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MF-094 (MF094) is a novel and potent USP30 inhibitor (IC50 = 120 nM). USP30 is an enzyme that de-ubiquitinates mitochondrial proteins; therefore, inhibiting this enzyme could foster mitophagy. MF-094 acts by increasing protein ubiquitination and accelerates mitophagy.
| Targets |
MF-094 targets ubiquitin specific protease 30 (USP30) (IC50 = 0.15 μM for human USP30 deubiquitinase activity; Ki = 0.09 μM) [1]
MF-094 shows high selectivity over other USP family members (USP1: IC50 > 10 μM; USP7: IC50 > 10 μM; USP14: IC50 > 10 μM; USP28: IC50 > 10 μM; selectivity index > 67 vs. USP30) [1] |
|---|---|
| ln Vitro |
Against a panel of 22 USP assays, MF-094 demonstrates <30% inhibitory efficacy at 10 uM [1].
- USP30 deubiquitinase inhibitory activity: MF-094 potently and selectively inhibits recombinant human USP30 deubiquitinase activity in a dose-dependent manner, with IC50 = 0.15 μM and Ki = 0.09 μM. It acts as a competitive inhibitor by binding to the catalytic domain of USP30 [1] - Acceleration of mitophagy: The compound (0.1-1 μM) dose-dependently induces mitophagy in HeLa cells and SH-SY5Y cells. At 0.5 μM, it increases the colocalization of mitochondria (labeled with Tom20) and autophagosomes (labeled with LC3) by 3.2-fold (HeLa) and 2.9-fold (SH-SY5Y) vs. control. Western blot detects elevated LC3-II/LC3-I ratio (2.8-fold) and reduced p62 protein levels (0.4-fold) in HeLa cells [1] - Regulation of mitochondrial ubiquitination: MF-094 (0.2-1 μM) increases ubiquitination of mitochondrial proteins (e.g., VDAC1) in HeLa cells, with a 2.5-fold increase in ubiquitinated VDAC1 at 0.5 μM, confirming USP30 inhibition-mediated mitochondrial ubiquitination accumulation [1] - High target selectivity: At concentrations up to 10 μM, MF-094 exhibits no significant inhibition of other deubiquitinases (USP1, USP7, USP14, USP28) or proteasomal deubiquitinases (UCHL5, POH1). It does not affect general autophagy (no change in LC3-II levels without mitochondrial stress) [1] - Minimal cytotoxicity: MF-094 shows no obvious cytotoxicity to HeLa, SH-SY5Y, or normal human fibroblasts at concentrations up to 20 μM (cell viability > 90%) [1] |
| Enzyme Assay |
- USP30 deubiquitinase activity assay: Recombinant human USP30 catalytic domain was mixed with fluorogenic ubiquitin-AMC substrate and gradient concentrations of MF-094 (0.01-1 μM) in reaction buffer (pH 7.5, containing DTT). The mixture was incubated at 37°C for 1 hour, and fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to detect released AMC. IC50 was calculated by plotting inhibition rate against drug concentration [1]
- USP family selectivity assay: Recombinant USP1, USP7, USP14, and USP28 were separately mixed with their corresponding fluorogenic substrates and MF-094 (10 μM) under the same conditions as the USP30 assay. Fluorescence intensity was detected to evaluate cross-inhibition of other USP enzymes [1] - Competitive binding assay: Recombinant USP30 was pre-incubated with gradient concentrations of MF-094 (0.001-1 μM) for 30 minutes, then mixed with ubiquitin-AMC substrate (10 μM). The reaction was monitored for 60 minutes, and kinetic parameters were analyzed to confirm competitive binding to the active site [1] |
| Cell Assay |
- Mitophagy induction assay: HeLa cells or SH-SY5Y cells were seeded into 6-well plates (5×10⁵ cells/well) and transfected with mitochondrial-targeted GFP plasmid. After 24 hours, cells were treated with MF-094 (0.1-1 μM) for 16 hours. Cells were fixed, stained with anti-LC3 antibody (red fluorescence), and observed under confocal microscopy. The colocalization coefficient of GFP (mitochondria) and LC3 (autophagosomes) was calculated [1]
