IDO1 (IC50 = 38 nM)

Strong IDO1 inhibitor IDO-IN-7 (analogue of NLG-919) has an IC50 of 38 nM. IDO-IN-7's binding mode to IDO1 is accessible through experimentation and demonstrates a direct coordination interaction with the ferric heme's sixth coordination site. In previous research, IDO-IN-7 was utilized as a reference drug to build immunostimulatory nanomicellar carriers and verify high-throughput screening assays for IDO1 inhibition[1].

In mice, a single oral administration of NLG919 reduces the concentration of plasma and tissue Kyn by ~ 50%. In mice bearing B16F10 tumors, NLG919 markedly enhances the antitumor responses of naive, resting pmel-1 cells to vaccination with cognate hgp100 peptide plus CpG-1826 in IFA.

Microscale thermophoresis (MST)[1]
Thermophoresis is the movement of a biomolecular complex in a temperature gradient depending on size, charge, and hydration shell that typically change upon ligand/target interaction. The MST experiment is based on the use of 16 capillary tubes that are filled with a fluorescent dye-labeled target protein and a serial titration of unlabeled ligand. Capillary tubes are then illuminated with an infrared laser that generates a temperature gradient. The protein/ligand complex migrates along this gradient causing changes in the observed fluorescence. These are used to generate a binding curve as a function of ligand concentration that is then analyzed to assess the Kd value.
Fluorescence labeling of rhIDO1 was performed following the protocol for N-hydroxysuccinimide (NHS) coupling of the dye NT647 to lysine residues. Briefly, 100 μL of a 9.95 μM solution of rhIDO1 protein in labeling buffer (130 mM NaHCO3, 50 mM NaCl, pH 8.2) was mixed with 100 μL of 39.8 μM NT647-NHS fluorophore in labeling buffer and incubated for 30 min at room temperature (RT) in the dark. Unbounded fluorophores were removed by size-exclusion chromatography with MST buffer (50 mM TRIS, 150 mM NaCl, 10 mM MgCl2, pH 7.4, 0.05% Tween20) as running buffer. The real concentration of each element of the sample, such as protein, heme group and RED dye, and the degree of labeling (DOL) were determined using extinction coefficient ɛ280 = 51,380 M−1 cm−1 for rhIDO1, ɛ405 = 159,000 M−1 cm−1 for rhIDO1 heme group and ɛ650 = 250,000 M−1 cm−1 for NT647 fluorophore, with a correction factor of Fcorr of 0.028 at 280 nm, using Cprot = [A280 – (A280 x Fcorr)/ɛ280 x l] and DOL resulted between 0.6 and 0.8 throughout all labeling reactions.
The stability of NT647-rhIDO1 and unmodified rhIDO1 protein was checked using circular dicroism. Spectra of both proteins were recorded using Jasco810 spectrophotometer with 1 mm path-length quartz cuvettes at room temperature (≈22 °C). Sensitivity was 100 millidegrees, and the scanning speed was 20 nm/min for an accumulation of 2 scans. CD data were collected between 180 and 260 nm for both samples at a concentration of 0.1 mg/ml in phosphate buffer (PPB; 50 mM K2HPO4, pH 7.4) Deconvolution of spectra was performed with CDNN 2.1 software. Results are reported in the supplementary materials (Table S1).
Compound screening was carried out using premium-coated capillary and MST buffer including 2% DMSO and 2 mM DTT. Compound stocks (50 mM) in DMSO were diluted in assay buffer to reach a final maximum concentration of 500 μM or 1 mM, depending on compound solubility. Compound pre-dilutions were prepared for MST experiments by 16-fold 1:1 serial dilutions in assay buffer containing 4% DMSO in PCR tubes (supplied by NanoTemper Technologies) to yield final volumes of 10 μL. A solution of NT647-rhIDO1 at a concentration of 90 nM was prepared and 10 μL of this solution was added to each compound dilution to reach a final NT647-rhIDO1 concentration of 45 nM and a reaction volume of 20 μL. These samples were loaded into 16 premium-coated capillary tubes and inserted in the chip tray of the MST instrument (Monolith NT.115) for thermophoresis analysis and the appraisal of Kd values. MST signals were recorded at MST 40% (compounds 7, 9, 10, 23, 28). Compounds not providing a binding curve with a good signal/noise ratio at 40% (8, 11–22, 24–27, 29) were tested at MST 80%. In both cases, a 20% LED power was used. Kd values were calculated from compound concentration-dependent changes in normalized fluorescence (Fnorm) of NT647-rhIDO1 after 21s of thermophoresis at MST 40% and after 4s at MST 80%. Each compound was tested in triplicate samples and data analyzed using MO Affinity Analysis software (NanoTemper Technologies). Confidence values (±) are indicated next to Kd value for each of tested compound. Specifically, confidence values define the range where the Kd falls with a 68% of certainty. The binding efficiency index (BEI) of each fragment was calculated with the following equations: (eq. 1) BEI = pKd/MW.

