protein synthesis ( IC50 = 532.5 nM ); RNA synthesis ( IC50 = 2.88 μM )
Eukaryotic ribosomes (reticulocyte ribosomes): Cycloheximide inhibits peptide synthesis on reticulocyte ribosomes [3]
- Protein synthesis machinery in eukaryotic cells: Cycloheximide acts as a protein synthesis inhibitor by targeting the translational process [1][4]
- TORC1 signaling pathway (indirect effect): Cycloheximide reactivates TORC1 signaling under nutrient-limiting conditions in budding yeast, leading to transcriptional upregulation of ribosome biogenesis genes [4]

In vitro activity: Cyclohexamide is a protein synthesis inhibitor that lessens the amount of phosphatidylserine exposure and loss of ΔΨm from apoptosis/necrosis that results from GSIV-induced host cell death.[1]

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1. In grouper fin cells (GF-1) infected with Giant seaperch iridovirus (GSIV), treatment with Cycloheximide (CHX) attenuated phosphatidylserine (PS) exposure and loss of mitochondrial membrane potential (ΔΨm) induced by apoptosis/necrosis; cell viability assays showed that GSIV infection caused a steady increase in dead GF-1 cells (11% at 2 dpi to 67% at 5 dpi), and Annexin V/PI staining revealed GSIV-induced apoptosis increased from 4% at 1 dpi to 29% at 5 dpi, with post-apoptotic necrosis apparent at 4-5 dpi; Cycloheximide suppressed these GSIV-induced cell death processes by inhibiting de novo protein synthesis[1]
2. In budding yeast under nutrient-limiting conditions (amino acid starvation, yeast metabolic cycle, meiosis), pretreatment with Cycloheximide induced rapid transcriptional upregulation of hundreds of ribosome biogenesis (ribi) genes; it also prevented translation of these newly transcribed ribi mRNAs, leading to an apparent decrease in translation efficiency (TE) of ribi genes; this effect was abolished when Cycloheximide pretreatment was omitted; the transcriptional upregulation of ribi genes by Cycloheximide required TORC1 signaling and was modulated by genetic background and drug delivery vehicle (ethanol vs. DMSO); Cycloheximide caused a decrease in ribi gene footprints and an increase in ribi mRNA levels in starved yeast cells, while non-ribi genes were relatively unaffected[4]
3. Cycloheximide inhibited peptide synthesis on reticulocyte ribosomes, with the mechanism of action related to interference with the eukaryotic translational process [3]

Cyclohexamide is a protein synthesis inhibitor that affects memory by changing memory formation modulators as a byproduct of inhibiting protein synthesis.[2]

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1. In ICR mice subjected to inhibitory avoidance training:
- When administered at 120 mg/kg subcutaneously 30 min prior to training with footshock intensities of 100, 150, 250, or 300 μA (1 s duration), Cycloheximide impaired memory in a footshock intensity-dependent manner, with more severe memory impairment at higher shock intensities (300 μA); saline control mice showed increasing memory latencies with higher shock intensities[2]
- When administered at doses of 30, 60, or 120 mg/kg subcutaneously 30 min prior to training with a 200 μA (1 s duration) footshock, Cycloheximide enhanced memory in an inverted-U dose-response manner, with the maximum enhancement at 30 mg/kg (P<0.05 vs. saline control)[2]

Cycloheximide (also known as naramycin A) is an inhibitor of eukaryotic protein synthesis, with in vivo IC50 values for protein synthesis of 532.5 nM and RNA synthesis of 2880 nM, respectively.

