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1G244 is a novel and potent DPP8/9 inhibitor (IC50s = 12 nM and 84 nM, respectively) with anticancer activity. It does not inhibit DPPIV and DPPII.
| Targets |
DPP8/9 (dipeptidyl peptidases 8 and 9) [1]
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| ln Vitro |
In five different multiple myeloma cell lines, 1G244 (0-100 μM; 72 hours; Delta47, U266, KMS-5, RPMI8226, or MM.1 S cells) formulations were found to lower viable cell counts in a desirable way [1]. 50 μM; 0-48 hours; MM.1 S cells) stimulates cells due to the detection of indoor forms of PARP and caspase-3 [1].
1G244 dose-dependently decreased the viable cell number of five multiple myeloma cell lines (Delta47, U266, KMS-5, RPMI8226, and MM.1S) at concentrations of 0-100 μM for 72 hours. Almost complete cell death of all cell lines was observed at 100 μM. However, 100 μM of 1G244 induced nonspecific cell death, so levels below 50 μM were used in subsequent experiments. MM.1S was the most susceptible among the five cell lines. [1] 1G244 also decreased viable cell number of three T-cell lymphoma cell lines (Karpas 299, H9, and HUT102) (Supplementary Fig. 1B). [1] In CD138-positive myeloma cells isolated from five patients, incubation with 50 μM 1G244 for 24 or 48 hours resulted in 91-97% of cells being regarded as non-viable. This included patient 2 whose myeloma cells had a deletion of chromosome 17p and were resistant to various chemotherapeutic and biologic agents such as bortezomib, lenalidomide, dexamethasone, cyclophosphamide, and doxorubicin. [1] Combination treatment: In MM.1S cells, 0.5 μM 1G244 alone displayed no cytotoxicity, but in combination with 20 nM bortezomib, the number of viable cells decreased significantly compared to bortezomib alone. Similar synergistic effects were observed in KMS-5 cells. [1] Mechanism: Western blot analysis showed that treatment of MM.1S cells with 50 μM 1G244 for 0-48 hours induced the cleaved (activated) forms of caspase-3 and PARP, indicating apoptosis. The pan-caspase inhibitor Z-VAD-FMK (100 μM) suppressed 1G244-induced cell death in MM.1S and KMS-5 cells. [1] |
| ln Vivo |
In a mouse xenograft tumor model, 1G244 (30 mg/kg; subcutaneous injection; once weekly; for 3 weeks; NOG female mice) effectively reduced the subcutaneous growth of MM.1S cells [1].
In a murine xenograft model, NOG mice were subcutaneously inoculated with 5 × 10^6 MM.1S cells. Three days after inoculation, 1G244 was administered subcutaneously at 30 mg/kg once a week. This treatment apparently suppressed the subcutaneous growth of MM.1S cells with no other obvious clinical symptoms in mice. [1] |
| Cell Assay |
Cell Viability Assay[1]
Cell Types: Delta47, U266, KMS-5, RPMI8226 or MM.1 S Cell Tested Concentrations: 0 μM, 1 μM, 5 μM, 10 μM, 50 μM or 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: There was a dose-dependent decrease in viable cell numbers in five multiple myeloma cell lines. Western Blot Analysis [1] Cell Types: MM.1 S cell Tested Concentrations: 50 μM Incubation Duration: 0 hrs (hours), 3 hrs (hours), 6 hrs (hours), 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) Experimental Results: caspase-3 and PARP proteins were diminished. For cell viability assays, cells were seeded onto 96-well culture plates. The number of viable cells was quantified using a colorimetric assay with WST-1 reagent. Specifically, 10 μl of Premix WST-1 per 100 μl of culture medium was added to each well, and cells were incubated under standard culture conditions for 1 hour. WST reduction was determined using an ELISA plate reader at an optical density of 450-650 nm. [1] For flow cytometry analysis of non-viable cells, cells were incubated with 7-Amino-Actinomycin (7-AAD) reagent for 15 minutes at room temperature in the dark. Cells were then analyzed on a flow cytometer. [1] For siRNA transfection, MM.1S cells were seeded at 1.0×10^5 cells/100 μL well onto 96-well plates. Cells were then transfected with 2 pmol of stealth siRNA (targeting DPP8 or DPP9) and Lipofectamine RNAiMAX reagent diluted in Opti-MEM medium, giving a final siRNA concentration of 20 nM. Cells were cultured for 72 hours. [1] For Western blot analysis, cells were lysed in a buffer containing 1% sodium dodecyl sulfate (SDS), 20 mM Tris-HCl pH 7.4, and protease inhibitors. Lysates were boiled in a reducing sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to a membrane. Membranes were hybridized with anti-PARP antibody, anti-caspase-3 antibody, and anti-actin antibody. Proteins were visualized with horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence. [1] |
