their ability to enhance molecular three-dimensional character, reduce total polar surface area, and improve pharmacokinetic properties.6–10 Indeed, Nunes et al. reported that replacing long-chain aliphatic alkyl linkers with spirocyclic pyridine scaffolds increased linker rigidity, reduced total polar surface area (TPSA), improved water solubility, and doubled the degradation activity (DC50) of IRAK4-targeting PROTACs.11 These findings suggest linker rigidity optimization significantly impacts PROTAC degradation activity and intracellular behavior.

Hematopoietic prostaglandin D synthase (H-PGDS) is an enzyme that catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2).12,13 Overexpression of H-PGDS is implicated in the progression of various pathological conditions, including allergic diseases and Duchenne muscular dystrophy (DMD).14–16 Although several H-PGDS inhibitors such as TFC-007 (ref. 17) and TAS-205 (ref. 18) have been developed, none have yet reached clinical application. To overcome this limitation, our group previously developed a series of PROTACs targeting H-PGDS, employing TFC-007 as the ligand for the protein of interest (POI) and pomalidomide as the E3 ligase ligand.19–23 Among these, PROTAC(H-PGDS)-7 (hereafter referred to as PROTAC-1) showed exceptionally potent degradation activity, with a DC50 of 17.3 pM.22 Structure–activity relationship (SAR) studies revealed that linker properties—particularly length and rigidity—have a critical influence on H-PGDS degradation efficiency. For example, shortening the linker from PEG5 to PEG0 between the ligands significantly enhanced degradation activity, whereas substituting a rigid piperazine ring with a flexible ethylene chain reduced activity (Fig. 1).22 These findings highlight the importance of fine-tuning linker architecture to optimize PROTAC performance.


image file: d5md00396b-f1.tif
Fig. 1 Previous structure–activity relationship (SAR) studies of PROTACs targeting H-PGDS and PROTACs designed in this study.

Despite these advances, the comprehensive impact of overall PROTAC flexibility on both degradation activity and cell permeability has not been thoroughly investigated. Notably, recent trends in PROTAC design have shifted toward incorporating moderately rigid motifs such as heterocycles (e.g., piperazine or piperidine) and alkyne bonds, rather than relying solely on linear alkyl or PEG-based linkers.

In this study, we comprehensively evaluated how differences in PROTAC flexibility influence H-PGDS binding affinity, degradation activity, intracellular accumulation, and ternary complex stability. To this end, we newly designed and synthesized three PROTACs with varying degrees of flexibility, introduced by incorporating spirocyclic rings or modifying the presence of carbonyl groups in the linker region (Fig. 1). Building upon the structure of PROTAC-1, PROTAC-2 was designed by replacing the carbonyl between piperazine and piperidine units with a methylene moiety, thereby slightly increasing rotational freedom between the ligands. PROTAC-3 featured an azaspiro[3.5]nonane ring, which imparts slightly less flexible than a piperazine ring, while PROTAC-4 incorporated an azaspiro[5.5]undecane scaffold, conferring greater rigidity. Through this systematic investigation, we aimed to clarify how linker flexibility affects membrane permeability and ternary complex stability, and how these properties collectively determine degradation activity. These findings provide valuable insights for the rational design of PROTACs with optimized cellular and biochemical performance.

Result and discussion

Synthesis of PROTACs

The synthetic route for PROTAC-2 is shown in Scheme 1a. Starting from compound 1,20 condensation with N,O-dimethylhydroxylamine afforded the corresponding Weinreb amide 2. Subsequent reduction with DIBAL yielded the aldehyde 3, which underwent reductive amination with a pomalidomide derivative to furnish PROTAC-2. The synthetic route for PROTAC-3 is outlined in Scheme 1b. Compound 4 was subjected to an aromatic nucleophilic substitution reaction with compound 5 to afford compound 6. Following TFA-mediated deprotection, condensation with compound 1 gave PROTAC-3 in 29% yield. The synthetic route for PROTAC-4 is shown in Scheme 1c. Starting from compound 4, aromatic nucleophilic substitution reaction with compound 7 yielded compound 8. Deprotection using 4 M HCl in 1,4-dioxane, followed by condensation with compound 1 afforded PROTAC-4 in 35% yield.
image file: d5md00396b-s1.tif
Scheme 1 Synthetic routes of (a) PROTAC-2, (b) PROTAC-3 and (c) PROTAC-4.

Docking studies

Prior to biological experiments, the predicted ternary complex structures of each PROTAC with CRBN and H-PGDS were analyzed using molecular docking simulations performed with MOE 2024.0601. Stable ternary complexes comprising CRBN (PDB: 4CI3), PROTAC, and H-PGDS (PDB: 5YWX) were successfully constructed (Fig. 2a). The superimposed structures of H-PGDS from each complex are shown in Fig. 2b, and the individual PROTAC conformations are presented in Fig. 2c. The corresponding Cα RMSD values, calculated using the PROTAC-1-based ternary complex as the reference, are shown in Fig. S1. Detailed protein–PROTAC interaction profiles for each predicted ternary complex are provided in Fig. S2–S5 of the SI.
image file: d5md00396b-f2.tif
Fig. 2 Predicted ternary complex of CRBN/PROTAC/H-PGDS obtained by docking simulations. (a) Predicted ternary complex structures formed by each PROTAC. CRBN and H-PGDS are shown in silver and brown. (b and c) Superposition of the ternary complex structures formed by each PROTAC (superposing H-PGDS). The ternary complexes containing PROTAC-1, PROTAC-2, PROTAC-3, and PROTAC-4 are shown in red, green, yellow, and pink, respectively. Panel b shows the RMSD values of the ternary complexes formed by each PROTAC with respect to the PROTAC-1-based ternary complex.

In general, the conserved hydrogen bonding interactions between CRBN and the pomalidomide moiety (notably with His380 and Trp382 (ref. 24)) were maintained across all PROTACs. While the interaction between the TFC-007 moiety and Trp104 of H-PGDS was not observed in the PROTAC-4 model, the spatial proximity between Trp104 and the pyrimidine nitrogen remained similar to that observed in other complexes (Fig. S6), suggesting a comparable binding geometry. With respect to structural stability, our previous study reported that a PEG5-containing PROTAC (PROTAC-PEG5), which displayed ∼100-fold lower H-PGDS degradation activity compared to the linker-free PROTAC-1,22 formed a ternary complex with a substantially higher Cα RMSD value (Fig. S1). This finding implies that linker-induced conformational flexibility may compromise complex stability and, consequently, degradation efficiency. In contrast, the ternary complexes formed by PROTAC-2, -3, and -4 exhibited consistently lower RMSD values than PROTAC-PEG5, indicating that their respective linker modifications did not significantly disrupt the ternary complex architecture. These results support the hypothesis that PROTAC-2, -3, and -4 possess H-PGDS degradation potential comparable to that of PROTAC-1.