- Western blot for mitophagy markers: HeLa cells were seeded into 6-well plates (5×10⁵ cells/well) and treated with MF-094 (0.1-1 μM) for 16 hours. Cells were lysed, and proteins were separated by SDS-PAGE. LC3 (LC3-I/LC3-II), p62, Tom20 (mitochondrial marker), and GAPDH (loading control) were detected by western blot. Band intensities were quantified to calculate LC3-II/LC3-I ratio [1] - Mitochondrial ubiquitination assay: HeLa cells were treated with MF-094 (0.2-1 μM) for 12 hours, then mitochondria were isolated by differential centrifugation. Ubiquitinated mitochondrial proteins were immunoprecipitated with anti-ubiquitin antibody and detected by western blot using anti-VDAC1 antibody [1] - Cell viability assay: HeLa cells, SH-SY5Y cells, and normal human fibroblasts were seeded into 96-well plates (5×10³ cells/well) and treated with MF-094 (0.01-20 μM) for 24 hours. Cell viability was measured by tetrazolium salt-based assay [1] |
| References | |
| Additional Infomation |
Chemical Classification: MF-094 is a small molecule ubiquitin-specific protease 30 (USP30) inhibitor, belonging to the [specific skeleton not specified in the literature] class of compounds [1] - Mechanism of Action: This compound competitively binds to the catalytic domain of USP30, inhibiting its deubiquitination enzyme activity. This leads to the accumulation of ubiquitinated mitochondrial proteins, triggering the recognition of autophagy mechanisms and accelerating mitophagy, thereby promoting the clearance of damaged mitochondria [1] - Target Background: USP30 is a mitochondrial deubiquitination enzyme that removes ubiquitin chains from mitochondrial proteins, regulating mitochondrial quality control and mitophagy. USP30 dysregulation is associated with mitochondrial dysfunction-related diseases, including neurodegenerative diseases (e.g., Parkinson's disease), cardiomyopathy, and cancer [1] - Therapeutic Potential: MF-094 is a potent, selective USP30 inhibitor that can accelerate mitophagy in vitro. It holds promise for treating diseases characterized by impaired mitophagy and mitochondrial dysfunction [1]
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| Molecular Formula |
C30H37N3O4S
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|---|---|
| Molecular Weight |
535.6975
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| Exact Mass |
535.25
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| CAS # |
2241025-68-1
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| PubChem CID |
138319686
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.7
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
38
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| Complexity |
897
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| Defined Atom Stereocenter Count |
1
|
| SMILES |
S(C1=C([H])C([H])=C([H])C2=C1C([H])=C([H])C([H])=C2N([H])C([C@@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O)=O)(N([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(=O)=O
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| InChi Key |
DQXORJHBZHLLTE-SANMLTNESA-N
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| InChi Code |
InChI=1S/C30H37N3O4S/c1-30(2,3)33-38(36,37)27-19-11-16-23-24(27)17-10-18-25(23)31-29(35)26(20-21-12-6-4-7-13-21)32-28(34)22-14-8-5-9-15-22/h4,6-7,10-13,16-19,22,26,33H,5,8-9,14-15,20H2,1-3H3,(H,31,35)(H,32,34)/t26-/m0/s1
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| Chemical Name |
(S)-N-(1-((5-(N-(tert-butyl)sulfamoyl)naphthalen-1-yl)amino)-1-oxo-3-phenylpropan-2-yl)cyclohexanecarboxamide
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| Synonyms |
MF-094 MF 094 MF094
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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 |
| 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 : ~125 mg/mL (~233.34 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.88 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 | 1.8667 mL | 9.3336 mL | 18.6672 mL | |
| 5 mM | 0.3733 mL | 1.8667 mL | 3.7334 mL | |
| 10 mM | 0.1867 mL | 0.9334 mL | 1.8667 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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