p97 ATPase ( IC50 = 0.11 μM ); p97 ATPase ( Ki = 0.22 μMM )
ML240 has an IC50 of 100 nM, making it a strong p97 inhibitor. In the UbG76V-GFP stabilization assay, ML240 exhibits activity (IC50, 0.9 μM). ML240 has a 0.22 μM Ki value and inhibits p97 in a competitive manner in relation to ATP. Furthermore, when tested at 20 μM, ML240 inhibits by more than 50% the labeling of just three protein kinase domains: DNAPK (DNA-dependent protein kinase), JAK1 JH2 (N-terminal pseudokinase domain of JAK1), and PIP5 K3 (phosphoinositide-3 kinase family). Independent of apical caspases 8 and 9, ML240 (1.1, 3.3, 10, or 20 μM) induces executioner caspases 3 and 7 and causes cell death[1].With GI50s of 0.76 and 0.5 μM after treatment for 24 hours, and 0.54 and 0.5 μM after treatment for 72 hours, respectively, ML240 is cytotoxic to HCT15 and SW403 cells[2].

ML240 has an IC50 of 100 nM, making it a strong p97 inhibitor. In the UbG76V-GFP stabilization assay, ML240 exhibits activity (IC50, 0.9 μM). ML240 has a 0.22 μM Ki value and inhibits p97 in a competitive manner in relation to ATP. Furthermore, when tested at 20 μM, ML240 inhibits by more than 50% the labeling of just three protein kinase domains: DNAPK (DNA-dependent protein kinase), JAK1 JH2 (N-terminal pseudokinase domain of JAK1), and PIP5 K3 (phosphoinositide-3 kinase family). Independent of apical caspases 8 and 9, ML240 (1.1, 3.3, 10, or 20 μM) induces executioner caspases 3 and 7 and causes cell death[1].With GI50s of 0.76 and 0.5 μM after treatment for 24 hours, and 0.54 and 0.5 μM after treatment for 72 hours, respectively, ML240 is cytotoxic to HCT15 and SW403 cells[2].

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ML240 inhibits the ATPase activity of p97 with an IC50 of 0.11 µM in an enzymatic assay. It competitively inhibits p97 with respect to ATP, with a Ki of 0.22 µM.[1]
ML240 stabilizes the p97-dependent reporter Ub^G76V-GFP in a cell-based degradation assay with an IC50 of 0.9 µM, indicating on-target inhibition in cells. It shows over 10-fold selectivity against the p97-independent proteasome substrate ODD-Luc (IC50 > 28 µM).[1]
ML240 impairs the endoplasmic reticulum-associated degradation (ERAD) pathway, as evidenced by accumulation of TCRα-GFP and CFTR reporters.[1]
ML240 potently stimulates accumulation of the lipidated autophagy marker LC3-II within minutes, indicating inhibition of autophagosome maturation.[1]
ML240 induces rapid activation of executioner caspases 3 and 7 and apoptosis in multiple colon cancer cell lines (e.g., HCT116, HT29). This activation is caspase-specific and not dependent on initiator caspases 8 or 9.[1]
ML240 exhibits broad antiproliferative activity against the NCI-60 panel of cancer cell lines, with a mean growth percent of -30.11 at 10 µM. It shows slightly greater potency towards immortalized (PHMLEB) and tumorigenic (PHMLER) cell lines compared to normal human mammary epithelial cells (HMEC).[1]
ML240 synergizes with the proteasome inhibitor MG132 in killing multiple colon cancer cell lines.[1]
ML240 rapidly induces the unfolded protein response (UPR), as shown by accumulation of ATF4.[1]
Kinase profiling using an activity-based proteomics platform (tested at 20 µM) showed that ML240 inhibited labeling of only three protein kinases by >50%: PIP5K3, JAK1 JH2 pseudokinase domain, and DNAPK, indicating high selectivity.[1]
Binding affinity screening against 43 CNS-relevant targets showed ML240 had significant binding only for the 5HT5a receptor (Ki = 2.5 µM).[1]

p97 ATPase inhibition assay: p97 ATPase activity was determined in an assay buffer containing 50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP, and 0.01% Triton X-100 to prevent compound colloid formation. ATPase activity was measured by adding a green reagent that detects inorganic phosphate release. IC50 values were determined from triplicate measurements.[1]
Mechanism of inhibition studies: To determine the mechanism, rates of ATP hydrolysis were evaluated at different ATP concentrations in the presence of ML240. Data were fitted to Michaelis-Menten equations to derive Ki values, confirming competitive inhibition with respect to ATP.[1]
Kinase profiling assay: An activity-based proteomics platform was used. Native cell lysates were incubated with ML240 (20 µM), followed by a broadly reactive ATP acyl-phosphate probe that covalently labels active kinase domains. Inhibition was measured by comparing labeling in treated versus control samples.[1]

HeLa cells that are stable in their expression of ODD-luciferase are seeded (5000 cells/well) onto a 96-well white solid bottom plate and allowed to grow for 16 hours. Following a one-hour treatment with DMEM containing MG132 (4 μM), cells are twice washed with 100 μL PBS. In the well, cycloheximide (50 μg/mL), ML240, and DMEM containing 2.5% FBS are added.One of the four 96-well plates that have been prepared is removed from the incubator at each time interval (70, 90, 120, or 150 minutes). Each well holds 50 μL of medium. Luciferin (50 μL of 1 mg/mL in PBS) is added and incubated for 5 minutes at room temperature with 500 rpm shaking. The Synergy HT Microplate Reader uses an integration time of 0.1 ms to determine luminosity intensity[2].