H-PGDS binding affinity

The binding affinity of each PROTAC toward H-PGDS was evaluated using a competitive fluorescence polarization (FP) assay. Changes in FP as a function of compound concentration are shown in Fig. 3, and the corresponding IC50 values are summarized. PROTAC-1 exhibited an IC50 of 45.1 ± 3.7 nM, whereas PROTAC-2, PROTAC-3, and PROTAC-4 showed IC50 values of 69.0 ± 4.0 nM, 32.1 ± 2.1 nM, and 36.1 ± 4.3 nM, respectively. These results indicate that the removal of the carbonyl group (PROTAC-2) and introduction of spirocyclic linkers (PROTAC-3 and PROTAC-4) exert only modest effects on H-PGDS binding affinity.
image file: d5md00396b-f3.tif
Fig. 3 Competitive fluorescence polarization (FP) assay of PROTACs against H-PGDS. FP values are normalized to the signal of H-PGDS with probe ligand only (set as 100%). Data are presented as mean ± SD (n = 3). IC50 values are summarized in the table.

H-PGDS degradation activity

The H-PGDS degradation activity of the three synthesized PROTACs (PROTAC-2 to -4) was assessed by western blot analysis in comparison with the parent compound, PROTAC-1. Initially, a time-course study (4–48 h) was conducted in KU812 cells treated with each PROTAC to evaluate degradation kinetics (Fig. S8). H-PGDS degradation was found to occur rapidly, and no marked differences among PROTACs were observed even at shorter incubation times. Based on these results, subsequent experiments were performed with a 24-hour treatment, and the outcomes are presented in Fig. 4. Western blotting was performed in triplicate (n = 3) (Fig. S9), and a representative result is shown in the Fig. 4. All PROTACs induced a concentration-dependent decrease in H-PGDS protein levels. The half-maximal degradation concentration (DC50) values for PROTAC-1, PROTAC-2, PROTAC-3, and PROTAC-4 were 0.094 ± 0.044 nM, 0.22 ± 0.070 nM, 0.15 ± 0.072 nM, and 0.19 ± 0.044 nM, respectively. At high concentrations (1 or 10 μM), a characteristic hook effect was observed. This phenomenon, commonly seen with PROTACs, occurs when excessive concentrations favor binary binding to either the E3 ligase or the target protein, thereby interfering with productive ternary complex formation required for ubiquitination and degradation. Notably, despite differences in linker flexibility, no significant variation in degradation efficiency was observed among the PROTACs, indicating that all exhibited comparable H-PGDS suppression activity.
image file: d5md00396b-f4.tif
Fig. 4 H-PGDS protein levels in KU812 cells as measured by western blot after 24 hours of incubation with PROTACs. The H-PGDS/β-actin ratios ware normalized to the DMSO control, which was set at 100%. A representative result of western blot analysis is shown (n = 3).

Intracellular accumulation and of PROTACs

Intracellular accumulation of PROTACs were evaluated by LC-MS/MS analysis to associate with their pharmacological effects. KU812 cells were treated with each PROTAC at a concentration of 1 nM and incubated for 24 hours. After incubation, cell pellets were collected, extracted with methanol, and analyzed by LC-MS/MS. As previously reported,21 all PROTACs were cleaved at the amide bond within the TFC-007 moiety by collision induced dissociation, producing a characteristic fragment structure, which is used as the selected product ion for LC-MS/MS quantification (Fig. 5a and individual fragments were depicted in S13). A calibration curve was generated from serial dilutions of standard samples to determine the intracellular amount of each PROTAC. The results revealed that PROTAC-3 and PROTAC-4, both incorporating azaspirocyclic linkers, exhibited enhanced cellular amount relative to PROTAC-1 (Fig. 5b). Notably, PROTAC-3 demonstrated the highest cellular amount, with an amount approximately twice that of the other compounds. These findings suggest that linker rigidity imparted by azaspirocyclic structures may improve intracellular accumulation via enhancement of membrane permeability or cellular retention.
image file: d5md00396b-f5.tif
Fig. 5 (a) Cleavage of chemical structures of PROTACs by collision induced dissociation in LC-MS/MS. (b) Intercellular amount of PROTACs. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test, comparing each compound to PROTAC-1. **p < 0.01, ****p < 0.0001; ns, not significant.

To explore physicochemical factors underlying these differences, log[thin space (1/6-em)]D (pH 7.4) and clog[thin space (1/6-em)]P values were determined (Fig. S10). Although moderate differences in lipophilicity were observed, no clear correlation with intracellular accumulation was found. Notably, PROTAC-3 displayed the highest intracellular level despite comparable log[thin space (1/6-em)]D and clog[thin space (1/6-em)]P values to PROTAC-2 and PROTAC-4, indicating that passive membrane permeability alone does not account for uptake. Other factors—such as cell-type–dependent uptake, conformational adaptability in different environments, or active transport processes—may also contribute.

MD simulation

Although PROTAC-3 exhibited H-PGDS degradation activity comparable to the other PROTACs, it showed approximately twice the intracellular accumulation. To further investigate this discrepancy, the stability of the ternary complex formed by each PROTAC was analyzed using molecular dynamics (MD) simulations. Initial ternary complex structures were constructed based on previously reported protocols,20 utilizing the crystal structures of CRBN (PDB: 4CI3) and H-PGDS (PDB: 5YWX). MD simulations were performed in aqueous solution for 200 ns using the Amber10:EHT force field. The RMSD values of each ternary complex are shown in Fig. 6. Among them, the ternary complex formed by PROTAC-3 exhibited the highest RMSD value, indicating that it was less stable than other ternary complexes. These results suggest that despite enhanced intracellular accumulation, the relatively lower stability of the ternary complex may limit the overall degradation efficiency of PROTAC-3.
image file: d5md00396b-f6.tif
Fig. 6 Plot of RMSD values during the MD simulation of PROTACs.