| Animal Protocol |
Animal/Disease Models: NOD/Shi -scid IL-2Rγnull (NOG) female mice (6-7 weeks; 19-21 g) were injected with MM.1 S cells [1]
Doses: 30 mg/kg Route of Administration: subcutaneous injection; Once a week; last for 3 weeks. Experimental Results: Dramatically inhibited the subcutaneousgrowth of MM.1 S cells in mouse xenograft model. For the in vivo efficacy study, NOD/Shi-scid IL-2Rγ-null (NOG) female mice (age 6-7 weeks, weight 19-21 g) were used. Mice were kept under specific pathogen-free conditions with a 12-hour day/night cycle. MM.1S cells (5 × 10^6) were inoculated subcutaneously on the left side of the back of NOG mice. Three days after inoculation, 1G244 (30 mg/kg) was administered subcutaneously once a week. Tumor growth was measured every third or fourth day with a caliper, and tumor volume was calculated using the formula: MD × TL^2 × 1/2, where MD is the maximum diameter and TL is the transverse length. Mice were sacrificed before the tumor volume reached 3,500 mm^3 for ethical reasons. [1] |
| ADME/Pharmacokinetics |
The document did not describe any ADME or pharmacokinetic properties of 1G244. [1]
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| Toxicity/Toxicokinetics |
Daily intravenous injection of 30 mg/kg 1G244 has been linked to severe cyanosis in rats on day 4 or 5 (based on reference 36). To avoid this, the authors administered 1G244 subcutaneously at 30 mg/kg once a week. No other obvious clinical symptoms were observed in mice with this once-a-week administration. [1]
At a concentration of 100 μM in vitro, 1G244 induced nonspecific cell death. [1] |
| References | |
| Additional Infomation |
1G244 is a specific DPP8/9 inhibitor. The study demonstrated that inhibition of DPP8/9 by 1G244 induced apoptotic cell death in multiple myeloma cells, as evidenced by caspase-3 and PARP cleavage, which was suppressed by a pan-caspase inhibitor. This is in contrast to its reported mechanism in acute myeloid leukemia (AML) where DPP8/9 inhibition induced pyroptosis. The authors concluded that DPP8 is a novel therapeutic target for multiple myeloma. [1]
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| Molecular Formula |
C29H30F2N4O2
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|---|---|
| Molecular Weight |
504.570913791656
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| Exact Mass |
504.233
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| CAS # |
847928-32-9
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| PubChem CID |
56658139
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
2.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
740
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C(C1C=CC(F)=CC=1)(C1C=CC(F)=CC=1)N1CCN(C(=O)C[C@H](N)C(N2CC3C=CC=CC=3C2)=O)CC1
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| InChi Key |
ZKIQFLSGMMYCGS-SANMLTNESA-N
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| InChi Code |
InChI=1S/C29H30F2N4O2/c30-24-9-5-20(6-10-24)28(21-7-11-25(31)12-8-21)34-15-13-33(14-16-34)27(36)17-26(32)29(37)35-18-22-3-1-2-4-23(22)19-35/h1-12,26,28H,13-19,32H2/t26-/m0/s1
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| Chemical Name |
(S)-2-Amino-4-{4-[bis-(4-fluorophenyl)-methyl]piperazin-1-yl}-1-(1,3-dihydro-isoindol-2-yl)-butane-1,4-dione
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| Synonyms |
1G-244 1G 2441G244
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~495.47 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.12 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.12 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9819 mL | 9.9094 mL | 19.8189 mL | |
| 5 mM | 0.3964 mL | 1.9819 mL | 3.9638 mL | |
| 10 mM | 0.1982 mL | 0.9909 mL | 1.9819 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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