2024 Volume 72 Issue 11 Pages 961-965
Proteolysis-targeting chimeras (PROTACs) have attracted attention as an innovative drug modality that induces the selective degradation of target proteins. This technology shows higher activity than conventional inhibitors and holds great potential in the field of drug discovery. Optimization of the linker is essential for PROTACs to achieve sufficient activity, particularly with regard to cell membrane permeability. However, the correlation between membrane permeability and the activity of PROTACs has not been fully explored. To address this, we established a new molecular design approach to remove the linker and optimize PROTAC structure. These PROTAC compound groups were used to analyze the correlation between membrane permeability and activity using LC-tandem mass spectrometry (LC-MS/MS). Results revealed that the degradation activity of PROTACs fluctuates with increasing membrane permeability and changes in response to linker optimization, while sufficient proteolytic activity can be retained. These findings demonstrate the importance of considering the balance between membrane permeability and activity in PROTAC design and provide a new strategy for developing more effective PROTACs.
Proteolysis-targeting chimeras (PROTACs) have emerged as an innovative drug modality that induces the selective degradation of target proteins.1,2) This technology leverages the cellular ubiquitin-proteasome system to eliminate specific proteins, representing a significant advancement over conventional inhibitors. PROTACs typically consist of three components: a ligand for the target protein, a ligand for an E3 ubiquitin ligase molecule, and a linker connecting the two.3–6) The linker plays a crucial role in determining the compound’s properties, including its activity and membrane permeability.7–15) The cell membrane permeability of PROTACs is highly dependent on their molecular weight and physicochemical properties. Many PROTACs have higher molecular weights than conventional small-molecule drugs, posing a challenge for cell membrane permeability. Therefore, efforts have been made to design PROTACs with optimized physicochemical properties by controlling their structural characteristics. For example, reports suggest that membrane permeability can be improved by increasing hydrophobicity and adjusting the number of hydrogen bond donors and acceptors in the molecules.
PROTACs have been developed to target hematopoietic prostaglandin D synthase (H-PGDS), which catalyzes the synthesis of prostaglandin D2 (PGD2).16,17) Overproduction of PGD2 is implicated in various diseases, including allergic conditions,18,19) sleep disorders,20) and Duchenne muscular dystrophy.21) This makes H-PGDS a promising therapeutic target. Several H-PGDS inhibitors have been developed, including TFC007, which binds to H-PGDS, and pomalidomide, which binds to cereblon. We have worked extensively on developing PROTACs that specifically target H-PGDS (PROTAC(H-PGDS)s).8,22–24) The PROTACs developed effectively degrade the H-PGDS protein through the ubiquitin-proteasome system and suppress PGD2 production. This novel approach using PROTACs offers a potential alternative to traditional H-PGDS inhibitors for treating allergic and inflammatory responses, as well as Duchenne muscular dystrophy. In our development of PROTAC(H-PGDS)s, PROTAC(H-PGDS)-7, in which the linker removed, showed greater activity (50% degradation concentration [DC50] = 17.3 pM) than any PROTAC(H-PGDS) compounds that comprise a linker8) (Figs. 1a and 1b). This suggests that designing PROTACs with a reduced molecular weight may be beneficial. However, shortening linker length does not always improve the degradative activity, and may even reduce it, especially if the linker is unsuitable for bringing the target protein and E3 ligase into proximity. Therefore, we evaluated the membrane permeability of PROTACs with varying linker lengths to examine the relationship between membrane permeability and linker length. This evaluation allowed us to identify the molecular properties that contribute to degradation activity.
Each PROTAC(H-PGDS) was prepared according to methods previously described.8) LC-MS analysis detected the amide-cleaved structure of the TFC007 moiety in PROTAC(H-PGDS)-7 (PEG0) (Fig. 1a). Furthermore, the same fragments were detected in PROTAC(H-PGDS)-4 (PEG3) and PROTAC(H-PGDS)-1 (PEG5), indicating that the amide structure can be cleaved and detected regardless of the linker used. Quantification was performed using the signal at m/z 401. Based on this signal, a calibration curve was plotted using stepwise-diluted samples, and the concentration of compound inside the cells was calculated.
Cellular uptake of the PROTAC(H-PGDS)s increased with increasing compound concentrations (0.1, 1 and 10 nM) in a dose-dependent manner (Fig. 2). Differences in the uptake of different compounds were also observed, with PEG0 showing greater uptake than PEG3 and PEG5. This difference in uptake was expected, as the PEG moiety is known to decrease cellular uptake25) and PEG0 is considered to have a more drug-like structure than PEG3 and PEG5.8)
Next, we evaluated the H-PGDS degradation activity and confirmed that the PROTAC(H-PGDS) compounds used in this study exhibited the same activity as previously reported.8) To assess correlation with drug efficacy, cells were incubated with compounds at concentrations of 1 and 10 nM. PEG0 showed greater degradation activity and cellular uptake than PEG3. At both concentrations, PEG0 uptake was approximately 2-fold greater than that of PEG3. However, degradation activity at 1 nM was not proportionally higher (Fig. 3). In our previous study, the hook effect was not observed over a concentration range of 1–10 nM. These results suggest that PEG0 and PEG3 may differ not only with regard to cellular uptake, but also in their pharmacological efficacy. There was little difference in the cellular uptake of PEG3 and PEG5 at 1 and 10 nM. However, degradation activity was higher for PEG3, suggesting that this difference is related to the pharmacological potency of the compounds.