Although all PROTACs demonstrated comparable H-PGDS degradation activity, PROTAC-3 exhibited a significantly higher intracellular amount—approximately twofold greater than that of the other derivatives (Fig. 5b). In contrast, MD simulation analysis revealed that the ternary complex formed by PROTAC-3 with CRBN and H-PGDS was the most structurally unstable among the tested PROTACs (Fig. 6). Furthermore, PROTAC-2 exhibited a significantly lower binding affinity for CRBN compared with the other PROTACs (Fig. S12); nevertheless, it demonstrated comparable degradation activity against H-PGDS. These findings suggest that the degradation efficiency is not determined solely by binding affinity to the target protein, but is also influenced by multiple factors, including intracellular accessibility (e.g., membrane permeability) and the stability of ternary complex formation.25 Indeed, it is well established that the formation and stability of the ternary complex are critical for target protein degradation, and that longer ternary complex residence times are generally associated with greater degradation efficiency.26 In this study, clear differences in membrane permeability and complex stability among PROTACs with similar degradation activity highlight the multifactorial nature of PROTAC performance. These results emphasize the necessity of comprehensive optimization strategies that consider multiple parameters, rather than focusing on a single design factor. In particular, the enhanced intracellular accumulation of PROTAC-3 is likely attributable to its unique structural features. PROTAC-3 incorporates a spirocyclic linker and lacks a carbonyl group in the linker region, a molecular design that reduces molecular flexibility and polarity. Such rigidification and polarity reduction are known to decrease conformational entropy and total polar surface area (TPSA), which can improve membrane permeability and pharmacokinetic profiles.11 In our study, differences in log[thin space (1/6-em)]D (pH 7.4) and clog[thin space (1/6-em)]P were minimal, and no clear correlation with intracellular levels was observed. Therefore, the elevated intracellular accumulation of PROTAC-3 cannot be attributed solely to passive membrane permeability. Instead, multiple factors are likely involved, including enhanced intracellular retention and metabolic stability, evasion of efflux pumps, cell-type-dependent uptake, conformational adaptability across environments, and active transport mechanisms (e.g., carrier-mediated transport or endocytosis). On the other hand, while rigidification may promote intracellular accumulation, it can also impair the geometric alignment between the target protein and the E3 ligase, thereby reducing ternary complex cooperativity and stability. This interpretation is consistent with our MD simulation results and with the report by Lan et al., which showed that rigidification of a BET-targeting PROTAC (HL1CON) through a β-gem-dimethyl moiety reduced both membrane permeability and ternary-complex cooperativity, ultimately diminishing degradation activity.27 These observations underscore that excessive rigidification can negatively impact PROTAC efficacy. Therefore, achieving an optimal balance between linker flexibility and rigidity is critical for modulating intracellular behavior and ternary complex formation. This study provides direct evidence that linker design exerts multifactorial effects on PROTAC function. Notably, although a trade-off between membrane permeability and ternary complex stability was observed, comparable degradation activity was ultimately achieved across all PROTACs. These findings imply that developing PROTACs capable of both efficient intracellular accumulation and stable ternary complex formation may lead to even greater degradation potency.

Conclusions

In this study, we investigated how differences in linker flexibility influence key properties of PROTACs targeting H-PGDS, including binding affinity, degradation activity, intracellular accumulation, and the ternary complex stability. To this end, three new PROTACs, PROTAC-2, PROTAC-3, and PROTAC-4 incorporating linkers with varying degrees of flexibility were designed, synthesized, and evaluated using multiple in vitro and computational assays. Our results revealed that variations in linker flexibility had minimal impact on both H-PGDS binding affinity and degradation activity. However, despite showing comparable degradation activity, PROTAC-3 exhibited approximately twice the intracellular amount of the other PROTACs, while forming the least stable ternary complex. These findings provide experimental evidence that PROTACs with similar degradation potency can differ significantly in membrane permeability and complex stability—two critical factors that collectively influence degradation efficiency. While we comprehensively evaluated the binary interactions of our PROTACs with both H-PGDS (by FP assay) and CRBN (by TR-FRET, see SI), we acknowledge that direct in vitro characterization of the ternary complexes (e.g., by SPR or FP-based ternary assays) was not conducted in this work. This remains a limitation primarily due to technical challenges in protein preparation and assay optimization. In future studies, we plan to establish in vitro ternary complex assays to complement our docking and MD simulations, which will provide a more detailed mechanistic understanding of ternary complex formation and stability.

The insights gained from this study offer valuable guidance for the rational design of PROTACs, particularly in the context of optimizing linker architecture. Moreover, the principles demonstrated here are not limited to H-PGDS-targeting PROTACs, but may be broadly applicable the development of PROTACs against diverse targets. We anticipate that the linker optimization strategies established in this study will contribute to the advancement of next-generation degraders and support the continued evolution of PROTAC-based drug discovery.

Experimental

Chemistry

General information. All chemicals were purchased from Sigma-Aldrich Co. LLC, Kanto Chemicals Co. Inc., Tokyo Chemical Industry Co. Ltd., Wako Pure Chemical Industries Ltd., and Ambeed Inc. were used without further purification. Reactions were followed by thin-layer chromatography (TLC) (60 F254, Merck), and spots were visualized by UV irradiation with a handheld UV lamp (254/365 nm) (UVP) and iodine vapor or ninhydrin reagent. Silica gel for column chromatography was Kanto Chemical 60N (spherical, neutral), NH silica gel (Chromatorex NH-DM1020, Fuji Silysia), or packed columns for medium pressure column chromatography (Hi-Flash column/Inject column Yamazen). Preparative HPLC was performed using a WP300 C18 column (5 μm, 20 mm × 250 mm, GL science) at a flow rate of 10 mL min−1 on a JASCO PU-4180 HPLC, and eluents were detected at 254 nm by a JASCO UV-2075. Analytical HPLC was performed using a J-Park Core C18 column (2.7 μm, 4.6 mm I.D. × 100 mL) at a flow rate of 1.2 mL min−1 (gradient: 10–90% MeCN–H2O containing 0.1% TFA, 10 min) and eluents were detected at 254 nm by an EXTREMA, JASCO (Tokyo, Japan).

