Rbin-1 (also known as ribozinoindole-1) is a potent and specific chemical inhibitor of eukaryotic ribosome assembly. It blocks the ATPase activity of recombinant full-length Mdn1 in vitro. In order to investigate the mechanism of action of Rbin-1, resistant clones that were able to proliferate in the presence of Rbin-1 were isolated from MDR-sup fission yeast cells through chemical mutagenization. The mdn1 gene contained one of five distinct point mutations, according to the results, in each of the 13 Rbin-1 resistant clones.

Physicochemical Properties

Molecular Formula	C13H12N4S
Molecular Weight	256.33
Exact Mass	256.078
Elemental Analysis	C, 60.92; H, 4.72; N, 21.86; S, 12.51
CAS #	328023-11-6
Related CAS #	328023-11-6
PubChem CID	5731061
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	479.6±47.0 °C at 760 mmHg
Flash Point	243.8±29.3 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.718
LogP	3.28
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	18
Complexity	322
Defined Atom Stereocenter Count	0
SMILES	S(C([H])([H])C(=C([H])[H])C([H])([H])[H])C1N=NC2=C(N=1)N([H])C1=C([H])C([H])=C([H])C([H])=C12
InChi Key	LPCWHNSRTRBKBO-UHFFFAOYSA-N
InChi Code	InChI=1S/C13H12N4S/c1-8(2)7-18-13-15-12-11(16-17-13)9-5-3-4-6-10(9)14-12/h3-6H,1,7H2,2H3,(H,14,15,17)
Chemical Name	3-(2-methylprop-2-enylsulfanyl)-5H-[1,2,4]triazino[5,6-b]indole
Synonyms	Ribozinoindole-1; Rbin-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	ATPase ( GI50 = 136±7 nM )
ln Vitro	In vitro activity: Rbin-1 is a potent and reversible inhibitor of eukaryotic ribosome biogenesis based on triazinoindole. Rbin-1 blocks the ATPase activity of recombinant full-length Mdn1. Rbin-1 and Rbin-2, two of the active analogs, reduce ATPase activity by 40% at 1 uM. Specifically, Rbin-2, which has a bromine substituent at position-7, exhibits a 10-fold increase in activity compared to Rbin-1 (GI50 = 14±1 nM (Rbin-2); 136±7 nM (Rbin-1), n = 4, mean±SD)[1].
ln Vivo
Enzyme Assay	Depending on the radioactive reagent's lifetime, volume ratios of 1:1000–1:300 are used when adding radioactive γ-P32-ATP to 600 mM MgATP (pH=7) solutions. The total volume of each reaction is 12 mL, which includes 4 mL of FPLC SEC buffer with 0.6 mM Na2SO4 and 2 mL of MgATP (final concentration=100 mM), 6 ml of protein from size exclusion chromatography fractions (final concentration 0-50 nM for different fractions, peak fractions are used for Rbin-1 and AMPPNP inhibition). Following a 30- to 60-minute incubation period at room temperature, the reactions are quenched with 12 mL of 0.2 M EDTA. On TLC PEI cellulose F plates, 1 mL of each reaction mixture is spotted. Formic acid (0.15 M) and lithium chloride (0.15 M) were present in the TLC buffer. Following that, the Typhoon Scanner 9400 is used to image the TLC plates. Utilizing ImageJ, compute the densitometric ratio between the spots that represent radioactive free phosphate and ATP to ascertain the percentage of ATP that has been hydrolyzed[1].
Cell Assay	Enzymatic ribosome assembly is strongly inhibited by ribozinoindoles. Mdn1 or the cellular processes that use this protein are the target of Rbin-1. It successfully prevents yeast cells from growing.
Animal Protocol
References	[1]. Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis. Cell. 2016 Oct 6;167(2):512-524.e14.
Additional Infomation	Ribozinoindole-1 is a triazinoindole that is 5H-[1,2,4]triazino[5,6-b]indole which is substituted at position 3 by a (2-methylprop-2-en-1-yl)thio group. A potent inhibitor of midasin which is an essential protein for eukaryotic ribosome biogenesis. It is a triazinoindole and an organic sulfide.

Solubility Data

Solubility (In Vitro)

DMSO: 19.5~39 mg/mL (76.1~152.1 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 1.67 mg/mL (6.52 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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: ≥ 1.67 mg/mL (6.52 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 16.7 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	3.9012 mL	19.5061 mL	39.0122 mL
	5 mM	0.7802 mL	3.9012 mL	7.8024 mL
	10 mM	0.3901 mL	1.9506 mL	3.9012 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.