PD-1/PD-L1 PPI; PD-L1 protein
BMS-8 targets the PD-1/PD-L1 immune checkpoint interaction (IC₅₀ = 7.2 μM for inhibiting PD-1/PD-L1 binding) [1]
BMS-8 acts on PD-L1 to induce its homodimerization, thereby interfering with PD-1/PD-L1 interaction [1]
BMS-8 binds to the cavity formed by two PD-L1 monomers, interacting with key residues including Ile54, Tyr56, Met115, Ala121, and Tyr123 on PD-L1 [2]

One of the PD-L1 monomers tends to have a more stable binding mode with BMS-8 than the other, and the small-molecule inducing PD-L1 dimerization was further stabilized by the non-polar interaction of Ile54, Tyr56, Met115, Ala121, and Tyr123 on both monomers and the water bridges involved in ALys124[2].

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BMS-8 inhibited the PD-1/PD-L1 interaction with an IC₅₀ value of 7.2 μM as determined by the AlphaLISA® assay [1]
- Molecular dynamics simulations showed that BMS-8 had a more stable binding mode with one PD-L1 monomer (conformation A) than the other (conformation B) in the PD-L1 dimer system; the core scaffold of BMS-8 maintained relatively stable RMSD values during simulation [2]
- Residue energy decomposition analysis revealed that BMS-8 interacted with PD-L1 dimer mainly through non-polar interactions with residues Ile54, Tyr56, Met115, Ala121, and Tyr123 on both PD-L1 monomers, and water bridges involving Lys124ₐ also stabilized the binding of BMS-8 to PD-L1 dimer [2]
- Metadynamics simulation indicated that the PD-L1 dimerization remained stable after the dissociation of BMS-8, suggesting that BMS-8-induced PD-L1 dimer formation is a key factor for its inhibitory activity [2]

In Vivo Antitumor Activity of NP19 [[an analog of BMS-8]] in an H22 Hepatoma Mouse Model[3]
Encouraged by the excellent in vivo antitumor efficacy of NP19 on the melanoma B16-F10 tumor model, and the fact that PD-1/PD-L1 inhibitors have broad spectrum of antitumor activities, we further evaluated the in vivo antitumor efficacy of compound NP19 using an H22 hepatoma tumor model in BALB/c mice. Each mouse was injected with 0.8 million H22 cells subcutaneously into the right flank. After tumors reached approximately 100 mm3 in volume, mice were randomized and treated by intraperitoneal (i.p.) injection of NP19 or a vehicle solution for 14 days. As shown in Figure 8, NP19 demonstrated significant in vivo antitumor efficacy with a TGI of 76.5% at the dose of 25 mg/kg (Figure 8A, 8B, 8C). In addition, NP19 did not cause an obvious body weight loss (Figure 8D), indicating that the compound was well tolerated.

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In Vivo Antitumor Activity of NP19 [an analog of BMS-8] in a B16-F10 Mouse Melanoma Model[3]
To determine whether the in vitro anti-PD-1/PD-L1 activity of the newly synthesized compounds can be translated into in vivo efficacy, we tested the antitumor activity of compound NP19 on a mice melanoma B16–F10 tumor model. NP19 was chosen for the in vivo efficacy study due to the ease of synthesis and less cytotoxicity (Table 9) when compared to the more potent compound NP2 or equally potent compound NP12. We treated BALB/c mice bearing melanoma tumors with vehicle control and NP19 (25 mg/kg, 50 mg/kg, 100 mg/kg) administered via intragastric gavage once a day for 15 days. As shown in Figure 6, after 15 days of treatment, the growth of melanoma tumors was inhibited dramatically following NP19 treatment.