1H and 13C NMR spectra were measured on an ECZ 600R spectrometer (JEOL) using deuterated solvents. Chemical shift values (ppm) were corrected for residual solvent signals as internal standards [DMSO-d6: 2.49 for 1H NMR, 39.5 for 13C NMR; CDCl3: 7.26 for 1H NMR, 77.0 for 13C NMR]. The splitting modes of the signals are as follows [singlet (s), doublet (d), triplet (t), quartet (q), double of doublets (dd), multiplet (m), broad (br)]. High-resolution mass spectrometry (HRMS) was measured by electrospray ionization using Shimadzu IT-TOF MS and LCMS-9050 (Shimadzu).

Compound 4 was prepared according to a reported method and subjected to in-house liquid chromatography-MS (LC-MS) for quality control.

Synthesis of PROTACs

Synthesis of compound 2. N,O-Dimethylhydroxylamine hydrochloride (80 mg, 0.817 mmol), HATU (310 mg, 0.0817 mmol), DIPEA (278 μL, 1.64 mmol) and compound 1 (228 mg, 0.545 mmol) were dissolved in N,N-dimethylformamide (3 mL). The mixture was stirred at room temperature for 12 hours. The reaction mixture was then diluted with CH2Cl2 and washed sequentially with saturated aqueous NaHCO3 and water. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatogram (NH silica gel, n-hexane[thin space (1/6-em)]:[thin space (1/6-em)]EtOAc = 70[thin space (1/6-em)]:[thin space (1/6-em)]30 to 20[thin space (1/6-em)]:[thin space (1/6-em)]80 gradient) to afford compound 2 (215 mg, 85%) as a pale yellow solid; 1H-NMR (600 MHz, CDCl3): δ 9.03 (s, 2H), 7.47–7.44 (m, 4H), 7.30 (t, J = 7.5 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 6.92 (d, J = 7.6 Hz, 2H), 3.73 (s, 3H), 3.70 (d, J = 12.1 Hz, 2H), 3.20 (s, 3H), 2.75 (td, J = 12.1, 1.8 Hz, 2H), 1.95–1.88 (m, 2H), 1.85 (d, J = 10.8 Hz, 2H), 1.62–1.61 (m, 1H); 13C-NMR (151 MHz, CDCl3): δ 175.9, 166.5, 161.2, 159.2, 152.5, 149.3, 129.8, 129.0, 126.0, 123.8, 122.1, 121.5, 116.9, 61.6, 49.4, 38.0, 32.3, 28.0; HRMS (ESI) m/z calcd for C25H28N5O4 [M + H]+, 462.2136; found, 462.2137.
Synthesis of compound 3. Compound 2 (50 mg, 0.108 mmol) was dissolved in THF (1 mL) and stirred under argon atmosphere at −78 °C for 20 minutes. DIBAL (163 μL, 0.163 mmol) was then added, and the mixture was stirred at −78 °C for 1 hours. The reaction mixture was quenched with MeOH (228 μL) and saturated aqueous NH4Cl (390 μL) and stirred at −78 °C for 30 min. After removing most of the solids, 1,4-dioxane was added and the mixture was stirred for several minutes. The remaining solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatogram (n-hexane[thin space (1/6-em)]:[thin space (1/6-em)]EtOAc = 50[thin space (1/6-em)]:[thin space (1/6-em)]50 to 20[thin space (1/6-em)]:[thin space (1/6-em)]80 gradient) to afford compound 3 (8.8 mg, 20%) as a yellow solid; 1H-NMR (600 MHz, DMSO-d6): δ 10.23 (s, 1H), 9.63 (s, 1H), 9.07 (s, 2H), 7.55 (d, J = 9.1 Hz, 2H), 7.48–7.45 (m, 2H), 7.30–7.27 (m, 1H), 7.25–7.23 (m, 2H), 6.96–6.93 (m, 2H), 3.56 (dt, J = 12.5, 3.8 Hz, 2H), 2.81–2.76 (m, 2H), 2.47–2.45 (m, 1H), 1.93–1.91 (m, 2H), 1.58 (ddd, J = 24.1, 10.8, 3.8 Hz, 2H); 13C-NMR (151 MHz, DMSO-d6): δ 204.7, 165.6, 161.1, 159.6, 152.6, 147.9, 130.2, 129.8, 125.6, 124.2, 121.6, 121.4, 116.1, 48.2, 46.9, 40.0; HRMS (ESI) m/z calcd for C23H23N4O3 [M + H]+, 403.1765; found, 403.1758.
Synthesis of PROTAC-2. Compound 3 (8.7 mg, 0.0216 mmol), 1H-isoindole-1,3(2H)-dione, 2-(2,6-dioxo-3-piperidinyl)-5-(4-piperidinyloxy) hydrochloride (9.0 mg, 0.0238 mmol), and DIPEA (22 μL, 0.130 mmol) were dissolved in dichloroethane (400 μL) and DMF (200 μL) and stirred at room temperature for 1.5 hours. NaBH(OAc)3 (11 mg, 0.0540 mmol) was then added, and the mixture was stirred for 13 hours. The reaction mixture was diluted with dichloroethane and washed sequentially with saturated aqueous NaHCO3. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatogram (CH2Cl2[thin space (1/6-em)]:[thin space (1/6-em)]MeOH = 100[thin space (1/6-em)]:[thin space (1/6-em)]0 to 85[thin space (1/6-em)]:[thin space (1/6-em)]15 gradient) to afford PROTAC-2 (5.7 mg, 36%) as a yellow solid; 1H-NMR (600 MHz, DMSO-d6): δ 11.09 (s, 1H), 10.22 (s, 1H), 9.08 (s, 2H), 7.70 (dd, J = 8.2, 7.2 Hz, 1H), 7.54 (d, J = 8.9 Hz, 2H), 7.48–7.45 (m, 2H), 7.36–7.32 (m, 2H), 7.29 (t, J = 7.5 Hz, 1H), 7.25–7.24 (m, 2H), 6.93 (d, J = 9.1 Hz, 2H), 5.09 (dd, J = 12.9, 5.5 Hz, 1H), 3.65 (d, J = 12.2 Hz, 2H), 3.30 (d, J = 4.1 Hz, 4H), 2.89–2.83 (m, 1H), 2.64–2.59 (m, 3H), 2.56–2.52 (m, 5H), 2.23 (d, J = 7.2 Hz, 2H), 2.03–2.00 (m, 1H), 1.81 (d, J = 11.5 Hz, 2H), 1.71–1.68 (m, 1H), 1.25–1.19 (m, 2H); 13C-NMR (151 MHz, DMSO-d6): δ 172.8, 170.0, 167.1, 166.3, 165.6, 161.0, 159.6, 152.6, 149.8, 148.3, 135.9, 133.7, 130.0, 129.8, 125.6, 124.2, 123.7, 121.7, 121.4, 116.5, 115.9, 114.8, 63.9, 53.1, 50.6, 49.0, 48.8, 32.4, 31.0, 30.2, 22.1; HRMS (ESI) m/z calcd for C40H40N8O6 [M + H]+, 729.3144, found 729.3144; purity: 98.9% (tR = 5.06 min).
