SAE/SUMO1(IC50= 3 nM);SAE/SUMO2(IC50= 11 nM)
SUMO-activating enzyme (SAE) – Heterodimer of SAE1 and UBA2 (IC₅₀ = 0.003 µM with SUMO1, 0.011 µM with SUMO2 in ATP-PPi exchange assay) [1]

In HCT116 cells, ML-792 (0.0007-5 μM; 4 hours) suppresses the activities of the SAE and SUMO-pathway[1].
ML-792 (0.001-10 μM; 72 hours) in MDA-MB-468, MDA-MB-231, HCT116, Colo-205, and A375 reduces cancer cell viability and inhibits cell proliferation[1].

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ML-792 is a potent and selective mechanism-based inhibitor of the SUMO-activating enzyme (SAE). In an ATP-inorganic pyrophosphate (PPi) exchange biochemical assay, it inhibited SAE activity with IC₅₀ values of 0.003 µM and 0.011 µM when SUMO1 or SUMO2 was used as the ubiquitin-like protein, respectively. It showed high selectivity over the closely related E1 enzymes NEDD8-activating enzyme (NAE, IC₅₀ = 32 µM) and ubiquitin-activating enzyme (UAE, IC₅₀ > 100 µM). Screening against a panel of 366 ATP-utilizing enzymes at 1 µM showed no significant inhibition. The compound inhibits SAE by forming a covalent adduct with SUMO in an ATP-dependent manner, catalyzed by SAE itself, as confirmed by mass spectrometry and immunoblotting with a specific anti-ML-792 antibody. [1]
In cellular assays using HCT116 colon carcinoma cells, ML-792 treatment led to a dose-dependent decrease in SAE (UBA2-SUMO) and E2 (UBC9-SUMO) thioester levels (EC₅₀ = 0.004 µM and 0.006 µM, respectively), as well as global SUMO conjugates (EC₅₀ = 0.019 µM). It also reduced SUMOylation of specific substrates TRIM28 (EC₅₀ = 0.016 µM) and RanGAP1 (EC₅₀ = 0.051 µM). Formation of the SUMO-ML-792 adduct in cells was confirmed (EC₅₀ = 0.003 µM). The inhibition was rapid (within 2 hours) and sustained for at least 48 hours. ML-792 did not affect the NEDD8 or ubiquitin pathways in cells. [1]
ML-792 treatment disrupted promyelocytic leukemia (PML) nuclear body (NB) organization and caused redistribution of the associated protein DAXX in HCT116 cells in a dose-dependent manner (EC₅₀ = 0.095 µM for PML NB size reduction and 0.007 µM for DAXX foci reduction). [1]
ML-792 decreased cancer cell viability in a dose-dependent manner across multiple tumor cell lines after 72-hour treatment in ATP-based viability assays, with EC₅₀ values ranging from 0.06 µM (MDA-MB-468) to 0.45 µM (A375). In many cell lines, viability loss plateaued below 100%. [1]
In 2D colony formation assays (anchorage-dependent), continuous treatment with ML-792 over 7-9 days showed potent anti-proliferative effects in HCT116 (EC₅₀ = 0.04 µM) and MDA-MB-231 (EC₅₀ = 0.11 µM) cells. It also substantially blocked colony formation in 3D soft-agar assays (anchorage-independent) with HCT116 cells (EC₅₀ = 0.03 µM). [1]
SK-MEL-28 melanoma cells engineered with a tetracycline-inducible MYC oncogene showed increased sensitivity to ML-792 upon MYC induction. The viability curve plateau decreased from 60% to 14% with MYC overexpression, and increased apoptotic markers (cleaved caspase-3, cleaved PARP) were observed. Small cell lung cancer (SCLC) cell lines with high endogenous MYC levels (NCI-H82, NCI-H524) also showed lower viability plateaus (33-42%) compared to lines with low MYC (59-62%). [1]
ML-792 treatment (0.5 µM for 16h) induced only modest and cell-line-specific transcriptional changes in Colo-205, HCT116, and MDA-MB-231 cells, as assessed by RNA-seq (only 17-102 genes changed >2-fold). Similarly, quantitative proteomic profiling (SILAC) in HCT116 cells treated for 24h revealed minimal changes (only ~30 out of >8000 proteins accumulated >2-fold). [1]
Continuous inhibition of the SUMO pathway by ML-792 in HCT116 cells did not result in accumulated DNA damage under normal conditions, as measured by comet assay or formation of γH2AX and 53BP1 foci. Furthermore, it did not affect the repair of DNA damage induced by cisplatin or hydroxyurea. [1]
ML-792 treatment induced severe mitotic defects. Time-lapse microscopy of HCT116 cells over 96 hours revealed multinucleation, impaired cytokinesis, mitotic slippage, and cell death during or after mitosis. Flow cytometry showed accumulation of cells with 8n DNA content (endoreduplication) at 24h and 48h. Analysis of mitotic stages in four cell lines (HCT116, MDA-MB-468, Colo-205, MDA-MB-231) after 24h treatment showed a significant decrease in the fraction of cells in anaphase/telophase in three of the lines, along with the appearance of thin DNA bridges connecting nuclei. These mitotic defects were confirmed to be on-target, as they were rescued by expression of an ML-792-resistant UBA2 (S95N M97T) mutant. [1]
In MCF-10A non-transformed mammary epithelial cells, ML-792 triggered viability loss and cell-cycle changes only under proliferating (subconfluent) conditions, not under contact-inhibited (confluent) conditions, despite equal SUMO pathway inhibition. [1]
Combination studies in HCT116 cells showed that ML-792 had additive effects, but no synergistic benefit, when combined with mitotic inhibitors vincristine, paclitaxel, or docetaxel. [1]