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In Vivo Pharmacokinetic Properties of NP19 [an analog of BMS-8][3]
As compound NP19 showed high potency in vitro, the pharmacokinetic (PK) profiles were next evaluated in male Sprague–Dawley rats by intravenous and oral administration. The key p.o. and i.v. administration PK parameters are summarized in Table 8. After a single i.v. administration with 1 mg/kg compound NP19, the half time (t1/2), the clearance rate (CL), and the apparent distribution volume (Vss) of NP19 are 1.5 ± 0.5 h, 0.9 ± 0.2 L/h/kg, and 2.1 ± 0.5 L/kg, respectively. When NP19 was administrated by the oral route at 10 mg/kg, the oral absorption (Tmax = 0.6 ± 0.2 h), long half-life (t1/2 = 10.9 ± 7.7 h), and oral bioavailability (F = 5%) were observed. In addition, no apparent adverse effects were observed in rats. NP19 showed a much longer half-life (10.9 h) following oral gavage as compared to the i.v. half-life (1.5 h); this may be due to the high lipophilicity (logP = 7.9) or poor aqueous solubility of NP19. As a result, NP19 exhibited flip-flop pharmacokinetics. Such flip-flop pharmacokinetics can sometimes occur for poorly water-soluble compounds such as Rebamipide, which has a t1/2 (p.o.)/t1/2 (i.v.) ratio of 13.5 due to poor water solubility (7.6 μg/mL). Another example is the lipophilic compound IAT (an antitubulin agent with 19 μg/mL of water solubility) reported by Chien-ming Li et al., which has a t1/2 (p.o.)/t1/2 (i.v.) ratio of ∼5, similar to NP19 [t1/2 (p.o.)/t1/2 (i.v.) = 7.1]. Due to the low oral bioavailability of the compound NP19, we presumed that a high dosage is needed to offer sufficient drug concentration to exhibit antitumor efficacy. Therefore, we further studied the in vivo activity of compound NP19.

All binding studies are performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (with v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-l-Ig/PD-Ll-His binding assay, inhibitors are pre-incubated with PD-Ll-His (10 nM final) for 15 m in 4 μL of assay buffer, followed by addition of PD-l-Ig (20 nM final) in 1 μL of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse are used. HTRF detection is achieved using europium crypate-labeled anti- Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies are diluted in HTRF detection buffer and 5 μL is dispensed on top of binding reaction. The reaction mixture is allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) is obtained using an En Vision fluorometer. Additional binding assays are established between PD-1-Ig/PD-L2-His (20, 5 nM, respectively), CD80-His/PD-Ll-Ig (100, 10 nM, respectively) and CD80-His/CTLA4-Ig (10, 5 nM, respectively).

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AlphaLISA® assay was used to measure the inhibitory effect of BMS-8 on PD-1/PD-L1 interaction: The assay system was established to detect the binding between PD-1 and PD-L1, and different concentrations of BMS-8 were added to the reaction system; the luminescence signal generated by the AlphaLISA® technology was detected, and the IC₅₀ value of BMS-8 for inhibiting PD-1/PD-L1 interaction was calculated based on the dose-response curve [1]
- Molecular docking simulation was performed to predict the binding mode of BMS-8 to PD-L1 dimer: The crystal structure of PD-L1 dimer (PDB ID:5J8O) was used as the receptor model, and the three-dimensional structure of BMS-8 was constructed and optimized; the docking process was carried out to obtain the binding conformation of BMS-8 in the PD-L1 dimer cavity, and the interaction between BMS-8 and key residues of PD-L1 was analyzed [2]
- Conventional molecular dynamics simulation was conducted on the BMS-8-PD-L1 dimer complex: The simulation system was built with the complex as the core, and the system was solvated and ionized; the simulation was run under specific force field conditions, and the RMSD, RMSF and other parameters of the complex, PD-L1 monomers and BMS-8 were calculated to evaluate the stability of the binding system [2]
- Metadynamics simulation was applied to study the unbinding process of BMS-8 from PD-L1 dimer: Collective variables (CV1 and CV2) were defined to describe the unbinding process, and the free energy landscape of the unbinding process was obtained; the conformational changes of BMS-8 and PD-L1 dimer during the unbinding process were analyzed by monitoring the distance between key residues of PD-L1 [2]

Particularly in a tumor microenvironment where lysis of tumor cells is a concern, the interaction between programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) plays a dominant role in the suppression of T cell responses. With an IC50 value of 18 nM, PD-1/PD-L1 inhibitor 2 is said to stop PD-L1 from interacting with PD-1.

