GAPDH-IN-1 (Compound F8) is a GAPDH inhibitor (IC50 of 39.31 μM for GAPDH enzyme activity). GAPDH-IN-1 forms a covalent adduct with aspartate in the active site, displacing the enzyme's cofactor NAD+ and simultaneously enhancing the reaction of cysteine-reactive probes with the catalytic cysteine.

Physicochemical Properties

Molecular Formula	C20H18N2O5
Molecular Weight	366.37
CAS #	252212-58-1
Appearance	Typically exists as solids at room temperature
SMILES	O1CC1COC1C=CC=C(C2=NN=C(C3=CC=CC(=C3)OCC3CO3)O2)C=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

ln Vitro	GAPDH-IN-1 (1.6-50 μM) dose-dependently increases the labeling of a 40 kDa protein probe[1]. GAPDH-IN-1 (50 μM, 4 h) covalently reacts with Asp35 in GAPDH, but not with cysteine residues, stimulating the subsequent reaction of Cys152 with an iodoacetamide-based (IA) probe[1]. GAPDH-IN-1 (25-50 μM, 24 h) enhances the response of the probe and GAPDH in HEK293 cells to a lesser extent than in cell lysates[1]. GAPDH-IN-1 (0-100 μM, 24 h) concentration-dependently inactivates GAPDH in HEK293 cell lysates with an IC50 of 39.31 μM[1]. GAPDH-IN-1 (0-100 μM) inhibits the viability of HEK293 cells with an IC50 of 50.64 μM[1]. GAPDH-IN-1-GAPDH adducts can be reduced by the addition of excess NAD+, as evidenced by the shift of the endogenous GAPDH band in silver-stained gels[1].
Cell Assay	Western Blot Analysis[1] Cell Types: HEK293 cells transiently expressing FLAG-tagged GAPDH or its cysteine-to-alanine mutants Tested Tested Concentrations: 50 μM Incubation Duration: 4 h Experimental Results: The band of FLAG-GAPDH was slightly shifted. Showed band shifts in all the cysteine mutants (C152A, C156A, and C247A).
References	[1]. Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe. ACS Chem Biol. 2022 Feb 18;17(2):340-347.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7295 mL	13.6474 mL	27.2948 mL
	5 mM	0.5459 mL	2.7295 mL	5.4590 mL
	10 mM	0.2729 mL	1.3647 mL	2.7295 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.