Inauhzin is a potent SIRT inhibitor, which effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Inauhzin also stabilizes p53, though not directly in vitro, by promoting p53 acetylation and blocking MDM2-mediated ubiquitylation of p53 in cells. Surprisingly, Inauhzin suppresses the growth of xenograft tumors derived from p53-harbouring H460 and HCT116 cells, inhibits cell proliferation, induces senescence and tumor-specific apoptosis, and does so without appearing to be harmful to normal tissues or the tumor-bearing SCID mice.
Physicochemical Properties
| Molecular Formula | C25H19N5OS2 |
| Molecular Weight | 469.581261873245 |
| Exact Mass | 469.103 |
| Elemental Analysis | C, 63.94; H, 4.08; N, 14.91; O, 3.41; S, 13.66 |
| CAS # | 309271-94-1 |
| Related CAS # | 309271-94-1 |
| PubChem CID | 5494506 |
| Appearance | White solid powder |
| Density | 1.5±0.1 g/cm3 |
| Boiling Point | 773.6±70.0 °C at 760 mmHg |
| Flash Point | 421.7±35.7 °C |
| Vapour Pressure | 0.0±2.7 mmHg at 25°C |
| Index of Refraction | 1.816 |
| LogP | 5.18 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 33 |
| Complexity | 690 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CCC(SC1=NN=C2C(NC3=C2C=CC=C3)=N1)C(N4C5=C(C=CC=C5)SC6=CC=CC=C46)=O |
| InChi Key | VHUOXERIKQWIJE-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C25H19N5OS2/c1-2-19(33-25-27-23-22(28-29-25)15-9-3-4-10-16(15)26-23)24(31)30-17-11-5-7-13-20(17)32-21-14-8-6-12-18(21)30/h3-14,19H,2H2,1H3,(H,26,27,29) |
| Chemical Name | 1-phenothiazin-10-yl-2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylsulfanyl)butan-1-one |
| Synonyms | Inauhzin |
| 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 | SIRT1; MDM-2/p53; IMPDH2 |
| ln Vitro | Inauhzin (10 µM) induces p53 levels as effectively as actinomycin D (10 nM) and mediates p53-dependent cytotoxicity through its particular functional groups in human lung carcinoma H460 cells. At 2 µM, inauhzin induces p53-dependent apoptosis as well as p53 level and activity. Moreover, inauhzin prevents p53 from being ubiquitylated and stabilizes it. In H460 cells, inauhzin acetylates p53, but not tubulin, which is impacted by SIRT1 knockdown[1]. The study found that inauhzin (0–2 µM) significantly increases the expression level and activity of p53 in HCT116p53+/+ cells and dose-dependently increases these same parameters in H460 cells. In the Nutlin-3 low-sensitive cells, inauhzin and Nutlin-3 exhibit synergistic cytotoxicity. Apoptosis dependent on p53 is synergistically induced by Inauhzin and Nutlin-3[2]. Inauhzin functions as a strong p53 activator and targets both SirT1 and IMP dehydrogenase 2 (IMPDH2)[3]. |
| ln Vivo | Inauhzin (30 mg/kg, i.p.) efficiently induces apoptosis and inhibits the growth of tumors in H460 xenografts that harbor p53[1]. About 70% less HCT116 tumor volume is present when inauhzin (30 mg/kg, i.p.) is administered. When administered in conjunction with 150 mg/kg of Nutlin-3, inauhzin (15 mg/kg) significantly enhances p53 induction, apoptosis, and tumor suppression in HCT116p53+/+ xenografts[2]. |
| Cell Assay | Cell growth is evaluated using the cell counting kit. In 96-well culture plates, cell suspensions are seeded at 5000 cells per well and incubated at 37°C for the entire night. After adding compounds to the plates, they are incubated for 72 hours at 37°C. WST-8 is added to each well at a final concentration of 10% to determine the amount of cell growth inhibition, and the absorbance of the samples is measured using a Microplate Reader at 450 nm[1]. |
| Animal Protocol | Female SCID mice that are five weeks old are kept in a BSL2 environment. Mice receive a subcutaneous inoculation of either 3×106 HCT116 or 5×106 H460 cells. Every other day, the growth of tumors is observed using two-dimensional electronic digital calipers. Tumor volume (mm3) = (length × width2)/2 is the formula used to calculate tumor volume. After 7 to 9 days, when the average tumor volume is about 100 mm3, animals are given an intraperitoneal injection of either inauhzin or a vehicle (5% DMSO). On the final day of treatment, the amount of tumor growth inhibition is determined. Tumors are taken and broken up in RIPA buffer together with a combination of protease inhibitors in order to identify p53 activation in vivo. Analysis of tumour lysates is done by IB. BrdU labeling is used to measure the proliferation of cells in tumors, and then immunostaining is applied. Mice are given an intraperitoneal injection of 200 mg/kg body weight of BrdU two hours prior to their sacrifice. TUNEL staining with the Fluorescein In situ cell death detection kit is used to investigate apoptosis[1]. |
| References |
[1]. A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53. EMBO Mol Med. 2012 Apr;4(4):298-312. [2]. Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth. Cancer Biol Ther. 2012 Aug;13(10):915-24. [3]. Reviving the guardian of the genome: Small molecule activators of p53. Pharmacol Ther. 2017 Oct;178:92-108. |
Solubility Data
| Solubility (In Vitro) | DMSO: 94~100 mg/mL (200.2~213 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1296 mL | 10.6478 mL | 21.2956 mL | |
| 5 mM | 0.4259 mL | 2.1296 mL | 4.2591 mL | |
| 10 mM | 0.2130 mL | 1.0648 mL | 2.1296 mL |