Pharmacokinetic Study in Male Sprague–Dawley Rats[3]
Male Sprague–Dawley rats (200–220 g) were used to study the pharmacokinetics of compound NP19 [an analog of BMS-8]. Diet was prohibited for 12 h before the experiment, but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral (10 mg/kg) or intravenous (1 mg/kg) administration of compound NP19. The compound was dissolved in 5% DMSO and 95% PEG-300 for intravenous administration or suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for oral administration. The samples were immediately centrifuged at 3000g for 10 min. The plasma as-obtained (100 μL) was stored at −20 °C until analysis. PK parameters were determined from individual animal data using noncompartmental analysis in DAS (Drug and statistics) software. Instruments and analytical conditions for PK studies: A UPLC-MS/MS system with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) interface, was used to analyze the blood samples. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). Masslynx 4.1 software was used for data acquisition and instrument control. Multiple reaction monitoring (MRM) modes of m/z 555.35 → 181.03 for NP19 and m/z 237 → 194.1 for carbamazepine were utilized to conduct quantitative analysis.

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In Vivo Efficacy Study in Mouse B16F10 Melanoma Model[3]
BALB/c mice, aged 6–8 weeks old, were used to study the inhibition effect of NP19 [an analog of BMS-8]on subcutaneous transplanted model of melanoma cells. Murine B16F10 melanoma cells growing in a logarithmic growth phase were suspended in PBS at a density of 2 × 106 per mL. Each mouse was inoculated subcutaneously with 200 μL containing 4 × 105 cells. After tumors reached approximately 100 mm3 in volume, mice were divided into four groups randomly (n = 10) and treated with NP19 (25, 50, 100 mg/kg) and vehicle, respectively. The drugs were administered via intragastric gavage once a day for 15 days. The vehicle group was administered with 0.5% sodium carboxymethyl cellulose (CMC-Na). Animal activity and body weight were monitored during the entire experiment period to assess acute toxicity. Mice were sacrificed 16 days after the initiation of the treatment, and the tumor tissue and major organ (liver, spleen, thymus, and kidney) samples were collected. The harvested tumor tissue and organs (liver, kidney) were fixed in 4% paraformaldehyde, processed into paraffin routinely, stained with hematoxylin and eosin (H&E), and captured by microscope. Tumor growth inhibition value (TGI) was calculated using the formula: TGI(%) = [1 – Wt/Wv] × 100%, where Wt and Wv are the mean tumor weight of treatment group and vehicle control.

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In Vivo Efficacy Study in Mouse H22 Hepatoma Tumor Model[3]
6–8 weeks old male BALB/c mice were used. A total of 8 × 105 H22 cells were inoculated into the right flank of each mouse according to protocols of tumor transplant research. NP19 [an analog of BMS-8]was dissolved in 5% DMSO, 40% PEG-200 and 55% saline solution to produce desired concentrations. Mice in control groups were injected intraperitoneally with 200 μL of vehicle solution only. Tumor volume was measured every 2 days with a traceable electronic digital caliper and calculated using the formula a × b2 × 0.5, where a and b represented the larger and smaller diameters, respectively. The mice were sacrificed after the treatments and tumors were excised and weighed.

[1]. Preparation of Biphenyl-Conjugated Bromotyrosine for Inhibition of PD-1/PD-L1 Immune Checkpoint Interactions. Int J Mol Sci. 2020 May 21;21(10):3639.

[2]. Computational Insight Into the Small Molecule Intervening PD-L1 Dimerization and the Potential Structure-Activity Relationship. Front Chem. 2019 Nov 12;7:764.

[3]. Discovery of Novel Resorcinol Dibenzyl Ethers Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction as Potential Anticancer Agents. J Med Chem. 2020 Aug 13;63(15):8338-8358.

Recently reports have indicated that small molecule compounds can block the PD-1/PD-L1 interaction by inducing PD-L1 dimerization. All these inhibitors share a common skeletal structure and interact with a cavity formed by two PD-L1 monomers. This unique interaction pattern provides clues for structure-based drug design but also exposes its limitations in discovering small molecule inhibitors with novel skeletal structures. This study uses conventional molecular dynamics and meta-dynamics simulations to predict the binding and dissociation mechanisms of existing small molecule inhibitors targeting PD-L1 dimerization, thus revealing their structure-activity relationships. During binding, representative inhibitors (BMS-8 and BMS-1166) show a more stable binding mode with one of the PD-L1 monomers than with the other. The small molecules inducing PD-L1 dimerization are further stabilized by nonpolar interactions of Ile54, Tyr56, Met115, Ala121, and Tyr123 on both monomers, as well as by a water bridge involving Alys124. The dissociation process prediction showed that PD-L1 dimerization remained stable after ligand dissociation. This indicates that the formation and stability of small molecules that induce PD-L1 dimerization are key factors for these ligands to exert inhibitory activity. Contact analysis, quantitative structure-activity relationship (QSAR) analysis based on R groups and molecular docking further showed that each linker on the core backbone of the ligand has a specific preference for pharmacophore elements when improving inhibitory activity through structural modification. In summary, the results of this study can guide the structural optimization and further discovery of novel small molecule inhibitors targeting PD-L1. [2]