Synthesis of compound 6. DMSO (2.7 mL) and DIPEA (461 μL, 2.71 mmol) were added to compound 4 (150 mg, 0.543 mmol) and compound 5 (157 mg, 0.597 mmol). The mixture was stirred at 90 °C for 17 hours. The reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound 6 (262 mg, quant) as an orange solid; 1H-NMR (600 MHz, DMSO-d6): δ 11.06 (s, 1H), 7.56–7.54 (m, 1H), 7.10 (d, J = 7.0 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 5.03 (dd, J = 12.9, 5.5 Hz, 1H), 3.93 (s, 4H), 3.29 (s, 4H), 2.88–2.82 (m, 1H), 2.44–2.48 (m, 1H), 2.56 (ddd, J = 17.1, 4.1, 2.4 Hz, 1H), 2.01–1.96 (m, 1H), 1.68–1.66 (m, 4H), 1.38 (s, 9H); 13C-NMR (151 MHz, DMSO-d6): δ 172.9, 170.1, 167.2, 166.6, 153.9, 148.0, 135.0, 133.3, 119.9, 111.6, 110.0, 78.7, 48.6, 40.1, 34.7, 33.6, 31.0, 28.1, 22.2; HRMS (ESI) m/z calcd for C25H31N4O6 [M + H]+, 483.2238; found, 483.2240.
Synthesis of PROTAC-3. TFA (1 mL) and CH2Cl2 (1 mL) was added to a solution of compound 6 (30 mg, 0.0622 mmol). The reaction mixture was stirred at room temperature for 1 hours and then concentrated under reduced pressure. The resulting crude product (39 mg, 0.0813 mmol), HATU (31 mg, 0.0813 mmol), HOAt (11 mg, 0.0813 mmol) and compound 1 (28 mg, 0.0542 mmol) were dissolved in N,N-dimethylformamide (300 μL), followed by the addition of DIPEA (92 μL, 0.542 mmol). The mixture was stirred at room temperature for 12 hours. After concentration under reduced pressure, the residue was filtered through a membrane filter and purified by preparative HPLC (gradient elution: 40–80% MeCN–H2O over 40 minutes) to afford PROTAC-3 as a yellow solid (12.4 mg, 29%); 1H-NMR (600 MHz, CDCl3): δ 9.03 (s, 2H), 7.48–7.45 (m, 6H), 7.30 (t, J = 7.4 Hz, 1H), 7.20 (dd, J = 17.6, 7.3 Hz, 3H), 6.92 (d, J = 7.7 Hz, 2H), 6.59 (d, J = 8.4 Hz, 1H), 4.92 (dd, J = 12.4, 5.3 Hz, 1H), 4.03 (s, 4H), 3.71 (s, 2H), 3.56 (d, J = 56.9 Hz, 4H), 2.88 (d, J = 16.3 Hz, 1H), 2.80–2.71 (m, 4H), 2.63 (s, 1H), 2.11 (dd, J = 7.8, 5.1 Hz, 1H), 1.98 (d, J = 12.0 Hz, 2H), 1.87 (s, 2H), 1.80 (d, J = 10.8 Hz, 4H); 13C-NMR (151 MHz, CDCl3): δ 173.1, 170.8, 168.2, 167.5, 167.0, 166.6, 161.1, 159.1, 152.5, 149.2, 147.9, 134.9, 133.8, 129.8, 129.0, 126.0, 124.2, 123.7, 122.0, 121.5, 120.1, 119.3, 117.1, 116.9, 89.6, 53.4, 51.8, 49.5, 49.3, 49.0, 42.6, 40.8, 39.0, 38.4, 36.4, 35.2, 34.4, 31.4, 29.7, 28.5, 28.0, 27.4, 22.7; HRMS (ESI) m/z calcd for C43H43N8O7 [M + H]+, 783.3249; found, 783.3247. HPLC purity: 96.4% (tR = 6.37 min).
Synthesis of compound 8. DMSO (2.7 mL) and DIPEA (277 μL, 1.63 mmol) were added to compound 4 (150 mg, 0.543 mmol) and compound 7 (152 mg, 0.597 mmol). The mixture was stirred at 90 °C for 17 hours. The reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound 8 (281 mg, quant) as a yellow solid; 1H-NMR (600 MHz, DMSO-d6): δ 10.32–11.66 (1H), 7.66 (dd, J = 8.4, 7.0 Hz, 1H), 7.33–7.30 (m, 2H), 5.07 (dd, J = 12.9, 5.5 Hz, 1H), 3.32 (s, 4H), 3.25 (t, J = 5.0 Hz, 4H), 2.89–2.83 (m, 1H), 2.63–2.51 (m, 2H), 2.03–1.99 (m, 1H), 1.61 (t, J = 5.2 Hz, 4H), 1.41 (t, J = 5.6 Hz, 4H), 1.38 (s, 9H); 13C-NMR (151 MHz, DMSO-d6): δ 172.9, 170.1, 167.1, 166.4, 154.0, 150.1, 135.8, 133.7, 123.9, 116.1, 114.4, 78.5, 48.8, 46.5, 41.1, 40.0, 34.9, 31.0, 29.3, 28.2, 22.1; HRMS (ESI) m/z calcd for C27H35N4O6 [M + H]+, 511.2551; found, 511.2552.
Synthesis of PROTAC-4. 4 M hydrogen chloride in 1,4-dioxane (2 mL) and a small amount of MeOH were added to a compound 8 (30 mg, 0.0588 mmol). The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. A portion of the resulting compound (19 mg, 0.0422 mmol), HATU (16 mg, 0.0422 mmol), HOAt (5.7 mg, 0.0422 mmol), and compound 1 (12.8 mg, 0.0281 mmol) were dissolved in N,N-dimethylformamide (300 μL), followed by the addition of DIPEA (50 μl, 0.281 mmol). The mixture was stirred at room temperature for 13 hours. After concentration under reduced pressure, the residue was filtered through a membrane filter and purified by preparative HPLC (gradient elution: 40–80% MeCN–H2O over 40 minutes) to afford PROTAC-4 as a yellow solid (7.9 mg, 35%); 1H-NMR (600 MHz, CDCl3): δ 9.03 (s, 2H), 8.12 (s, 1H), 8.00 (s, 1H), 7.58 (dd, J = 8.3, 7.1 Hz, 1H), 7.47–7.43 (m, 4H), 7.38 (d, J = 7.0 Hz, 1H), 7.31–7.28 (m, 1H), 7.21 (d, J = 7.6 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 6.93–6.90 (m, 2H), 4.96 (dd, J = 12.5, 5.5 Hz, 1H), 3.70 (d, J = 10.7 Hz, 2H), 3.62–3.60 (m, 2H), 3.51 (s, 2H), 3.36–3.25 (m, 4H), 2.90–2.60 (m, 6H), 2.13–2.09 (m, 1H), 2.00–1.93 (m, 2H), 1.81–1.76 (m, 5H), 1.62–1.55 (m, 6H); 13C-NMR (151 MHz, CDCl3): δ 173.1, 170.9, 168.2, 167.3, 166.7, 166.5, 161.2, 159.2, 152.5, 150.6, 149.1, 135.6, 134.1, 129.8, 129.1, 126.0, 123.7, 123.5, 122.1, 121.5, 117.2, 117.0, 116.8, 115.5, 53.4, 49.5, 49.1, 47.1, 41.3, 38.4, 37.5, 37.1, 35.4, 34.5, 31.4, 29.9, 28.5, 27.9, 22.7; HRMS (ESI) m/z calcd for C45H47N8O7 [M + H]+, 811.3562; found, 811.3562; purity: 85.9% (tR = 6.48 min).

