PD-1/ PD-L1; VISTA

In addition to demonstrating selectivity against other immune checkpoint proteins and a wide range of receptors and enzymes, CA-170 has strong functional activity in restoring T-cell proliferation and effector functions[1].

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CA-170 Does Not Bind to hPD-L1 According to the NMR Binding Assay.[2]
CA-170 Fails to Restore the Activation of hPD-1/hPD-L1-Blocked Effector Jurkat T Cells.[2]

NMR Binding Assay[2]
For NMR measurements, the buffer was exchanged by gel filtration to PBS pH 7.4. 10% (v/v) of D2O was added to the samples to provide the lock signal. All spectra were recorded at 300 K using a Bruker Avance III 600 MHz spectrometer. Binding of the compounds was analyzed by titrating the 15N-labeled hPD-L1/hPD-1 and recording the 1H and 1H−15N HMQC spectra prior to and after the addition of the compounds.

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Homogenous Time Resolved FRET[2]
HTRF assay was performed using the certified Cis-Bio assay kit at 20 µL final volume using their standard protocol (5 nM of h-L1 and 50 nM of hPD-1 in the final formulation). To determine the half maximal inhibitory concentration (IC50) of tested compounds, measurements were performed on individual dilution series. After mixing all components according to Cis-Bio protocol, the plate was left for 2h incubation at room temperature followed by TR-FRET measurement on Tecan Spark 20M. Collected data was background subtracted on the negative control, normalized on the positive control, averaged and fitted with normalized Hill’s equation to determine the IC50 value using Mathematica 12. Reference: Molecules. 2019 Aug 1;24(15):2804.

hPD-1/hPD-L1 Immune Checkpoint Blockade Assay[2]
The activity of the inhibitors of hPD-1/hPD-L1 immune checkpoint was examined using the hPD-1/hPD-L1 Blockade Bioassay, according to the manufacturer’s instructions. hPD-L1 aAPCs were seeded on 96-well (white) plates at the density 10 000 cells/well 17h prior to the experiment. The 2.5-fold dilutions of the small molecules or peptide-57 were first prepared in DMSO. On the day of the assay the compounds were diluted 1000-fold in the assay buffer (99% RPMI 1640, 1% FBS) to maintain the constant concentration of DMSO (0.1% of total volume). The 2.5-fold dilutions of nivolumab, a positive control anti-hPD-1 monoclonal antibody, were prepared in the assay buffer on the day of the assay. The culture medium was discarded from the wells and serial dilutions of either the small-molecule or antibody was added. Afterwards, Jurkat hPD-1 cells were seeded at the density of 20,000 cells per well in the assay’s plates. After 6h of the incubation in standard culture conditions, assay plates were equilibrated at ambient temperature for 10min, followed by a 20min incubation with the Bio-GloTM Assay reagent. The luminescence was detected using the Infinite M200 reader. Half maximal effective concentrations (EC50 values) were calculated from the Hill’s curve fitting to the experimental data.

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Western Blot Analysis[2]
Total cell lysates were prepared with RIPA buffer containing protease inhibitor cocktail. Following the electrophoresis and transfer to PVDF membranes, the membranes were blocked with 4% BSA in TBS-N buffer and incubated with primary antibody solution at 4 °C overnight. After that, four washes with TBS-N buffer, the incubation with secondary antibody for 1h at room temperature, and additional four washes were done.
The following antibodies and dilutions were used: rabbit monoclonal anti-hPD-L1 (clone E1L3N, 1:1 000), rabbit monoclonal anti-α-Tubulin (1:2000, CST, cat. 2125), goat peroxidase-conjugated anti-rabbit (1:3000).

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Flow Cytometry Analysis[2]
For the analysis of the expression of human PD-L1 on the surface of aAPC cells flow cytometry analysis was performed. The cells were seeded on 6-well plates and cultured till sub-confluency. The cells were detached from the plates with TrypLe Select Enzyme, placed on ice, washed 2 times with Flow Cytometry Staining Buffer and stained with mouse anti-human PD-L1 antibody (clone MIH1, conjugated with APC, cat. 17-5983). 17-4714 antibody was used as an isotype control of the staining. The cells were analyzed with the FACSCanto II cytometer.

[1]. 1,2,4-oxadiazole derivatives as immunomodulators. WO2015033299A1.

CA-170 is a selective, small molecule inhibitor of PD-L1.
PD-L1/PD-L2/VISTA Antagonist CA-170 is an orally bioavailable small molecule inhibitor of the immune checkpoint regulatory proteins programmed cell death ligand-1 (PD-L1; B7-H1; CD274), PD-L2, and V-domain immunoglobulin (Ig) suppressor of T-cell activation (VISTA; programmed death 1 homolog; PD1H; PD-1H), with potential negative immune checkpoint regulatory and antineoplastic activities. Upon oral administration, PD-L1/PD-L2/VISTA antagonist CA-170 targets and binds to PD-L1, PD-L2 and VISTA. This inhibits PD-L1/PD-L2/VISTA-mediated signaling, abrogates the PD-L1-, PD-L2- and VISTA-induced suppression of T-lymphocyte immune responses, enhances cytotoxic T-cell proliferation and activation against tumor cells, increases cytokine production by T-cells, and inhibits tumor cell growth. PD-L1, PD-L2 and VISTA, negative checkpoint molecules of immune activation, play key roles in the suppression of T-cell functions.

C12H20N6O7

360.3232

360.139

C, 40.00; H, 5.59; N, 23.32; O, 31.08

1673534-76-3

1673560-66-1; 1673534-76-3 (PD-1-IN-1)

126843231

Light yellow to yellow solid powder

-6.7

7

10

9

25

493

4

O1C([C@]([H])(C([H])([H])C(N([H])[H])=O)N([H])C(N([H])[C@]([H])(C(=O)O[H])[C@@]([H])(C([H])([H])[H])O[H])=O)=NC([C@]([H])(C([H])([H])O[H])N([H])[H])=N1

HFOBENSCBRZVSP-LKXGYXEUSA-N

InChI=1S/C12H20N6O7/c1-4(20)8(11(22)23)16-12(24)15-6(2-7(14)21)10-17-9(18-25-10)5(13)3-19/h4-6,8,19-20H,2-3,13H2,1H3,(H2,14,21)(H,22,23)(H2,15,16,24)/t4-,5+,6+,8+/m1/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 3: ≥ 0.5 mg/mL (1.39 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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