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1. Peptide synthesis inhibition assay on reticulocyte ribosomes: Reticulocyte ribosome preparations were incubated with Cycloheximide at varying concentrations (no specific concentrations provided) in a reaction system supporting peptide synthesis; the rate of peptide chain elongation or total peptide synthesis was measured (detection method not specified) to evaluate the inhibitory effect of Cycloheximide on ribosome-mediated peptide synthesis; the assay confirmed that Cycloheximide interfered with peptide synthesis on reticulocyte ribosomes, though the exact inhibitory mechanism was not fully elucidated in this assay[3]

GF-1 cell monolayers (4.0 mL, 10⁵ cells/mL) grown on 60-mm Petri dishes are cultured for a minimum of 20 hours, followed by two PBS rinses. The cells are then treated with cycloheximide (CHX, 0.33 µg/mL) and BKA (20 µM) ANT inhibitor for 0–5 days. Lastly, the GSIV K1 strain (multiplicity of infection [m.o.i. ] = 5) is infected for 0–5 days after the initial step (dpi). Following the incubation period, every sample is taken out of the medium and given a PBS wash. After that, cells are incubated in 100 µL of staining solution for 10 to 15 minutes.

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1. GSIV-infected GF-1 cell viability and apoptosis/necrosis assay:
- Cell culture: GF-1 cells were cultured under standard conditions and infected with GSIV at a multiplicity of infection (MOI) not specified; cells were treated with Cycloheximide (concentration not specified) or vehicle control at appropriate time points post-infection.
- Viability assessment: Cell viability was measured at 2, 3, 4, and 5 days post-infection (dpi) using a viability assay (method not specified), with dead cell percentages calculated (11% at 2 dpi to 67% at 5 dpi in infected, untreated cells).
- Apoptosis/necrosis detection: Annexin V/PI staining was performed at 1-5 dpi, followed by flow cytometry (FACS) analysis to quantify apoptotic cells (4% at 1 dpi to 29% at 5 dpi) and post-apoptotic necrotic cells (apparent at 4-5 dpi).
- Mitochondrial membrane potential (ΔΨm) measurement: JC-1 dye was used to detect ΔΨm loss in infected cells at 1-3 dpi (42% at 1 dpi, 98% at 3 dpi); the effect of Cycloheximide on ΔΨm loss and PS exposure was evaluated by comparing treated and untreated infected cells[1]
2. Budding yeast translation efficiency (TE) and mRNA level assay:
- Cell culture: Budding yeast cells were cultured in replete or nutrient-limiting (amino acid starvation) medium; cells were pretreated with Cycloheximide (concentration not specified) or vehicle control before sample collection.
- Ribosome profiling and RNA-seq: Ribosome footprints and total mRNA were isolated from yeast cells at different time points during the yeast metabolic cycle, meiosis, or amino acid starvation; sequencing was performed to quantify ribi and non-ribi gene footprints and mRNA levels, and TE (ribosome footprints/mRNA) was calculated.
- Polysome gradient analysis: mRNA position in polysome gradients was evaluated to confirm TE changes; the effect of Cycloheximide on ribi gene transcription was assessed by monitoring NOP2 mRNA abundance over time (faster and greater accumulation with ethanol vs. DMSO as delivery vehicle); reporter gene assays with NOP2 promoter/coding sequence/UTR elements were used to confirm that Cycloheximide influenced ribi TE via transcriptional activation (only when NOP2 promoter was driving expression)[4]

Mice: In this experiment, male ICR mice that are around two months old are utilized. IP cycloheximide is given in concentrations of 0, 30, 60, or 120 mg/kg (saline controls). Injections of cycloheximide are given half an hour before training. Mice amnesia is commonly studied at a dose of 120 mg/kg. Observe that rats receive much lower amnestic Cycloheximide doses (1-3 mg/kg) than mice do, which is consistent with a comparable difference in the LD50s of mice and rats. As measured 30–60 minutes after injection, cycloheximide doses of 120–150 mg/kg cause about 95% inhibition of brain protein synthesis, while doses of 30 mg/kg cause about 80% inhibition. Rats: Methoxyflurane anesthesia is used to perform unilateral carotid artery ligation on 7-day-old Sprague Dawley rat pups. The right common carotid artery is permanently ligated with 4-0 silk after a midline incision in the neck. Every animal received surgery for a maximum of five minutes. Rats are returned to their mother for recovery and feeding for two hours after surgery. The pups are then placed in an airtight chamber that is partially submerged in a temperature-controlled water bath to maintain a constant 36°C inside during the 100-minute hypoxic exposure period (8% O₂, 92% N₂). The rat pups in the HI with Cycloheximide treatment group receive an intraperitoneal injection of Cycloheximide at a dose of 0.6 mg/kg at0,6,12, or 24 hours of recovery, while the HI control group receives an equal volume of normal saline. Once the rat pups have been returned to their dam, they are sacrificed. The entire brain tissue is removed under deep intraperitoneal pentobarbital anesthesia (60 mg/kg) for triphenyl tetrazolium chloride (TTC) and flow cytometry at 48 and 72 hours after HI, respectively.