Biology

Reagents. Tissue culture plastics were purchased from Greiner Bio-One (Tokyo, Japan). Penicillin–streptomycin mixed solution were from Nacalai (Kyoto, JAPAN). Roswell Park Memorial Institute-1640 (RPMI-1640) medium. Fetal bovine serum (FBS) was from Thermo Fisher Scientific (Waltham, MA, USA).
Binding affinity assay for H-PGDS. The fluorescence polarization-based (FP-based) binding assay was performed using prostaglandin D synthase (hematopoietic-type) FP-Based Inhibitor Screening Assay Kit Green (600007) (Cayman Chemical) in accordance with the manufacturer's instructions. In brief, the binding assays were performed in nonbinding black 384-well and used a recombinant human H-PGDS protein (Fig. S7), glutathione, and fluorescence probe in assay buffer to produce a final volume of 47.5 μL. Then, 2.5 μL of test compounds made up as stocks in DMSO was added, and the plate was incubated for 1 h at room temperature. Each was tested against H-PGDS in triplicate at final test compound concentrations (5.00, 2.50, 1.25 μM, 625, 313, 156, 78.1, 39.1, 19.5, 9.80, 4.90, and 2.45 nM). Plates were then read with excitation wavelengths (470 nm) and emission wavelengths (530 nm) on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). The measurements of fluorescence polarization of a molecule (mP) are taken in the fluorescence polarization mode. The percentage of inhibition of test compounds was calculated according to the following equation
image file: d5md00396b-t1.tif
where mPsample is the value of the wells containing test compounds and mP100% is the value of the maximum binding well. The concentration of test compounds that reduces the mP value by 50% (IC50) was estimated from a graph plotted the mP value versus the concentration of the compounds on semi-log axis.
Cell culture. Human chronic myelogenous leukemia KU812 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 μg mL−1 penicillin–streptomycin mix. KU812 cells were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) Cell Bank (JCRB0104).
Western blotting. KU812 cells were treated with the indicated concentrations of compounds for 24 hours. Whole cells were lysed with SDS lysis buffer (20 mM Tris-HCl at pH 8.0, 10% glycerol, 1% SDS) and immediately boiled for 10 min to obtain clear lysates. Protein concentrations were measured using the BCA method (Pierce, Washington, USA). Lysates containing equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Merck) for western blot analysis using the appropriate antibodies. Immunoreactive proteins were visualized using the Clarity Western ECL substrate (Bio-Rad, California, USA); light emission intensity was quantified using a ChemiDoc MP Imaging System equipped with Image Lab™ Software (Bio RAD, California, USA). The antibodies used in this study were rabbit anti-H-PGDS polyclonal antibody (1[thin space (1/6-em)]:[thin space (1/6-em)]2000 dilution), mouse anti-β-actin mAb (A2228, Sigma-Aldrich, 1[thin space (1/6-em)]:[thin space (1/6-em)]2000 dilution). Numbers below the H-PGDS panels represent H-PGDS/actin, normalized by designating the expression from the vehicle control condition as 100%.

Computational analysis

i) Ternary complex modeling. Molecular docking simulations were performed using Molecular Operating Environment (MOE) 2024.0601. Ternary complexes consisting of CRBN (PDB: 4CI3), H-PGDS (PDB: 5YWX), and each PROTAC were constructed according to Docking Method 5 described in the literature.28 In contrast, the ternary complex of PROTAC-PEG5 was constructed according to Method 4B.28 Docking calculations were carried out using the Amber10:EHT force field. Complexes were selected based on the following criteria: RMSD < 2.5 Å, RMSD_RF_PP < 670 Å2, ligand_E < 3600 kcal mol−1, and ligand_E_PP < [value] Å2. Additionally, two-dimensional interaction models were generated using the following thresholds: hydrogen bonding energy < −0.5 kcal mol−1, ionic interaction energy < −0.5 kcal mol−1, and interatomic distances > 4.5 Å. Glutathione was incorporated into the simulation system in the same orientation as observed in the crystal structure of H-PGDS (PDB: 5YWX). All docking calculations were conducted under default settings unless otherwise specified.
ii) Molecular dynamics simulation. Molecular dynamics (MD) simulations were carried out using MOE 2024.0601 with the Amber10:EHT force field. The ternary complex models selected through docking were solvated by adding water molecules, and a periodic boundary condition was applied, ensuring that the simulation box extended at least 10 Å beyond the edge of the protein in all directions. The system was neutralized with NaCl at a final concentration of 0.1 mol L−1. The temperature during simulation was controlled using NAMD.

Each simulation followed a four-step protocol:

1. Minimization step: 100 ps at 0 K.

2. Heating step: 100 ps with a linear increase in temperature from 10 K to 300 K, applying tethering forces ranging from 0.5 to 100 Å.

