Apostatin-1 (Apt-1) is a novel and potent TRADD inhibitor. TRADD is an adaptor protein acting as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). Pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

Physicochemical Properties

Molecular Formula	C19H27N3OS
Molecular Weight	345.5022
Exact Mass	345.187
CAS #	2559703-06-7
Related CAS #	2559703-06-7
PubChem CID	154824620
Appearance	White to off-white solid
Density	1.2±0.1 g/cm3
Index of Refraction	1.630
LogP	3.63
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	5
Heavy Atom Count	24
Complexity	440
Defined Atom Stereocenter Count	0
SMILES	S(C1=NC([H])([H])C([H])([H])N1C([H])([H])[H])C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C(N([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O
InChi Key	OXBMBNASDMMXSA-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H27N3OS/c1-22-14-13-20-19(22)24-17(15-9-5-4-6-10-15)18(23)21-16-11-7-2-3-8-12-16/h4-6,9-10,16-17H,2-3,7-8,11-14H2,1H3,(H,21,23)
Chemical Name	N-cycloheptyl-2-[(1-methyl-4,5-dihydroimidazol-2-yl)sulfanyl]-2-phenylacetamide
Synonyms	Apt-1; Apostatin-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	RIPK1
ln Vitro	Apostatin-1 (Apt-1) is a TRADD inhibitor that affects the ubiquitination of RIPK1 and beclin 1 by interacting with the pocket on the N-terminal TRAF2 binding domain of TRADD (TRADD-N). In vitro, apostatin-1 successfully prevents bortezomib- and RIPK1-dependent apoptosis (RDA).[1]
ln Vivo	Apostatin-1 (Apt-1) (20 mg/kg, IP, once) prolongs the survival of mice with systemic inflammation by inhibiting inflammatory responses.
Cell Assay	Velcade (50 nM) and Apt-1 (10 μM) were used to induce RIPK1-dependent apoptosis (RDA) in Jurkat cells, and the effects on cell viability were assessed after the treatment for 24 hours.MEFs were treated with mTNF (1 ng/mL) and 5Z-7- Oxozeaenol (0.5 μM), and Apt-1 was added at various concentrations for 8 hours.
References	[1]. Modulating TRADD to restore cellular homeostasis and inhibit apoptosis. Nature. 2020 Nov;587(7832):133-138.

Solubility Data

Solubility (In Vitro)

DMSO: 69~83.3mg/mL (~199.7~241.2 mM) Ethanol: ~9 mg/mL (~26.1 mM)

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	2.8944 mL	14.4718 mL	28.9436 mL
	5 mM	0.5789 mL	2.8944 mL	5.7887 mL
	10 mM	0.2894 mL	1.4472 mL	2.8944 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.