SMER3 specifically inhibits the yeast E3 ubiquitin ligase SCF^Met30. It disrupts the interaction between the F-box protein Met30 and the SCF core component Skp1, thereby preventing the assembly or promoting the disassembly of the SCF^Met30 complex. The compound shows high selectivity over the closely related SCF^Cdc4 ligase.[1]

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].

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In a yeast-based high-throughput screen using a ChemBridge DiverSet library of 30,000 compounds, SMER3 was identified as a small-molecule enhancer of rapamycin (SMER). It exhibited synthetic sickness/lethality with sub-optimal concentrations of rapamycin, inhibiting yeast cell growth in combination but showing little toxicity alone at the tested concentrations.[1]
Treatment of yeast cells with SMER3 (30 µM for 30 minutes) led to dissociation of Met30 from Skp1, as determined by co-immunoprecipitation experiments.[1]
In a quantitative mass spectrometry (SILAC) analysis, treatment of yeast cells expressing HBTH-tagged Skp1 with 20 µM SMER3 for 30 minutes specifically reduced the abundance of Met30, but not other identified F-box proteins, in Skp1-bound complexes.[1]
SMER3 treatment (6 hours) induced a cell cycle arrest phenotype in yeast that resembled the phenotype of genetic inhibition of Met30, but was distinct from the phenotype induced by inhibition of Cdc4 or general SCF core components.[1]
Deletion of the MET4 gene, a transcriptional activator downstream of SCF^Met30, conferred partial resistance to the growth inhibitory effects of 4 µM SMER3 in liquid culture growth curve analysis.[1]

In Vitro Ubiquitination Assay: Components of the SCF^Met30 complex were co-expressed in insect cells and purified using a GST tag fused to Skp1. Met4 substrate was bound to the complex and co-purified. Similarly, the SCF^Cdc4 complex and its phosphorylated substrate Sic1 were purified. The purified ligase-substrate complexes for both SCF^Met30 and SCF^Cdc4 were combined and pre-incubated with DMSO or indicated concentrations of SMER3 for 20 minutes at room temperature. The ubiquitination reaction was initiated by adding E1 enzyme, E2 enzyme (Cdc34), ubiquitin, and ATP. The reaction was allowed to proceed for 25 minutes, with an aliquot taken at 5 minutes to account for the faster kinetics of SCF^Cdc4. Reaction products were analyzed by immunoblotting. SMER3 inhibited Met4 ubiquitination by SCF^Met30 in a dose-dependent manner but had no significant effect on Sic1 ubiquitination by SCF^Cdc4.[1]
Differential Scanning Fluorimetry (DSF): The Met30-Skp1 complex and Skp1 alone were purified from insect cells. Proteins were mixed with SMER3 and Sypro Orange dye in 384-well plates. Fluorescence signals were monitored using a thermal cycler system as the temperature increased. The melting temperature (Tm) shift was analyzed. SMER3 altered the Tm of the Met30-Skp1 complex but not that of Skp1 alone, indicating direct binding to the complex.[1]
Drug Affinity Responsive Target Stability (DARTS): Yeast cell lysates containing endogenous Met30-RGS6H were incubated with SMER3 or DMSO control, followed by digestion with thermolysin protease. The extent of proteolysis was analyzed by immunoblotting. SMER3 protected Met30 from protease digestion, indicating stabilization upon drug binding. Similar protection was observed for the recombinant Met30 F-box domain produced by in vitro transcription/translation.[1]