Cellular assay[1]
P1.HTR, a highly transfectable clonal variant of mouse mastocytoma P815 was cultured in Iscove's Modified Dulbecco's Medium supplemented with 10% FCS. P1.HTR cells were transfected by electroporation with plasmid constructs coding for murine IDO1 (P1.IDO1). A stable transfectant cell line was obtained by puromycin selection. Cells at the concentration of 0.1 × 106 cell/ml were incubated with 30 μM of compounds for 16 h. Control was represented by cells incubated with an equivalent volume of DMSO (the vehicle in which compounds were solubilized). After the incubation, supernatants of cell cultures were recovered and kynurenine concentration was detected by HPLC. Dose-response curves were built through the same cellular assay, incubating P1.IDO1 cells with serial dilutions of molecules, starting from 30 μM. All the experiments were conducted in triplicate and repeated almost two times. Results are represented as the mean ± standard deviation of the kynurenine fold change (l-Kyn FC), meaning the ratio between kynurenine concentration secreted in the supernatant of the compound-treated versus vehicle-treated cells.

In vivo DOX treatment and tumor measurement[2]
To compare the inhibition of tumor growth by DOX, mouse breast tumor model was established by injecting normal 4T1 cells as above described, and DOX was injected into them at the dose of 5.0 mg/kg for 5 times at intervals of 3 days each, when the tumor volume reach about 50 mm3. Tumor volume was measured after every 4 days using an electronic caliper, and the mean tumor volumes (mm3) of the DOX and control groups were used to plot tumor growth curves.

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In vivo application of DOX and NLG919 combination[2]
BALB/c mice bearing 4T1 tumors of ~50 mm3 volume were administered vehicle control, DOX, NLG919, or a combination of DOX and NLG919 after every 3 days for 5 times; the administration dosage of DOX and NLG919 were 5.0 mg/kg (i.v.) and 20 mg/kg (orally), respectively. The tumor volume in each mouse group was measured as described earlier after every 3 days up to 21 days. The Kyn (nM)/Trp (µM) ratios in the tumor homogenates and plasma of mice in each group were assessed. Then, the tumor tissues were subjected to the hematoxylin and eosin stain (H&E) and proliferating cell nuclear antigen (PCNA) staining and analyzed by IHC.

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Dissolved in water at 3 mg/mL; 6 mg/day injected via IP, or administered subcutaneously at 1 mg/dose twice a day via injection plus 360 μg/day via an SC osmotic pump.

Mice bearing large established B16F10 tumor

[1]. Fragment-based approach to identify IDO1 inhibitor building blocks. Eur J Med Chem. 2017 Dec 1;141:169-177.

Indoleamine 2,3-dioxygenase 1 (IDO1) is attracting a great deal of interest as drug target in immune-oncology being highly expressed in cancer cells and participating to the tumor immune-editing process. Although several classes of IDO1 inhibitors have been reported in literature and patent applications, only few compounds have proved optimal pharmacological profile in preclinical studies to be advanced in clinical trials. Accordingly, the quest for novel structural classes of IDO1 inhibitors is still open. In this paper, we report a fragment-based screening campaign that combines Water-LOGSY NMR experiments and microscale thermophoresis approach to identify fragments that may be helpful for the development of novel IDO1 inhibitors as therapeutic agents in immune-oncology disorders.[1]

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Purpose: Breast cancer has become a major public health threat in the current society. Anthracycline doxorubicin (DOX) is a widely used drug in breast cancer chemotherapy. We aimed to investigate the immunogenic death of breast tumor cells caused by DOX, and detect the effects of combination of DOX and a small molecule inhibitor in tumor engrafted mouse model.[2]
Methods: We used 4T1 breast cancer cells to examine the anthracycline DOX-mediated immunogenic death of breast tumor cells by assessing the calreticulin exposure and adenosine triphosphate and high mobility group box 1 release. Using 4T1 tumor cell-engrafted mouse model, we also detected the expression of indoleamine 2,3-dioxygenase (IDO) in tumor tissues after DOX treatment and further explored whether the specific small molecule IDO1 inhibitor NLG919 combined with DOX, can exhibit better therapeutic effects on breast cancer. [2]
Results: DOX induced immunogenic cell death of murine breast cancer cells 4T1 as well as the upregulation of IDO1. We also found that treatment with NLG919 enhanced kynurenine inhibition in a dose-dependent manner. IDO1 inhibition reversed CD8+ T cell suppression mediated by IDO-expressing 4T1 murine breast cancer cells. Compared to the single agent or control, combination of DOX and NLG919 significantly inhibited the tumor growth, indicating that the 2 drugs exhibit synergistic effect. The combination therapy also increased the expression of transforming growth factor-β, while lowering the expressions of interleukin-12p70 and interferon-γ. [2]
Conclusion: Compared to single agent therapy, combination of NLG919 with DOX demonstrated better therapeutic effects in 4T1 murine breast tumor model. IDO inhibition by NLG919 enhanced the therapeutic efficacy of DOX in breast cancer, achieving synergistic effect.[2]

C18H22N2O

282.38

282.173

C, 76.56; H, 7.85; N, 9.92; O, 5.67

1402836-58-1

66558287

White to khaki solid powder

1.3±0.1 g/cm3

524.6±33.0 °C at 760 mmHg

271.1±25.4 °C

0.0±1.4 mmHg at 25°C

1.676

3.28

1

2

3

21

355

0

YTRRAUACYORZLX-UHFFFAOYSA-N

InChI=1S/C18H22N2O/c21-18(13-6-2-1-3-7-13)10-16-14-8-4-5-9-15(14)17-11-19-12-20(16)17/h4-5,8-9,11-13,16,18,21H,1-3,6-7,10H2

1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol

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 (8.85 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 25.0 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: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear EtOH 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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Solubility in Formulation 6: ≥ 2.5 mg/mL (8.85 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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