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Ub^G76V-GFP and ODD-Luc degradation assay: A dual-reporter stable HeLa cell line expressing the p97-dependent Ub^G76V-GFP and the p97-independent ODD-Luc (oxygen-dependent degradation domain of HIF1α fused to luciferase) was used. Cells were treated with compounds, and reporter stabilization was measured. For ODD-Luc, Western blot analysis was performed in parallel to confirm results weren't due to luciferase inhibition.[1]
Antiproliferative/Cell Viability Assay: Colon cancer cell lines (e.g., HCT15, SW403) were treated with compounds for 24 or 72 hours. Cellular viability was determined using a luminescent cell viability assay kit that measures ATP content.[1]
Apoptosis/Caspase Activation Assay: Cells were treated with ML240, and caspase-3/7 activity was measured in cell lysates using a fluorogenic substrate. Specificity was confirmed using the pan-caspase inhibitor Z-VAD(OMe)FMK.[1]
Western Blot Analysis for Pathway Evaluation: Cells were treated with compounds, fractionated into cytosolic and nuclear plus membrane fractions, and analyzed by immunoblotting for markers like ubiquitin conjugates, LC3-II, TCRα-GFP, CFTR, ATF4, CHOP, p21, p27, PARP cleavage, etc.[1]
ERAD Reporter Assay (TCRα-GFP and CFTR): HEK293 cells stably expressing TCRα-GFP or transfected with CFTR cDNA were treated with compounds. Protein accumulation was assessed by Western blot of cellular fractions.[1]
siRNA Depletion: U2OS cells were transfected with p97 siRNA or control siRNA for 72 hours. Degradation of UPS substrates (p21, p27, etc.) was monitored by Western blot after cycloheximide chase.[1]

In vitro pharmacokinetic properties were assessed.
Aqueous Solubility: ML240 showed poor solubility across pH ranges tested: 0.35 µg/mL at pH 5.0, 0.33 µg/mL at pH 6.2, and 0.27 µg/mL at pH 7.4.[1]
Parallel Artificial Membrane Permeability Assay (PAMPA): Effective permeability (P_e) values were 357 x 10^-6 cm/s at pH 5.0, 628 x 10^-6 cm/s at pH 6.2, and < 60.3 x 10^-6 cm/s at pH 7.4.[1]
Plasma Protein Binding (PPB): ML240 was highly bound to plasma proteins: 99.53% (human, 1 µM), 99.71% (human, 10 µM), 99.73% (mouse, 1 µM), 99.63% (mouse, 10 µM).[1]
Plasma Stability (PS): ML240 was 100% stable in both human and mouse plasma after 3 hours of incubation.[1]
Hepatic Microsomal Stability (HMS): 54.2% of ML240 remained after 1 hour in human microsomes, and 0.63% remained in mouse microsomes.[1]

Hepatocyte Toxicity: The LC₅₀ of ML240 towards Fa2N-4 immortalized human hepatocytes was 8.5 µM.[1]
Plasma Protein Binding: As above, indicates high binding which may influence free drug concentration.[1]

[1]. Structure-activity relationship study reveals ML240 and ML241 as potent and selective inhibitors of p97 ATPase. ChemMedChem, 2013 Feb, 8(2):297-312.

[2]. Selective, reversible inhibitors of the AAA ATPase p97. Probe Reports from the NIH Molecular Libraries Program. April 14, 2011.

ML240 is a member of the class of quinazolines that is quinazoline which is substituted at positions 2, 5 and 8 by 2-amino-1H-benzimidazol-1-yl, benzylnitrilo and methoxy groups, respectively. It is a ATP-competetive inhibitor of AAA ATPase p97, also known as valosin-containing protein (VCP). It has a role as an antineoplastic agent. It is a member of quinazolines, a member of benzimidazoles, a secondary amino compound, an aromatic amine, an aromatic ether and a primary amino compound.

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ML240 is a potent and selective ATP-competitive inhibitor of the AAA+ ATPase p97 (VCP). It was developed from a quinazoline scaffold via structure-activity relationship (SAR) optimization.[1]
Unlike its analog ML241, ML240 uniquely inhibits the autophagy pathway and rapidly induces apoptosis via executioner caspase-3/7 activation, independent of initiator caspases 8 and 9.[1]
The differential activity between ML240 and ML241 suggests that rapid apoptosis may be linked to simultaneous inhibition of p97 functions in both the ubiquitin-proteasome and autophagy pathways, or inhibition of a novel p97 function.[1]
ML240 is proposed as a promising chemical probe for studying p97 biology and as a starting point for developing novel cancer chemotherapeutic agents.[1]

C23H20N6O

396.45

396.17

C, 69.68; H, 5.09; N, 21.20; O, 4.04

1346527-98-7

49830258

Light yellow to yellow solid powder

1.4±0.1 g/cm3

696.6±65.0 °C at 760 mmHg

375.1±34.3 °C

0.0±2.2 mmHg at 25°C

1.718

3.75

2

6

5

30

558

0

O(C([H])([H])[H])C1=C([H])C([H])=C([H])C2=C1N=C(N=C2N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N1C(N([H])[H])=NC2=C([H])C([H])=C([H])C([H])=C12

NHAMBLRUUJAFOY-UHFFFAOYSA-N

InChI=1S/C23H20N6O/c1-30-19-13-7-10-16-20(19)27-23(28-21(16)25-14-15-8-3-2-4-9-15)29-18-12-6-5-11-17(18)26-22(29)24/h2-13H,14H2,1H3,(H2,24,26)(H,25,27,28)

2-(2-aminobenzimidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine

ML240; ML 240; ML-240

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)

DMSO : 12.5~79 mg/mL ( 31.5~199.3 mM )
Ethanol : < 1 mg/mL

Solubility in Formulation 1: 2.5 mg/mL (6.31 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (6.31 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (6.31 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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 (Please use freshly prepared in vivo formulations for optimal results.)