Yeast Growth Inhibition and Synergy with Rapamycin: Yeast cells were cultured in standard medium. For the primary screen, cells were treated with a sub-optimal concentration of rapamycin along with compounds from the library. Growth was assessed visually or spectrophotometrically. SMER3 was identified as a compound that caused a "no growth" phenotype only in the presence of rapamycin.[1]
Growth Curve Analysis: Yeast cells (wild-type or met4Δ mutant) were treated with 4 µM SMER3 or DMSO vehicle in liquid culture. Cell density (OD₅₉₅) was measured automatically every 30 minutes over time to generate growth curves.[1]
Spot Assay for Genetic Interaction: Temperature-sensitive yeast mutants (e.g., met30-6, cdc4-3, cdc53-1, skp1-25) were grown to mid-log phase at permissive temperature (25°C). Serial dilutions of cells were spotted onto agar plates with or without 2.5 nM rapamycin. Plates were incubated at permissive temperatures (28°C or 30°C, depending on the mutant) to assess growth sensitivity.[1]
Met4 Ubiquitination In Vivo: Yeast cells were cultured to mid-log phase (0.8 x 10⁷ cells/mL) and treated with indicated concentrations of SMER3 or rapamycin for 45 minutes. Total protein was extracted and analyzed by Western blot using an anti-Met4 antibody. Ubiquitinated forms of Met4 appear as higher molecular weight bands due to reduced proteasomal degradation.[1]
Co-immunoprecipitation for Protein-Protein Interaction: Yeast strains expressing endogenously 13Myc-tagged Met30 were treated with 30 µM SMER3 or DMSO for 30 minutes at 30°C. Cells were lysed, and 13Myc-Met30 was immunoprecipitated. The immunocomplexes were analyzed by Western blot for the presence of Skp1.[1]
SILAC-based Quantitative Mass Spectrometry: A yeast strain expressing endogenously HBTH-tagged Skp1 was grown in two cultures containing "heavy" (¹³C/¹⁵N) or "light" (¹²C/¹⁴N) isotopes of arginine and lysine. The heavy culture was treated with DMSO, and the light culture was treated with 20 µM SMER3 for 30 minutes at 30°C. Cells were then cross-linked with 1% formaldehyde for 10 minutes. Cell lysates were prepared under denaturing conditions (8M urea), mixed in equal amounts, and HBTH-Skp1-bound complexes were sequentially purified using nickel and streptavidin resins under denaturing conditions. Purified complexes were digested with trypsin, and peptides were analyzed by LC-MS/MS. Relative protein abundance was determined by comparing peptide peak intensities from light (SMER3-treated) and heavy (DMSO-treated) samples.[1]
Cell Cycle Arrest Morphology Analysis: Temperature-sensitive mutants were shifted to the restrictive temperature (37°C) for 4 hours. For Skp1 depletion, expression was repressed in dextrose medium for 12 hours. For drug treatment, cells were treated with SMER3 for 6 hours. Cell morphology was then examined by microscopy.[1]

In the initial yeast screen, at the concentrations used, SMER3 showed little toxicity by itself but exhibited synthetic sickness/lethality when combined with rapamycin.[1]

[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.

LSM-42773 is an aromatic ketone.

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SMER3 was discovered as a "small-molecule enhancer of rapamycin" (SMER) from an unbiased, phenotype-based chemical genetic screen in yeast. It establishes a functional link between the TOR signaling pathway and the SCF^Met30-mediated sulfur amino acid sensing/metabolic network. The study demonstrates the feasibility of developing specific inhibitors for individual SCF ubiquitin ligase complexes, despite their structural similarities. SMER3 represents a first-generation chemical tool for studying SCF biology and suggests potential strategies for combination therapy with rapamycin in diseases like cancer, although its direct mammalian targets and therapeutic potential require further elucidation.[1]

C11H4N4O2

224.17

224.033

67200-34-4

568763

Light yellow to green yellow solid powder

1.7±0.1 g/cm3

437.2±55.0 °C at 760 mmHg

296 °C(dec.)

218.2±31.5 °C

0.0±1.0 mmHg at 25°C

1.779

0.9

0

6

0

17

350

0

SFSSAKVWCKFRHE-UHFFFAOYSA-N

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

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

SMER3 SMER 3 SMER-3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~12.5 mg/mL (~55.76 mM)

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.

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 (Please use freshly prepared in vivo formulations for optimal results.)