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SMER3

Alias: SMER3 SMER 3 SMER-3
Cat No.:V5826 Purity: ≥98%
SMER3, a Rapamycin enhancer, is a selective Skp1-Cullin-F-box (SCF)Met30 ubiquitin ligase inhibitor.
SMER3
SMER3 Chemical Structure CAS No.: 67200-34-4
Product category: New1
This product is for research use only, not for human use. We do not sell to patients.
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Product Description
SMER3, a Rapamycin enhancer, is a selective Skp1-Cullin-F-box (SCF)Met30 ubiquitin ligase inhibitor. SMER3 enhances the growth inhibitory activities of Rapamycin by inhibiting SCFMet30.
Biological Activity I Assay Protocols (From Reference)
ln Vitro
Met4 ubiquitination in yeast cells is inhibited when the cells are exposed to SMER3 (0-60 μM). SMER3 can lessen the growth inhibition of met4Δ cells [1]. Met4 ubiquitination by SCFMet30 was reduced by adding SMER3 to the ligase process in a dose-dependent manner. SMER3 does not change the amounts of Skp1 or Met30 protein, but it strongly inhibits the binding of Met30 to Skp1 [1].
References

[1]. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol. 2010 Jul;28(7):738-42.

Additional Infomation
LSM-42773 is an aromatic ketone.
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C11H4N4O2
Molecular Weight
224.17
Exact Mass
224.033
CAS #
67200-34-4
PubChem CID
568763
Appearance
Light yellow to green yellow solid powder
Density
1.7±0.1 g/cm3
Boiling Point
437.2±55.0 °C at 760 mmHg
Melting Point
296 °C(dec.)
Flash Point
218.2±31.5 °C
Vapour Pressure
0.0±1.0 mmHg at 25°C
Index of Refraction
1.779
LogP
0.9
Hydrogen Bond Donor Count
0
Hydrogen Bond Acceptor Count
6
Rotatable Bond Count
0
Heavy Atom Count
17
Complexity
350
Defined Atom Stereocenter Count
0
InChi Key
SFSSAKVWCKFRHE-UHFFFAOYSA-N
InChi Code
InChI=1S/C11H4N4O2/c16-9-6-4-2-1-3-5(6)7-8(9)13-11-10(12-7)14-17-15-11/h1-4H
Chemical Name
13-oxa-10,12,14,16-tetrazatetracyclo[7.7.0.02,7.011,15]hexadeca-1(16),2,4,6,9,11,14-heptaen-8-one
Synonyms
SMER3 SMER 3 SMER-3
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)
Solubility Data
Solubility (In Vitro)
DMSO : ~12.5 mg/mL (~55.76 mM)
Solubility (In Vivo)
Solubility in Formulation 1: 1.25 mg/mL (5.58 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 12.5 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 4.4609 mL 22.3045 mL 44.6090 mL
5 mM 0.8922 mL 4.4609 mL 8.9218 mL
10 mM 0.4461 mL 2.2305 mL 4.4609 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.

Calculator

Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

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An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
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  • The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
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  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
g/mol

Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:
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Definitions of molecular mass, molecular weight, molar mass and molar weight:
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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

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  • The answer appears in the Volume (to add to vial) box
In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