3. Equilibration step: 1000 ps at constant temperature (300 K).

4. Production step: 200 ns at 300 K.

Simulations were run with a time step of 2 fs, and the trajectory was saved every 100 ps. Following simulation, results were analyzed in MOE 2024.0601. From each production trajectory, the most stable structure was selected based on potential energy and ligand interactions for further structural evaluation.


LC-tandem mass spectrometry (LC-MS/MS). For quantification of PROTACs in cells, cells were pelleted by centrifuge and mixed with 100 μL of methanol to extract PROTACs. After removal of protein precipitant by centrifugation, the extracts were filtered and subjected to LC-MS/MS analysis using Ultimate3000 UHPLC with TSQ-Quantiva (Thermo Fisher Scientific, Waltham, MA, U.S.A.). A Triart Bio C18 column (3 μm, 3 × 100 mm) (YMC, Kyoto, Japan) was used for LC at 50 °C. Mobile phase A consisted of 10 mM ammonium formate in water, and mobile phase B consisted of 10 mM ammonium formate in H2O[thin space (1/6-em)]:[thin space (1/6-em)]acetonitrile (1[thin space (1/6-em)]:[thin space (1/6-em)]9, v/v). The flow rate was 0.35 mL min−1, and the injected sample volume was 5 μL. The gradient program of the mobile phase was initiated by 50% B and increased to 100% B at 2 min, followed by maintaining 100% B for 1.2 min, and immediate restoration to initial condition (50% B), and equilibrated for 1 min. The mass spectrometer was operated in the heated electrospray ionization mode with following ion source parameter: spray voltage: 3500 V, sheath gas: 40 Arb, aux gas: 10 Arb, sweep gas: 1 Arb, transfer temp: 350 °C, vaporizer temp: 250 °C. The data were acquired in the selective reaction monitoring mode (collision energy: 35 V; collision gas: Ar, 1.5 mTorr; mass transition (parent ion/product ion [m/z]): 743.22/401.21 for PROTAC-1, 729.16/387.13 for PROTAC-2, 783.30/401.21 for PROTAC-3, and 811.30/401.21 for PROTAC-4). The selectivity of the mass transition was confirmed by the no detectable signals of all PROTACs in DMSO-treated cells (SI14). Along with sample analysis, calibration standards (0.3–100 nM mixture of PROTAC-1, 2, 3, and 4 in methanol) were also analyzed. TraceFinder 4.1 (Thermo Fisher Scientific) was used for peak detection and smoothing. Following the quantification of the peak area, the calibration curve was analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, U.S.A.) and calculated concentrations of each PROTACs in the cell extracts. The calibration curve of PROTAC-1 is depicted in SI15 as an example. The intracellular amount of PROTACs were then calculated as the total amount of PROTACs within the total volume of cell extracts.
log[thin space (1/6-em)]D measurement. Analytical UPLC was performed using an ACQUITY UPLC® BEH C18 column (1.7 μm, 2.1 mm I.D. × 50 mm) at a flow rate of 0.5 mL min−1. A solution A: 20 mM CH3COONH4/MeCN = 95/5, B: MeCN, and a gradient of 40% of solution A to 100% of solution B was measured in 3 min. Eluents were detected at 254 nm by a UPLC system (Waters). Five standard compounds (methyl paraben (1.96), ethyl paraben (2.47), propyl paraben (3.04), butyl paraben (3.57), heptyl paraben (4.83)) were analyzed by UPLC (log[thin space (1/6-em)]D in parentheses). The retention times of the samples were compared with those of the standard compounds, and log[thin space (1/6-em)]D was calculated.
Binding affinity assay for CRBN. The homogeneous time-resolved fluorescence (HTRF) assay was performed using HTRF Cereblon Binding Kits (Revvity, #64BDCRBNPEG) in accordance with the manufacturer's instructions. Briefly, serially diluted compounds were incubated with GST-tagged wild-type human CRBN protein (Fig. S11), XL665-labelled thalidomide and europium cryptate labeled GST antibody in white 96-well low volume plates (Revvity, 66PL96025) at room temperature for 3 h. The plate was read using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). The HTRF signals were collected at 620 nm and 665 nm and calculated using the following equation: signal 665 nm/signal 620 nm × 10[thin space (1/6-em)]000. KaleidaGraph was used to plot dose–response curves and calculate IC50 values for each compound.

Author contributions

H. O. and K. S. performed the experiments and analyzed results. H. O. and Y. D. designed the research and wrote the paper. All authors discussed the results and commented on the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

Supplementary information is available. See DOI: https://doi.org/10.1039/D5MD00396B.

Raw data were generated at National Institute of Health Sciences. Derived data supporting the findings of this study are available from the corresponding author Y. D. on request.

Acknowledgements

This study was supported in part by AMED under grant numbers 25ama221127 to Y. D. This work was also supported by the Japan Society for the Promotion of Science (KAKENHI, grants JP21K05320 and JP23H04926 to Y. D., JSPS Fellows grant JP24KJ1716 to H. O.).