The in vitro inhibitory activity of ML-792 against SAE and related E1 enzymes was assessed using an ATP-PPi exchange assay. The compound was serially diluted in a 96-well plate containing assay buffer (50 mM HEPES pH 7.5, 25 mM NaCl, 5 mM MgCl₂, 0.05% BSA, 0.01% Tween-20, 1 mM TCEP). A reaction mixture containing SAE enzyme (2 nM), ATP (1 mM), and PPi (0.1 mM, containing trace [³²P]PPi) was added. Reactions were initiated by adding SUMO1 or SUMO2 (1 µM) and incubated at 37°C for 60 minutes. Reactions were stopped with trichloroacetic acid. The stopped reactions were transferred to a dot-blot apparatus with activated charcoal filter paper, washed, dried, and quantified using a phosphorimager. IC₅₀ values were calculated from background-corrected counts. Assays for NAE and UAE were conducted under similar conditions with appropriate enzyme and substrate concentrations. [1]
Formation of the covalent SUMO-ML-792 adduct was confirmed by intact protein mass spectrometry. ML-792 (10 µM) was incubated with SAE (4 µM), ATP (100 µM), MgCl₂ (10 mM), and SUMO1 (4 µM) in HEPES buffer at 37°C for 60 min. The reaction was stopped with EDTA, acidified, and analyzed by LC-MS. A control reaction without ATP was also run. [1]

Cell Line: Human melanoma cell line A37; human colon carcinoma cells HCT116 and Colo-205; human breast cancer cells MDA-MB-468 and MDA-MB-231.
Concentration: 0.001, 0.01, 0.1, 1, 10 μM
Incubation Time: 72 hours
Result: The viability effect was shown to be dose-dependent, with EC50 values ranging from 0.06 μM in MDA-MB-468 cells to 0.45 μM in A375 cells.

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Cellular SUMO pathway inhibition was assessed by western blotting for thioester levels and global SUMOylation. HCT116 cells were treated with ML-792 for 4 hours, lysed, and analyzed by non-reducing SDS-PAGE and immunoblotting with antibodies against UBA2, UBC9, SUMO2/3, and specific SUMOylated proteins (TRIM28, RanGAP1). An antibody against the ML-792-SUMO adduct was also used. Band intensities were quantified, normalized to α-tubulin, and used to calculate EC₅₀ values. [1]
PML nuclear body and DAXX localization were analyzed by immunofluorescence. HCT116 cells grown on coverslips were treated with ML-792, fixed, permeabilized, and stained with anti-PML or anti-DAXX antibodies, followed by fluorescent secondary antibodies and Hoechst 33342. Images were captured by fluorescence microscopy, and PML NB size or DAXX foci number per nucleus were quantified using image analysis software. [1]
Cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay. Cells were plated in 96-well plates, treated with a dilution series of ML-792 for 72 hours, and luminescence was measured. For combination studies, HCT116 cells in 384-well plates were treated with ML-792 and other compounds (e.g., mitotic inhibitors) alone or in combination for 72 hours before viability assessment. [1]
2D colony formation assays were performed by plating a low number of cells (500 HCT116 or 1000 MDA-MB-231) in 12-well plates and treating them continuously with ML-792 for 7 days. Colonies were fixed, stained with crystal violet, imaged, and total colony area was quantified. [1]
3D soft-agar colony formation assays were performed by suspending HCT116 cells in growth medium containing 0.4% agar and plating them onto a solidified layer of 1% agar in 24-well plates. Plates were treated with ML-792 or DMSO and incubated for 10-14 days. Colony number and area were scored. [1]
For cell cycle analysis, cells were treated with ML-792, fixed in ethanol, stained with propidium iodide and RNase A, and analyzed by flow cytometry to determine DNA content distribution. [1]
Time-lapse microscopy was used to track cell fate. HCT116 cells treated with DMSO or ML-792 were placed in a chamber on an automated microscope and imaged every 5 minutes for over 96 hours. Images were processed, and individual cell entries into and exits from mitosis were manually recorded and analyzed. [1]
Mitotic stage quantification was performed by fixing treated cells, staining DNA with Hoechst 33342, and visually classifying mitotic cells (based on nuclear morphology under fluorescence microscopy) into prophase/prometaphase, metaphase, or anaphase/telophase stages. At least 140 mitotic cells were scored per sample. [1]
RNA sequencing for transcriptomic analysis was performed on cells treated with DMSO or ML-792 for specified times (e.g., 8h, 16h, 48h). Total RNA was isolated, library was constructed, and sequenced. Data was analyzed for differential gene expression and alternative splicing. [1]
Proteomic profiling was done using SILAC (Stable Isotope Labeling by Amino acids in Cell culture). HCT116 cells were metabolically labeled with "light" or "heavy" isotopes of lysine and arginine for 9 days. Labeled cells were then treated with DMSO or ML-792 for 24 hours, mixed 1:1 based on cell count, lysed, digested with trypsin, fractionated, and analyzed by LC-MS/MS. Protein ratios were quantified. [1]
DNA damage was assessed using the alkaline comet assay. HCT116 cells were treated with ML-792 alone or in combination with DNA damaging agents (cisplatin, hydroxyurea). Cells were harvested, embedded in agarose on slides, lysed, subjected to alkaline electrophoresis, neutralized, fixed, stained with SYBR Green, and imaged. Tail moment was analyzed. Immunofluorescence for DNA damage markers (γH2AX, 53BP1, BRCA1) was also performed on treated cells. [1]

