Physicochemical Properties
| Molecular Formula | C40H56N12O9 |
| Molecular Weight | 848.947648048401 |
| Exact Mass | 848.429 |
| CAS # | 167698-69-3 |
| PubChem CID | 156612994 |
| Appearance | Typically exists as solid at room temperature |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 1056.1±75.0 °C at 760 mmHg |
| Flash Point | 592.5±37.1 °C |
| Vapour Pressure | 0.0±0.3 mmHg at 25°C |
| Index of Refraction | 1.646 |
| LogP | 4.91 |
| Hydrogen Bond Donor Count | 10 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 24 |
| Heavy Atom Count | 61 |
| Complexity | 1610 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C([C@H](CC(C)C)NC([C@H](CCC/N=C(\N)/N)NC(=O)OCC1C=CC=CC=1)=O)N[C@H](C(NCC(NCC(NC1C=CC2C(C)=CC(=O)OC=2C=1)=O)=O)=O)CCC/N=C(\N)/N |
| InChi Key | BOVMHLIAYDDKAY-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C40H56N12O9/c1-23(2)17-30(51-36(57)29(12-8-16-46-39(43)44)52-40(59)60-22-25-9-5-4-6-10-25)37(58)50-28(11-7-15-45-38(41)42)35(56)48-20-32(53)47-21-33(54)49-26-13-14-27-24(3)18-34(55)61-31(27)19-26/h4-6,9-10,13-14,18-19,23,28-30H,7-8,11-12,15-17,20-22H2,1-3H3,(H,47,53)(H,48,56)(H,49,54)(H,50,58)(H,51,57)(H,52,59)(H4,41,42,45)(H4,43,44,46) |
| Chemical Name | benzyl N-[5-(diaminomethylideneamino)-1-[[1-[[5-(diaminomethylideneamino)-1-[[2-[[2-[(4-methyl-2-oxochromen-7-yl)amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate |
| 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 | SARS-CoV PLpro |
| ln Vitro | Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the betacoronavirus genus can cause severe respiratory illnesses, accompanied by pneumonia, multiorgan failure, and ultimately death. CoVs have the ability to transgress species barriers and spread swiftly into new host species, with human-to-human transmission causing epidemic diseases. Despite the severe public health threat of MERS-CoV, there are currently no vaccines or drugs available for its treatment. MERS-CoV papain-like protease (PLpro) is a key enzyme that plays an important role in its replication. In the present study, we evaluated the inhibitory activities of doxorubicin (DOX) against the recombinant MERS-CoV PLpro by employing protease inhibition assays. Hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC–peptide bond in the presence of DOX showed an IC50 value of 1.67 μM at 30 min. Subsequently, we confirmed the interaction between DOX and MERS-CoV PLpro by thermal shift assay (TSA), and DOX increased ΔTm by ~20 °C, clearly indicating a coherent interaction between the MERS-CoV PL protease and DOX. The binding site of DOX on MERS-CoV PLpro was assessed using docking techniques and molecular dynamic (MD) simulations. DOX bound to the thumb region of the catalytic domain of the MERS-CoV PLpro. MD simulation results showed flexible BL2 loops, as well as other potential residues, such as R231, R233, and G276 of MERS-CoV PLpro. Development of drug repurposing is a remarkable opportunity to quickly examine the efficacy of different aspects of treating various diseases. Protease inhibitors have been found to be effective against MERS-CoV to date, and numerous candidates are currently undergoing clinical trials to prove this. Our effort follows a in similar direction[1]. |
| Enzyme Assay |
MERS-CoV PLpro Inhibition Assay[1] IC50 values for different concentrations and durations of DOX were determined using the plate-based flat bottom black test 384. Briefly, hydrolyzed fluorogenic peptide Z-Arg-Leu-Arg-Gly-Gly peptide linkage 7-amido–methylcoumarin (Z-RLRGG-AMC) significantly increased the fluorescence of the AMC fraction, allowing the conversion to be measured accurately. Reactions were performed in a total volume of 50 µL, which contained the following components: 20 mM Tris buffer, pH 8.0, 30 µM Z-RLRGG-AMC, and varying concentrations of inhibitor (DOX) (1.84 pM–183.98 µM). Assays were initiated with the addition of PLpro to produce a final enzyme concentration of 60 nM. Plates were shaken gently for 30 s, and fluorescence from the release of AMC from peptide recorded for 60 min on Biotek HT Microplate Reader (γexcitation = 360 nm; γemission = 460 nm and gain = 40) and the IC50 value was calculated by the dose–response-inhibition function in Graphpad Prism with inhibitor vs. normalized response equation. The experiment was repeated three times. Positive control wells, representing 100% inhibition, included 10 μg/mL DOX; negative control wells, showing zero inhibition, included vehicle contained only buffer. Thermal Shift Assay (TSA)[1] Doxorubicin binding to MERS CoV PLpro protein was estimated by differential scanning fluorimetry (DSF) using an Applied Biosystems™ PCR machine as previously described. We also tested acyclovir’s (known antiviral agent) possible interaction against MERS-CoV PLpro as DOX. Briefly, reaction buffer was used as a control and MERS CoV-PLpro protein was diluted into it to a final concentration of 2 mol/L before being treated at 25 °C for 30 min with 0.29–18.4 μM of DOX. A temperature gradient of 1 °C/minute between 25 °C and 95 °C was used to record the fluorescence using SYPRO orange. The melting temperature (Tm) was determined with TSA CRAFT software, which is robust and less sensitive to noise (Lee et al., 2019). TSA-CRAFT, implement the curve fitting through the Boltzmann equation to determine the melting temperature (Tm). |
| Cell Assay |
SDS-PAGE[1] SDS polyacrylamide gel electrophoresis using BioRad Mini-protean: protein samples were mixed with a buffer containing 2X Laemmlli Buffer in a total volume of 20 μL. Samples were heated at 95 °C in a water bath for five minutes and immediately cooled in ice before electrophoresis on SDS-PAGE initially at 60 V, and after the protein passed the stacking gel it was run at 100 V. After electrophoreses, gels were stained with Coomassie blue staining solution and destained in a gentle agitation with a destaining solution of 50% H2O, 10% acetic acid and 40% methanol. Pictures of SDS-PAGE gels were taken and cropped using the Gel-Doc System and made publication-ready in Microsoft PowerPoint without any color or contrast correction. Western Blotting[1] Western blotting was performed for monoclonal-His antibody to confirm the presence of N-terminal 6xHis-Tag, as described previousl. Whole cell protein (10 μL) isolated by sonication was mixed with 2X Laemmli dye and electrophoresed on 10% SDS−polyacrylamide gel, after which it was transferred onto nitrocellulose membranes using a transblot turbo machine, then blocked in Every Blot Blocking Buffer at room temperature for 10 min. The membranes were washed five times with TBST buffer at 5 min intervals, then incubated overnight (4 °C) with primary poly-His antibody. After 5 consecutive washes at 5-minute intervals with TBST, the membranes were then rinsed with TBST 5 times for 5 min each after being incubated with secondary antibody for 2 hours at room temperature. Bands were scanned using Luminata™ Western Chemiluminescent horse radish peroxidase substrates, followed by Gel-Doc System. |
| References | [1].Identification of Doxorubicin as Repurposing Inhibitory Drug for MERS-CoV PLpro. Molecules. 2022 Nov; 27(21): 7553. |
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 | 1.1779 mL | 5.8896 mL | 11.7793 mL | |
| 5 mM | 0.2356 mL | 1.1779 mL | 2.3559 mL | |
| 10 mM | 0.1178 mL | 0.5890 mL | 1.1779 mL |