Notes and references

  1. K. M. Sakamoto, K. B. Kim, A. Kumagai, F. Mercurio, C. M. Crews and R. J. Deshaies, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 8554–8559 CrossRef CAS PubMed.
  2. A. R. Schneekloth, M. Pucheault, H. S. Tae and C. M. Crews, Bioorg. Med. Chem. Lett., 2008, 18, 5904–5908 CrossRef CAS PubMed.
  3. M. Hinterndorfer, V. A. Spiteri, A. Ciulli and G. E. Winter, Nat. Rev. Cancer, 2025, 25(7), 493–516 Search PubMed.
  4. J. Lin, Z. Chen, D. Zhang, N. Zhang, H. Chen and D. S. Guo, Macromol. Rapid Commun., 2025, e2401051 CrossRef PubMed.
  5. N. Shibata, K. Nagai, Y. Morita, O. Ujikawa, N. Ohoka, T. Hattori, R. Koyama, O. Sano, Y. Imaeda, H. Nara, N. Cho and M. Naito, J. Med. Chem., 2018, 61, 543–575 CrossRef CAS PubMed.
  6. Z. Chen, B. Hu, R. K. Rej, D. Wu, R. K. Acharyya, M. Wang, T. Xu, J. Lu, H. Metwally, Y. Wang, D. McEachern, L. Bai, C. L. Gersch, M. Wang, W. Zhang, Q. Li, B. Wen, D. Sun, J. M. Rae and S. Wang, J. Med. Chem., 2023, 66, 12559–12585 CrossRef CAS PubMed.
  7. Y. Dong, T. Ma, T. Xu, Z. Feng, Y. Li, L. Song, X. Yao, C. R. Ashby, Jr. and G. F. Hao, Acta Pharm. Sin. B, 2024, 14, 4266–4295 CrossRef CAS PubMed.
  8. X. Han, C. Wang, C. Qin, W. Xiang, E. Fernandez-Salas, C. Y. Yang, M. Wang, L. Zhao, T. Xu, K. Chinnaswamy, J. Delproposto, J. Stuckey and S. Wang, J. Med. Chem., 2019, 62, 941–964 CrossRef CAS PubMed.
  9. M. J. Meyers, S. A. Long, M. J. Pelc, J. L. Wang, S. J. Bowen, B. A. Schweitzer, M. V. Wilcox, J. McDonald, S. E. Smith, S. Foltin, J. Rumsey, Y. S. Yang, M. C. Walker, S. Kamtekar, D. Beidler and A. Thorarensen, Bioorg. Med. Chem. Lett., 2011, 21, 6545–6553 CrossRef CAS PubMed.
  10. A. D. Takwale, S. H. Jo, Y. U. Jeon, H. S. Kim, C. H. Shin, H. K. Lee, S. Ahn, C. O. Lee, J. Du Ha, J. H. Kim and J. Y. Hwang, Eur. J. Med. Chem., 2020, 208, 112769 CrossRef CAS PubMed.
  11. J. Nunes, G. A. McGonagle, J. Eden, G. Kiritharan, M. Touzet, X. Lewell, J. Emery, H. Eidam, J. D. Harling and N. A. Anderson, ACS Med. Chem. Lett., 2019, 10, 1081–1085 CrossRef CAS PubMed.
  12. E. Christ-Hazelhof and D. H. Nugteren, Biochim. Biophys. Acta, 1979, 572, 43–51 CrossRef CAS PubMed.
  13. Y. Urade, N. Fujimoto, M. Ujihara and O. Hayaishi, J. Biol. Chem., 1987, 262, 3820–3825 CrossRef CAS PubMed.
  14. T. Matsuoka, M. Hirata, H. Tanaka, Y. Takahashi, T. Murata, K. Kabashima, Y. Sugimoto, T. Kobayashi, F. Ushikubi, Y. Aze, N. Eguchi, Y. Urade, N. Yoshida, K. Kimura, A. Mizoguchi, Y. Honda, H. Nagai and S. Narumiya, Science, 2000, 287, 2013–2017 CrossRef CAS PubMed.
  15. I. Mohri, K. Aritake, H. Taniguchi, Y. Sato, S. Kamauchi, N. Nagata, T. Maruyama, M. Taniike and Y. Urade, Am. J. Pathol., 2009, 174, 1735–1744 CrossRef CAS PubMed.
  16. S. Rittchen and A. Heinemann, Cell, 2019, 8, 619 CrossRef CAS PubMed.
  17. T. Nabe, Y. Kuriyama, N. Mizutani, S. Shibayama, A. Hiromoto, M. Fujii, K. Tanaka and S. Kohno, Prostaglandins Other Lipid Mediators, 2011, 95, 27–34 CrossRef CAS PubMed.
  18. H. Aoyagi, D. Kajiwara, K. Tsunekuni, K. Tanaka, K. Miyoshi and N. Hirasawa, Eur. J. Pharmacol., 2020, 875, 173030 CrossRef CAS PubMed.
  19. Y. Murakami, H. Osawa, T. Kurohara, Y. Yanase, T. Ito, H. Yokoo, N. Shibata, M. Naito, K. Aritake and Y. Demizu, RSC Med. Chem., 2022, 13, 1495–1503 RSC.
  20. H. Osawa, T. Kurohara, T. Ito, N. Shibata and Y. Demizu, Bioorg. Med. Chem., 2023, 84, 117259 CrossRef CAS PubMed.
  21. H. Yokoo, H. Osawa, K. Saito and Y. Demizu, Chem. Pharm. Bull., 2024, 72, 961–965 CrossRef CAS PubMed.
  22. H. Yokoo, N. Shibata, A. Endo, T. Ito, Y. Yanase, Y. Murakami, K. Fujii, K. Hamamura, Y. Saeki, M. Naito, K. Aritake and Y. Demizu, J. Med. Chem., 2021, 64, 15868–15882 CrossRef CAS PubMed.
  23. H. Yokoo, N. Shibata, M. Naganuma, Y. Murakami, K. Fujii, T. Ito, K. Aritake, M. Naito and Y. Demizu, ACS Med. Chem. Lett., 2021, 12, 236–241 CrossRef CAS PubMed.
  24. E. S. Fisher, K. Böhm, J. R. Lydeard, H. Yang, M. B. Stadler, S. Cavadini, J. Nagel, F. Serluca, V. Acker, G. M. Lingaraju, R. B. Tichkule, M. Schebesta, W. C. Forrester, M. Schirle, U. Hassiepen, J. Ottl, M. Hild, R. E. J. Beckwith, J. W. Harper, J. L. Jenkins and N. H. Thomä, Nature, 2014, 512, 49–53 CrossRef PubMed.
  25. N. L. Tran, G. A. Leconte and F. M. Ferguson, Curr. Protoc., 2022, 2, e611 CrossRef PubMed.
  26. R. P. Wurz, H. Rui, K. Dellamaggiore, S. Ghimire-Rijal, K. Choi, K. Smither, A. Amegadzie, N. Chen, X. Li, A. Banerjee, Q. Chen, D. Mohl and A. Vaish, Nat. Commun., 2023, 14, 4177 CrossRef CAS PubMed.
  27. H. Lan, O. Hsia, M. Nakasone, A. Wijaya, A. Ciulli, C. Butts and V. Aggarwal, ChemRxiv, 2024, preprint,  DOI:10.26434/chemrxiv-2024-5dbnb.
  28. M. L. Drummond, C. A. Williams, R. D. Klug-McLeod, R. J. Baell and J. A. Watson, J. Chem. Inf. Model., 2020, 60, 2570–2583 CrossRef PubMed.

This journal is © The Royal Society of Chemistry 2025
Click here to see how this site uses Cookies. View our privacy policy here.