These results revealed that PEG0 was superior in terms of cellular uptake and its intracellular mechanisms, including achieving correct ternary complex formation and distribution within cells. Although intracellular concentrations of PROTAC(H-PGDS)s were similar between compounds (Fig. 3), their degradation activities differed according to their intracellular mechanisms (e.g., PEG3 and PEG5). The measurements reported here have limitations related to the inclusion of non-specific bound compounds, such as those attached to the cell membrane, although differences in cellular uptake among different PROTACs were clearly observed.
The intracellular concentration of PEG0 was determined relative to the total amount added to the medium at concentrations of 0.1, 1, and 10 nM. The amount of PEG0 in cells increased with the concentration of PROTACs, although the rate of cellular uptake was approximately 14% at any concentration (Fig. 4). The introduction of a PEG linker has limitations as it decreases the uptake of compounds because of non-specific binding. Therefore, we determined the amount of intracellular PEG0 by comparing cell culture conditions using medium with and without fetal bovine serum (FBS; Fig. 4). Although a dose-dependent increase in cellular uptake was observed, the rate of compound uptake increased by approximately 40% at each concentration of PEG0, suggesting that more than 20% of the added compound was captured by non-specific binding to FBS. Under these conditions, a reduction in the expression of H-PGDS was observed, depending on the cellular level of PEG0 (Fig. 4c).
We evaluated the membrane permeability of PROTACs with different PEG linker lengths to elucidate the molecular properties that contribute to degradation activity and to examine the relationship between membrane permeability and linker length. The cell membrane permeability of PROTACs largely depends on their molecular weight and physicochemical properties. Many PROTACs have higher molecular weights than conventional low-molecular-weight drugs, which poses a challenge for cell membrane permeability. The drug-like properties of PROTACs can be dramatically improved by linker modifications that alter the number of hydrogen bond donors (HBD), lipophilicity, molecular weight, rotatable bonds, and polar surface area.12) Appropriate linker modifications can enhance the cell permeability of PROTACs. For example, cationic rigid linkers containing piperidine or piperazine moieties can enhance rigidity, water solubility, and cell permeability. Notably, structures of PROTACs in clinical trials often incorporate these types of linkers. While our study primarily focused on the impact of PEG linker length, we recognize that linker type is equally important in optimizing PROTAC performance. Future studies on PROTACs targeting different proteins will benefit from a more detailed investigation into the interplay between linker composition and degradation activity.
The findings of this study regarding membrane permeability changes related to linker length and conversion, and their effects on degradation activity, are important. Notably, we showed that removing the linker can improve membrane permeability. Furthermore, we identified sets of PROTACs that exhibit similar membrane permeability but different intracellular degradation activities, and showed that linker length also influences intracellular degradation mechanisms and should be optimized as a bifunctional moiety. These findings provide a basis for further structure optimization studies and may inform new design strategies.
All compounds were synthesized and analyzed according to methods previously described.8)
Cell CultureHuman chronic myelogenous leukemia cells (KU812) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB0104; Osaka, Japan) and cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and 100 µg/mL penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan).
Western BlottingKU812 cells were pre-cultured in cell culture medium supplemented with 0, 1, or 10% FBS. In this experiment, cells were treated with the compound of interest for 6 h. In all other cases, cells were incubated with the compound for 24 h. Cells were lysed with sodium dodecyl sulfate (SDS) lysis buffer (0.1 M Tris–HCl at pH 8.0, 10% glycerol, 1% SDS) and immediately heated for 5 min to obtain clear lysates. Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Pierce Biotechnology, Waltham, MA, U.S.A.). Lysates containing equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany) for Western blotting analysis. The antibodies used in this study were anti-H-PGDS rabbit polyclonal antibody (1 : 2000 dilution) and anti-β-actin mouse monoclonal antibody (1 : 2000 dilution; Sigma-Aldrich). Immunoreactive proteins were visualized using the Clarity Western Enhanced Chemiluminescence Substrate (Bio-Rad, Hercules, CA, U.S.A.) and light emission intensity was quantified using a ChemiDoc MP Imaging System equipped with Image Lab™ Software (Bio-Rad). Whole-cell lysates were analyzed via Western blotting with the indicated antibodies. Numbers below the H-PGDS panels represent H-PGDS/actin, normalized by designating expression in the vehicle control condition as 100%.
LC-Tandem Mass Spectrometry (LC-MS/MS)For quantification of PROTAC(H-PGDS)s (PEG0, 3, and 5) in cells, cells were pelleted by centrifuge and mixed with 100 µL of methanol to extract PROTAC(H-PGDS)s. 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 acetonitrile. The flow rate was 0.35 mL/min, 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 (ESI) 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 (m/z): 743.22/401.21 for PEG0, 946.41/401.21 for PEG3, and 1034.46/401.21 for PEG5). Along with sample analysis, calibration standards (0.01–100 nM mixture of PEG0, 3, and 5 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 PROTAC(H-PGDS)s (PEG0, 3, 5) in cells.
This study was supported by AMED (Grant numbers 24mk0121286, 24mk0101220, 24ama221127, and 24ak0101185 to Y.D.), the Japan Society for the Promotion of Science (JSPS, KAKENHI, Grants JP22K15257 and JP24K18260 to H.Y., and Grants JP21K05320 and JP23H04926 to Y.D.), JST (ACT-X, Grant JPMJAX222L to H.Y.), Takeda Science Foundation (H.Y.), Kowa Life Science Foundation (H.Y.), Astellas Foundation for Research on Metabolic Disorders (H.Y.), the Watanabe Foundation (H.Y.), and JSPS (KAKENHI, Grant JP24KJ1716 to H.O.).
The authors declare no conflict of interest.
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