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1. Inhibitory avoidance training and memory testing in ICR mice:
- Experimental animals: Male ICR mice (no specific weight/age provided).
- Drug preparation: Cycloheximide was dissolved in a suitable vehicle (not specified) to prepare doses of 30, 60, 120 mg/kg; saline was used as the control vehicle.
- Administration route and timing: Subcutaneous injection 30 min prior to inhibitory avoidance training.
- Training protocol: Mice were subjected to inhibitory avoidance training with footshock intensities of 100, 150, 200, 250, or 300 μA (1 s duration).
- Memory testing: Memory was assessed 48 h after training by measuring avoidance latencies; the effect of Cycloheximide on memory was evaluated by comparing latencies between drug-treated and saline-treated groups[2]

Interactions
Chlordane has been reported to cause a rapid loss of activity in cyclohexylimide. Pretreatment of rats with spironolactone and ethinylestradiol protects them from the toxicity of the bactericide cyclohexylimide. Non-human Toxicity Values Oral LD50 in rats: 2 mg/kg Intraperitoneal LD50 in rats: 3700 μg/kg Subcutaneous LD50 in rats: 2500 μg/kg Intravenous LD50 in rats: 2 mg/kg For more non-human toxicity values (complete data) for cyclohexylimide (out of 10), please visit the HSDB record page.

[1]. Virus Res . 2016 Feb 2:213:37-45.

[2]. Behav Brain Res . 2012 Aug 1;233(2):293-7.

[3]. J Biol Chem . 1971 Jan 10;246(1):174-81.

[4]. Nucleic Acids Res. 2019 Jun 4;47(10):4974-4985.

Cyclohexylimide may be developmentally toxic depending on state or federal labeling requirements. Cyclohexylimide is a colorless crystalline solid. It is used as a bactericide and anticancer drug. (EPA, 1998) Cyclohexylimide is a dicarboxylimide with the chemical formula 4-(2-hydroxyethyl)piperidine-2,6-dione, in which a hydrogen atom on the carbon atom connecting the hydroxyl group is replaced by a 3,5-dimethyl-2-oxocyclohexyl group. It is an antibiotic produced by Streptomyces griseus. It has multiple functions, including as a bacterial metabolite, protein synthesis inhibitor, neuroprotective agent, anticoronavirus agent, and ferroptosis inhibitor. It belongs to the piperidinone class of compounds and is a piperidinium antibiotic, antibiotic bactericide, dicarboxylimide, secondary alcohol, and cyclic ketone. It is functionally related to piperidinium-2,6-dione. Cyclohexylimide has been reported in Streptomyces, Streptomyces powdery, and Streptomyces griseus, and relevant data are available. Cycloheximide is an antibiotic substance isolated from streptomycin-producing Streptomyces griseus strains. Its mechanism of action is to inhibit elongation during protein synthesis. The study investigated the stimulation of ribonucleoprotein complex translocation to the cytoplasm after administration of cycloheximide to rats. After administration of 2 mg/kg cycloheximide to rats, the total ribonucleoprotein complex content in the liver decreased within 2 hours. The overall decrease was due to increased protein translocation to the cytoplasm, rather than decreased synthesis. Other results indicated that gene transcription continued during the protein synthesis inhibition period, and gene products were translocated to the cytoplasm for translation. Cycloheximide (2-5 mg/kg body weight) completely inhibited protein synthesis in rat liver within 30 minutes, and nucleoprotein labeling was also strongly inhibited. Under these conditions, the AMT and orotic acid-(14)C labeling of nucleolar 45S precursor rRNA remained unchanged for at least 4 hours, indicating that the synthesis and processing rate of 45S precursor rRNA was not initially significantly altered. In the early stages of cyclohexylimide's action, the 45S precursor rRNA processing pathway undergoes dramatic changes. The orientation of ribosomal precursor rRNA along the alternative processing pathway is strictly controlled by the continuous supply of key proteins. Cyclohexylimide is a potent inhibitor of protein synthesis in fungi and animals. It leads to increased adrenal RNA, increased glucocorticoid production, and decreased pyruvate utilization in separated adipose tissue.

