Cereblon
PD-1/PD-L1 interaction (IC50 = 39.2 ± 5.8 nM for PROTAC PD-1/PD-L1 degrader-1 (P22) in HTRF binding assay) [1]

PROTAC PD-1/PD-L1 degrader-1 (1–10 M; 24 hours) decreases PD-L1 expression in a dose-dependent manner by 21% and 35% at 1 M and 10 M, respectively[1].
PROTAC PD-1/PD-L1 degrader-1 (1-10 μM; 24 hours) decreases PD-L1 expression in a dose-dependent manner by 21% and 35% at 1 μM and 10 μM, respectively[1].

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- PROTAC PD-1/PD-L1 degrader-1 (P22) inhibited PD-1/PD-L1 interaction with an IC50 of 39.2 ± 5.8 nM as determined by HTRF binding assay. [1]
- In cytotoxicity assays, PROTAC PD-1/PD-L1 degrader-1 (P22) showed negligible anti-proliferative effects on human non-small cell lung cancer cell lines A549 (IC50 > 10 μM), H1299 (IC50 > 10 μM), mouse melanoma cell line B16-F10 (IC50 > 10 μM), human breast cancer cell line MDA-MB-231 (IC50 > 10 μM), and Jurkat T cells (IC50 > 10 μM), indicating that it does not directly inhibit cancer cell growth. [1]
- In a Hep3B/OS-8/hPD-L1 and CD3 T cell co-culture model, PROTAC PD-1/PD-L1 degrader-1 (P22) significantly promoted IFN-γ secretion in a dose-dependent manner. At 3.3 μM, IFN-γ secretion was increased by 215%, which was 1.69-fold more remarkable than that induced by Keytruda (127%). [1]
- Flow cytometry assay on MDA-MB-231 cells (high PD-L1 expression, 66.8% positive rate) showed that treatment with 10 μM PROTAC PD-1/PD-L1 degrader-1 (P22) for 24 h reduced cell-surface PD-L1 expression by more than 14% compared to DMSO-treated group. [1]
- Western blot analysis on MDA-MB-231 whole cell lysates demonstrated that PROTAC PD-1/PD-L1 degrader-1 (P22) reduced PD-L1 protein levels in a dose-dependent manner: 21% reduction at 1 μM and 35% reduction at 10 μM after 24 h treatment. BMS-1198 (warhead) and pomalidomide (E3 ligase ligand) did not induce PD-L1 degradation. [1]
- Mechanistic studies using protease inhibitor MG132 and lysosomal inhibitor bafilomycin (Baf) revealed that PROTAC PD-1/PD-L1 degrader-1 (P22)-mediated PD-L1 degradation was restored by bafilomycin but not by MG132, suggesting a lysosome-dependent degradation pathway. [1]

- The ability of PROTAC PD-1/PD-L1 degrader-1 (P22) to inhibit PD-1/PD-L1 interaction was evaluated using a homogeneous time-resolved fluorescence (HTRF) binding assay. The assay kit contained PD-1 and PD-L1 proteins. The experiment was performed according to the manufacturer's guidelines. The biochemical data were presented as IC50 values calculated from dose-dependent curves. [1]

