Cycloheximide

Cycloheximide (Naramycin A), an antifungal antibiotic found in Streptomyces griseus, is an eukaryote protein synthesis inhibitor, with IC₅₀ values of 532.5 nM and 2880 nM for protein synthesis and RNA synthesis in vivo, respectively. Cycloheximide binds to the E-site of the 60S subunit of the ribosome during the protein synthesis process in eukaryotic cells, blocking the ribosome translocation process mediated by eEF2 and preventing the synthesis of new proteins. Cycloheximide suppresses ferroptosis and inhibits autophagy. Cycloheximide is promising for research of cancer and neuro-related diseases^{[1][2][3][4][5][6][7][8]}. IC50 & Target:IC50: 532.5 nM (protein synthesis), 2.88 μM (RNA synthesis)^[1] In Vitro:Cycloheximide (200 µM, 2 or 10 min) inhibits eEF2-mediated tRNA translocation in vitro^[1].
Cycloheximide (35 μM, 1 or 4 h) inhibits de novo protein synthesis in rabbit aortic smooth muscle cells and J774A.1 macrophages^[4].
Cycloheximide (0.5-1 μg/mL, 1-3 days) inhibits the proliferation of C6 glioma cells, induces cell cycle arrest at G1 and S phases, and promotes cell differentiation in C6 glioma cells^[5].
Notes:
1. Passage number, cell state, and cell density can significantly affect experimental outcomes. It is crucial to optimize the cell condition before conducting experiments (HCT 116 adherent cells tend to have smaller morphology and are prone to aggregation at high densities; therefore, ensure an even cell plating is achieved).
2. Different cell lines may exhibit varying sensitivities to Cycloheximide, which could result in discrepancies in the detection outcomes. It is recommended to consult the literature and perform preliminary experiments to determine optimal drug treatment times, concentrations, and antibody conditions, ensuring that the experimental protocol is both reasonable and feasible.
3. Drugs should be prepared fresh for each use. Ultrasonic heating may be employed to assist solubilization until the solution appears clear and transparent. Any remaining solution after the initial experiment should be aliquoted and stored at -20°C.
4. The detection indicators MDM2 and c-Myc proteins may display double bands, potentially indicating the occurrence of post-translational modifications:
a. C-Myc undergoes multiple post-translational modifications (including phosphorylation, acetylation, ubiquitination, and glycosylation at several sites). Consequently, discrepancies between the actual detected molecular weight and the predicted band size can occur, along with the appearance of double bands^[6].
b. MDM2 has multiple phosphorylation sites, with a predicted molecular weight of 55 kDa. However, factors such as the presence of various isoforms, post-translational modifications, and the cleavage effect from p53 activation can result in measured molecular weights of approximately 90 kDa and 60 kDa, potentially leading to the observation of multiple bands^[7].
In Vivo:Cycloheximide (30, 60 or 120 mg/kg, injected before 200 μA electric shock training) has a significant effect on the latency of the memory test in mice (P<0.001). In the saline controls, this level of electric shock leads to significantly higher latencies in the test trial than those at training. Cycloheximide (30 mg/kg, injection) leads to significantly higher latencies in the test trial than those seen in the saline control group. The latency of mice injected with higher doses of Cycloheximide in the test trial is comparable to that of the saline group, the higher doses neither enhanced nor impaired memory under these conditions, resulting in an inverted U-shaped dose-response curve for Cycloheximide to enhance memory^[2].
Cycloheximide (200 mg/kg, i.p., 20 min prior to the administration of KA) inhibits the phosphorylation of extracellular signal-regulated protein kinase (p-ERK), c-Jun N-terminal kinase 1 (p-JNK1), and calcium/calmodulin-dependent protein kinase II (p-CaMK II), as well as the increased expression of c-Fos and c-Jun proteins induced by Kainic acid (KA) (HY-N2309) in primary hippocampal neurons of mice^[8].