M2I-1 (full name: Mad2 Inhibitor-1) is a first-in-class small molecule inhibitor that targets the binding of Mad2 to Cdc20, an essential protein-protein interaction (PPI) within the mitotic SAC (spindle assembly checkpoint). In FP assays, it can also suppress the Mad2-F-Mbp1 interaction. Computational and biochemical analyses revealed that M2I-1 disrupts the conformational dynamics of Mad2, which is essential for the formation of complexes with Cdc20. Research on cells showed that M2I-1 reduces the SAC response, suggesting that the substance may be active in cells. This study thus identifies the formation of the SAC-specific complex between Mad2 and Cdc20 as a protein-protein interaction that is amenable to small molecule targeting.

Physicochemical Properties

Molecular Formula	C19H24N4O4S
Molecular Weight	404.48
Exact Mass	404.151
Elemental Analysis	C, 56.42; H, 5.98; N, 13.85; O, 15.82; S, 7.93
CAS #	312271-03-7
Related CAS #	312271-03-7
PubChem CID	2883762
Appearance	Solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.622
LogP	5.02
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	28
Complexity	639
Defined Atom Stereocenter Count	0
SMILES	O=C(/C(C(N1)=O)=C\C2=CC=C(N(CC(C)C)CC(C)C)C([N+]([O-])=O)=C2)NC1=S
InChi Key	BWEKPQUKWLNUKX-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H24N4O4S/c1-11(2)9-22(10-12(3)4)15-6-5-13(8-16(15)23(26)27)7-14-17(24)20-19(28)21-18(14)25/h5-8,11-12H,9-10H2,1-4H3,(H2,20,21,24,25,28)
Chemical Name	5-[[4-[bis(2-methylpropyl)amino]-3-nitrophenyl]methylidene]-2-sulfanylidene-1,3-diazinane-4,6-dione
Synonyms	M2I-1; M2I1; M2I 1; Mad2 Inhibitor-1
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	Mad2
ln Vitro	M2I-1, the initial small molecule inhibitor, is crucial for preventing Mad2 from binding to Cdc20, making it a PPI within the SAC.[1]
ln Vivo
Enzyme Assay	M2I-1, also known as Mad2 Inhibitor-1, is a novel class of small molecule inhibitor that inhibits Mad2's binding to Cdc20, a crucial protein-protein interaction (PPI) that occurs within the mitotic spindle assembly checkpoint (SAC). The disruption of Mad2's conformational dynamics is crucial for the formation of complexes with Cdc20. In FP experiments, it can also inhibit the Mad2-F-Mbp1 interaction.
Cell Assay	M2I-1 was found to interfere with Mad2's conformational dynamics, which is essential for the formation of complexes with Cdc20, based on biochemical and computational analyses. M2I-1 was found to reduce the SAC response in cellular studies, suggesting that the substance may be active in cells.
Animal Protocol
References	[1]. Mad2 Inhibitor-1 (M2I-1): A Small Molecule Protein-Protein Interaction Inhibitor Targeting the Mitotic Spindle Assembly Checkpoint. ACS Chem. Biol., 2015, 10 (7), pp 1661-1666.

Solubility Data

Solubility (In Vitro)

DMSO: ~80 mg/mL ( 197.78 mM) Water: Insoluble Ethanol: Insoluble

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.18 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. Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% saline：≥ 2.5 mg/mL (~6.2 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4723 mL	12.3616 mL	24.7231 mL
	5 mM	0.4945 mL	2.4723 mL	4.9446 mL
	10 mM	0.2472 mL	1.2362 mL	2.4723 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.