- In vitro antiproliferative activity of PROTAC PD-1/PD-L1 degrader-1 (P22) was determined using the CCK-8 assay. Cell lines (A549, H1299, B16-F10, MDA-MB-231, Jurkat) were incubated at 37°C in a humidified 5% CO2 incubator for 24 h in 96-microwell plates. Then, 100 μL of culture medium with 0.1% DMSO containing different concentrations of the test compound was added to each well and incubated at 37°C for another 48 h. The optical density was detected with a microplate reader at 450 nm. IC50 values were calculated according to the dose-dependent curves. [1]
- For IFN-γ release assay, Hep3B cells engineered to stably express OS-8 (anti-CD3 single chain variable fragment) and human PD-L1 (hPD-L1) were used. Fresh PBMCs were isolated, and CD3+ T cells were isolated using a human T cell isolation kit (negative selection). Hep3B-OS8-hPDL1 cells were harvested and treated with mitomycin C at 37°C for 1.5 h and washed 4 times with PBS. Hep3B-OS8-hPDL1 and T cells (2.5×10^4 in 50 μL and 5×10^4 in 100 μL complete media, respectively) were added to 96-well plates, followed by addition of 4× final concentration of test compound in 50 μL complete media, and co-cultured at 37°C in 5% CO2 incubator for 72 h. Supernatants (150 μL) were harvested after 72 h to determine IFN-γ levels by ELISA. [1]
- For flow cytometry, cells (MDA-MB-231, A549, B16F10, H1299) were plated in six-well plates and treated with PROTAC PD-1/PD-L1 degrader-1 (P22) at indicated concentrations (10 μM for 24 h). Cells were stained for 30 min at 4°C using anti-human-PD-L1-PE antibody or isotype control. Flow cytometry was performed on a flow cytometer, and data were analyzed using software. [1]
- For western blotting, cells (MDA-MB-231) were plated in 6-well plates and treated with PROTAC PD-1/PD-L1 degrader-1 (P22) at indicated concentrations (1 μM and 10 μM for 24 h). Cells were collected, washed with cold PBS, and lysed with RIPA buffer. Protein lysates were denatured at 100°C in SDS-PAGE loading buffer for 5 min, separated by 10% SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with anti-PD-L1 antibody overnight at 4°C. Protein expression levels were normalized to GAPDH. Images were captured, and grayscale of each band was evaluated through ImageJ software. [1]

[1]. Discovery of novel resorcinol diphenyl ether-based PROTAC-like molecules as dual inhibitors and degraders of PD-L1. Eur J Med Chem. 2020;199:112377.

- PROTAC PD-1/PD-L1 degrader-1 (P22) is based on the BMS-1198 warhead and contains a rigid piperazine linker, which contributed to its high inhibitory activity (IC50 = 39.2 nM) compared to compounds with flexible/straight linkers. [1]
- The degradation of PD-L1 by PROTAC PD-1/PD-L1 degrader-1 (P22) was found to be lysosome-dependent rather than proteasome-dependent, as evidenced by restoration of PD-L1 levels upon co-treatment with bafilomycin (lysosomal inhibitor) but not with MG132 (proteasome inhibitor). This mechanism is similar to that reported for SA-49 and ENDTACs. [1]
- The authors suggest that PROTAC PD-1/PD-L1 degrader-1 (P22) may serve as a starting point for exploring the degradation of PD-L1 by PROTAC-like strategy, though it showed only modest degradation efficiencies (35% reduction at 10 μM). [1]

C59H58CLN7O11

1076.59

1075.388

C, 65.82; H, 5.43; Cl, 3.29; N, 9.11; O, 16.35

2447066-37-5

2447066-37-5

146673162

White to off-white solid powder

6.3

2

13

16

78

2220

0

CC1=C(C=CC=C1C2=CC3=C(C=C2)OCCO3)COC4=C(C=C(C(=C4)OCC5=CC(=CC=C5)C#N)CN6CCCCC6C(=O)N7CCN(CC7)C(=O)CCCC(=O)NC8=CC=CC9=C8C(=O)N(C9=O)C1CCC(=O)NC1=O)Cl

CJIXMPCTSMEQPG-UHFFFAOYSA-N

InChI=1S/C59H58ClN7O11/c1-36-40(10-5-11-42(36)39-17-19-48-51(30-39)76-27-26-75-48)35-78-50-31-49(77-34-38-9-4-8-37(28-38)32-61)41(29-44(50)60)33-66-21-3-2-14-47(66)58(73)65-24-22-64(23-25-65)54(70)16-7-15-52(68)62-45-13-6-12-43-55(45)59(74)67(57(43)72)46-18-20-53(69)63-56(46)71/h4-6,8-13,17,19,28-31,46-47H,2-3,7,14-16,18,20-27,33-35H2,1H3,(H,62,68)(H,63,69,71)

5-[4-[1-[[5-chloro-2-[(3-cyanophenyl)methoxy]-4-[[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy]phenyl]methyl]piperidine-2-carbonyl]piperazin-1-yl]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]-5-oxopentanamide

PD-L1 degrader P22; CUN-01077; CUN 01077; CUN01077

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~210 mg/mL (~195.1 mM)

Solubility in Formulation 1: ≥ 1.4 mg/mL (1.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.4 mg/mL (1.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)