Biological Data
  • Two unsupervised data analyses classify five SMERs into three different groups based on their gene expression profiles a, Chemical structures of SMER1 to SMER5. b, Two dimensional (2-D) hierarchical clustering reveals that the expression profile of SMER1 is similar to that of rapamycin, whereas the profiles of SMERs 2, 4 and 5 are indistinguishable from that of DMSO (solvent) control. The profile of SMER3 is distinct. Each row corresponds to a gene, and each column corresponds to an experimental sample. c, Principal component analysis is consistent with hierarchical clustering. Light blue: DMSO; blue: SMER1; cyan: SMER2; red: SMER3; sage: SMER4; chartreuse: SMER5; green: rapamycin. Replicates were obtained from independent small molecule treatments in separate experiments.[1].Mariam Aghajan, et al. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol. 2010 Jul;28(7):738-42.
  • SMER3 targets SCFMet30 a, Biochemical evidence for SCFMet30 inhibition by SMER3 but not rapamycin. Yeast cells were cultured in YPDA medium to mid-log 0.8×107 cells/ml, treated with indicated concentrations of SMER3 or rapamycin for 45 min, and total protein was extracted for Western blot analyses (Supplementary Information). Met4 ubiquitination in vivo can be directly assessed by immunoblotting because ubiquitinated forms of Met4 are not subjected to proteasomal degradation and can thus be detected due to a characteristic mobility shift on denaturing gels30. Asterisk (*) denotes a non-specific band immuno-reactive to the anti-Met4 antibody (generous gift from Mike Tyers). b, SMER3 resistance in met4Δ cells. Yeast cells were treated with either vehicle (DMSO) or 4 μM SMER3 and growth curve analysis was performed with an automated absorbance reader measuring O.D. at 595 nm every 30 min (Supplementary Information). Cell growth was measured in liquid because SMER3 activity is undetectable on agar. c, Genetic interaction between SCFMet30 and TOR. Temperature sensitive mutants as indicated were grown at 25°C to mid-log phase in YPDA medium and serial dilutions were spotted onto plates with or without 2.5 nM rapamycin. The plates were incubated at the permissive temperatures for the mutants: 28°C for cdc34-3, cdc53-1, cdc4-3 and met30-6 because these mutants exhibited fitness defects at 30°C even without rapamycin, or 30°C (standard growth temperature) for met30-9 and skp1-25 because these alleles are not temperature sensitive until at 37°C. d, SMER3 specifically inhibits SCFMet30 E3 ligase in vitro. Components of SCFMet30 were co-expressed in insect cells and the complex was purified based on a GST-tag fused to Skp1.[1].Mariam Aghajan, et al. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol. 2010 Jul;28(7):738-42.
  • Molecular mechanism for the specificity of SCFMet30 inhibition by SMER3 a, Protein-protein interaction between Met30 and Skp1 is diminished by SMER3 in vivo. Yeast strains expressing endogenous 13Myc-tagged Met30 were either untreated, or treated with solvent control (DMSO) or 30μM SMER3 for 30 minutes at 30°C. 13MycMet30 was immunopurified and immuncomplexes were analyzed for Skp1 binding by Western blot analysis. b, SMER3 specifically targets SCFMet30 in vivo as determined by quantitative mass spectrometry. A yeast strain expressing endogenous HBTH-tagged Skp1 was grown in medium containing either heavy (13C/15N) or light (12C/14N) arginine and lysine to metabolically label proteins. The “heavy” culture was treated with solvent control (DMSO) and the “light” culture with 20μM SMER3 for 30 minutes at 30°C. Cells were incubated with 1% formaldehyde to cross-link and stabilize protein complexes in vivo for 10 minutes at 30°C. Cell lysates were prepared under denaturing conditions in 8M urea, mixed at equal amounts, and HBTHSkp1-bound complexes were sequentially purified on Ni2+ and streptavidin sepharose under fully denaturing conditions. Tryptic peptides of the purified complexes were analyzed by LC-MS/MS. Relative abundance of proteins was determined by measuring the peptide peak intensities. Abundance ratios for SCF components identified by multiple quantifiable peptides are shown as SILAC ratios of “light” (SMER3-treated) over “heavy” (DMSO-treated) peptide intensities. c, SMER3 specificity for SCFMet30 vs. SCFCdc4 as verified by cell cycle arrest morphology. Temperature sensitive mutants were shifted to 37°C for 4 hours. The Skp1 depletion phenotype was observed after repression of Skp1 expression in dextrose medium for 12 hours. SMER3 treatment of cells was for 6 hours. d, SMER3 directly binds to Met30-Skp1, but not Skp1 alone as determined by differential scanning fluorimetry (DSF).[1].Mariam Aghajan, et al. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol. 2010 Jul;28(7):738-42.
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