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The development of monoclonal antibodies (mAbs) has revolutionized cancer immunotherapy, which can inhibit the interaction between immune checkpoint molecules (such as programmed death receptor 1 (PD-1)) and their ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, while small molecule drugs are expected to overcome these drawbacks. BMS-8 is an effective small molecule drug that induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our detection system showed that BMS-8 could inhibit PD-1/PD-L1 interaction with an IC50 value of 7.2 μM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 through computer simulation. As a result, we successfully prepared a biphenyl-coupled bromotyrosine (X) with an IC50 value of 1.5 μM, which was about five times higher than that of BMS-8. We further prepared an amino acid conjugate of X (amino-X) to elucidate the correlation between the docking mode of amino-X and the IC50 value. The results showed that the degree to which amino-X detached from BMS-8 in the PD-L1 homodimer pocket was related to the IC50 value. This observation provides us with insights into how to further understand how to derivatize compound X to obtain better inhibitory effects. [1]

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This study describes a method for modifying gold electrodes with the compound BMS-8, which can interact with the immune checkpoint protein programmed death ligand 1 (PD-L1). The results showed that we confirmed the presence of sPD-L1 at concentrations ranging from 10⁻¹⁸ to 10⁻⁸ M using electrochemical impedance spectroscopy (EIS), with a limit of detection (LOD) of 1.87 × 10⁻¹⁴ M (S/N = 3.3). Furthermore, we detected sPD-L1 at a concentration of 10⁻¹⁴ M using cyclic voltammetry (CV). We also validated the functionalization of the electrode using high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements. We investigated the selectivity of this electrode for other proteins: programmed death receptor 1 (PD-1), differentiation cluster 160 (CD160), and B-cell and T-cell attenuation factor (BTLA) at a concentration of 10⁻⁸ M. Distinguishing between PD-L1 and PD-1 was achieved by analyzing the frequency dispersion of the capacitance effect on the modified gold electrode surface. The concentrations of PD-L1 and PD-1 ranged from 10⁻¹⁸ to 10⁻⁸ M. Significant differences in heterogeneity were observed between PD-L1 and PD-1. Pseudocapacitance studies indicated a strong and specific interaction between BMS-8 and the PD-L1 protein. https://pubmed.ncbi.nlm.nih.gov/33517203/

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BMS-8 is a potent small molecule drug that can inhibit the interaction between PD-1 and PD-L1 immune checkpoints, overcoming the disadvantages of monoclonal antibody drugs such as poor tumor penetration and high production costs[1]
- BMS-8 induces PD-L1 homodimerization, which is the main mechanism by which it inhibits the binding of PD-1/PD-L1[1]
- BMS-8 structural modifications can refer to the structure-activity relationship revealed by quantitative structure-activity relationship (QSAR) analysis based on R groups; BMS-8 each connection point on the core backbone has a specific preference for pharmacophore elements when improving inhibitory activity[2]

C27H28BRNO3

494.43

493.125

C, 65.59; H, 5.71; Br, 16.16; N, 2.83; O, 9.71

1675201-90-7

117941742

White to off-white solid powder

1.3±0.1 g/cm3

616.9±55.0 °C at 760 mmHg

326.9±31.5 °C

0.0±1.9 mmHg at 25°C

1.620

6.28

1

4

7

32

596

0

BrC1=C(C=CC(=C1)CN1CCCCC1C(=O)O)OCC1C=CC=C(C2C=CC=CC=2)C=1C

QRXBPPWUGITQLE-UHFFFAOYSA-N

InChI=1S/C27H28BrNO3/c1-19-22(10-7-11-23(19)21-8-3-2-4-9-21)18-32-26-14-13-20(16-24(26)28)17-29-15-6-5-12-25(29)27(30)31/h2-4,7-11,13-14,16,25H,5-6,12,15,17-18H2,1H3,(H,30,31)

1-[[3-bromo-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl]methyl]piperidine-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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