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Therapeutic Uses
Antibiotics, antifungals; protein synthesis inhibitors
Drugs (Veterinary Drugs): Antibiotics, antifungals, trichomonads…particularly effective against canine malignant lymphoma.
Cyclohexylimide…was used to treat cryptococcal infections (Cryptococcus neoformans) before the advent of amphotericin B.

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1. Cycloheximide (CHX) is a glutarimide antibiotic and a classic eukaryotic protein synthesis inhibitor, primarily targeting translation on ribosomes; it does not inhibit prokaryotic protein synthesis [3]
2. Cycloheximide inhibits GSIV-induced mitochondrial-mediated apoptosis/necrosis in GF-1 fish cells by inhibiting the synthesis of newborn proteins, and its effect is synergistic with that of bonclamide (BKA, an adenine nucleotide translocase inhibitor), which can reduce PS exposure and ΔΨm loss [1]
3. The effect of cycloheximide on mouse memory is bidirectional (impairment and enhancement) and depends on the dose and the intensity of the training foot shock: high dose (120 mg/kg) at high shock intensity (300 μA) impairs memory, while low dose (30 mg/kg) at moderate shock intensity (200 μA) results in an inverted U-shaped effect. The dose-response mechanism enhances memory; this suggests that cycloheximide modulates memory by altering memory formation regulators, which is a secondary consequence of protein synthesis inhibition rather than a direct interference with the protein synthesis required for training initiation for memory formation [2]. 4. Under nutritional restriction, cycloheximide induces TORC1-dependent upregulation of ribosomal genes while inhibiting their translation, thereby distorting the measurement of mRNA levels and translation efficiency (TE) in budding yeast; this highlights a key consideration when using cycloheximide in experiments measuring TE or mRNA levels, and cycloheximide pretreatment should be avoided to obtain accurate TE data [4]. 5. Cycloheximide-induced transcription of yeast ribosomal genes is associated with nuclear output of transcription factors (Dot6, Tod6, Stb3) after drug treatment, with Stb3 showing the strongest response [4].

C15H23NO4

281.35

281.162

C, 64.04; H, 8.24; N, 4.98; O, 22.75

66-81-9

6197

White to off-white solid powder

1.1±0.1 g/cm3

491.8±10.0 °C at 760 mmHg

111-116 °C

251.2±19.0 °C

0.0±2.8 mmHg at 25°C

1.499

Streptomyces

0.56

2

4

3

20

404

4

O=C(CC(C[C@H]([C@H]1C([C@@H](C)C[C@H](C)C1)=O)O)C2)NC2=O

YPHMISFOHDHNIV-FSZOTQKASA-N

InChI=1S/C15H23NO4/c1-8-3-9(2)15(20)11(4-8)12(17)5-10-6-13(18)16-14(19)7-10/h8-12,17H,3-7H2,1-2H3,(H,16,18,19)/t8-,9-,11-,12+/m0/s1

4-[(2R)-2-[(1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl]-2-hydroxyethyl]piperidine-2,6-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.39 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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