[1]. Probing the roles of SUMOylation in cancer cell biology by using a selective SAE inhibitor. Nat Chem Biol. 2017 Nov;13(11):1164-1171.

ML-792 ((1R,2S,4R)-4-((5-(1-(3-bromobenzyl)-1H-pyrazol-3-carbonyl)pyrimidin-4-yl)amino)-2-hydroxycyclopentyl)methanesulfonamide) is a small-molecule, mechanism-based inhibitor of SUMO-activated enzyme (SAE). It is the first reported highly selective SAE inhibitor with nanomolar activity. Its mechanism involves the formation of a covalent adduct with SUMO in an ATP-dependent reaction catalyzed by SAE itself, similar to the NAE inhibitor pevonedistat (MLN4924). [1] ML-792 is considered a powerful chemical tool for studying SUMOylation biology at the endogenous level, overcoming the limitations of gene knockdown/knockout and previous less potent/non-specific inhibitors. [1] This study shows that SUMOylation is crucial for cancer cell proliferation, mitotic progression, and precise chromosome segregation. Inhibition of SUMOylation by ML-792 leads to mitotic failure, internal replication, and DNA bridge formation. [1]
Cells with high MYC oncogene activity (inducible or endogenous) showed increased sensitivity to ML-792, confirming a synthetic lethal interaction and suggesting the potential therapeutic value of SAE inhibitors in MYC-amplified tumors. [1]
The targeting activity of ML-792 was confirmed by rescuing SUMOylation loss and mitotic defects through the expression of the ML-792-resistant UBA2 mutant (S95N M97T). [1]

C21H23BRN6O5S

551.413522005081

550.063

C, 45.74; H, 4.20; Br, 14.49; N, 15.24; O, 14.51; S, 5.81

1644342-14-2

86566743

White to off-white solid powder

1.7±0.1 g/cm3

815.1±75.0 °C at 760 mmHg

446.7±37.1 °C

0.0±3.1 mmHg at 25°C

1.740

1.71

3

10

9

34

801

3

BrC1=CC=CC(=C1)CN1C=CC(C(C2=CN=CN=C2N[C@H]2C[C@@H]([C@@H](COS(N)(=O)=O)C2)O)=O)=N1

PZCKLTWSXFDLLP-OGWOLHLISA-N

InChI=1S/C21H23BrN6O5S/c22-15-3-1-2-13(6-15)10-28-5-4-18(27-28)20(30)17-9-24-12-25-21(17)26-16-7-14(19(29)8-16)11-33-34(23,31)32/h1-6,9,12,14,16,19,29H,7-8,10-11H2,(H2,23,31,32)(H,24,25,26)/t14-,16-,19+/m1/s1

((1R,2S,4R)-4-((5-(1-(3-bromobenzyl)-1H-pyrazole-3-carbonyl)pyrimidin-4-yl)amino)-2-hydroxycyclopentyl)methyl sulfamate

ML-792; ML 792; ML792

2934.99.03.00

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)

DMSO : ~100 mg/mL ( ~181.35 mM )

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.77 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.77 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 20.8 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: ≥ 2.08 mg/mL (3.77 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.

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