Versatile Synthetic Route to Cycloheximide and Analogues That Potently Inhibit Translation Elongation

Park, Yongho; Koga, Yumi; Su, Cindy; Waterbury, Amanda L.; Johnny, Christopher L.; Liau, Brian B.

doi:10.1002/ange.201901386

Cited by 5 publications

(6 citation statements)

References 40 publications

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“…We then sought to explore how Reg3g level could be regulated by tacrolimus. Cycloheximide a protein synthesis inhibitor (Park et al, 2019) was applied to determine whether the degradation of Reg3g protein was impacted by tacrolimus. Interestingly, the time‐course analysis showed that tacrolimus treatment did not result in detectable changes in the degradation rate of Reg3g protein (Figure S2), thereby suggesting that tacrolimus might not reduce Reg3g level by causing protein degradation.…”

Section: Resultsmentioning

confidence: 99%

Regenerating islet‐derived protein 3 gamma (Reg3g) ameliorates tacrolimus‐induced pancreatic β‐cell dysfunction in mice by restoring mitochondrial function

Zhou

Xie

et al. 2022

British J Pharmacology

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Background and Purpose Tacrolimus a first‐line medication used after transplantation can induce β‐cell dysfunction, causing new‐onset diabetes mellitus (NODM). Regenerating islet‐derived protein 3 gamma (Reg3g), a member of the pancreatic regenerative gene family, has been reported to improve type 1 diabetes by promoting β‐cell regeneration. We aim to investigate the role of Reg3g in reversing tacrolimus‐induced β‐cell dysfunction and NODM in mice. Experimental Approach Circulating REG3A (the human homologue of mouse Reg3g) in heart transplantation patients treated with tacrolimus was detected. The glucose‐stimulated insulin secretion and mitochondrial functions, including mitochondria membrane potential (MMP), mitochondria calcium, ATP production, oxygen consumption rate and mitochondrial morphology were investigated in β‐cells. Additionally, effects of Reg3g on tacrolimus‐induced NODM in mice were analysed. Key Results Circulating REG3A level in heart transplantation patients with NODM significantly decreased compared with those without diabetes. Tacrolimus down‐regulated Reg3g via inhibiting STAT3‐mediated transcription activation. Moreover, Reg3g restored glucose‐stimulated insulin secretion suppressed by tacrolimus in β‐cells by improving mitochondrial functions, including increased MMP, mitochondria calcium uptake, ATP production, oxygen consumption rate and contributing to an intact mitochondrial morphology. Mechanistically, Reg3g increased accumulation of pSTAT3(Ser727) in mitochondria by activating ERK1/2‐STAT3 signalling pathway, leading to restoration of tacrolimus‐induced mitochondrial impairment. Reg3g overexpression also effectively mitigated tacrolimus‐induced NODM in mice. Conclusion and Implications Reg3g can significantly ameliorate tacrolimus‐induced β‐cell dysfunction by restoring mitochondrial function in a pSTAT3(Ser727)‐dependent manner. Our observations identify a novel Reg3g‐mediated mechanism that is involved in tacrolimus‐induced NODM and establish the novel role of Reg3g in reversing tacrolimus‐induced β‐cell dysfunction.

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Section: Resultsmentioning

confidence: 99%

Regenerating islet‐derived protein 3 gamma (Reg3g) ameliorates tacrolimus‐induced pancreatic β‐cell dysfunction in mice by restoring mitochondrial function

Zhou

Xie

et al. 2022

British J Pharmacology

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“…Cycloheximide (CHX) is a commonly used protein synthesis inhibitor [29] and a CHX‐chase assay was conducted using CHX (Sigma, USA). After the si‐MEG3 was transfected into BV2 cells, the cells were mixed with 20 μg/ml CHX and the protein level of HuR was measured via Western blot at 0, 10, 20, and 40 min.…”

Section: Methodsmentioning

confidence: 99%

lncRNA MEG3 restrained the M1 polarization of microglia in acute spinal cord injury through the HuR/A20/NF‐κB axis

et al. 2022

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The M1 polarization of microglia and neuroinflammation restrict the treatment of acute spinal cord injury (ASCI), and long non‐coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) expression is lessened in ASCI. However, the function and mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI are unclear. The expressions of lncRNA MEG3 in ASCI mouse spinal cord tissues and lipopolysaccharide (LPS)‐treated primary microglia and BV2 cells were quantified through a quantitative real‐time polymerase chain reaction. In‐vitro assays were conducted to explore the function of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. RNA degradation, RNA immunoprecipitation, RNA pull‐down, cycloheximide‐chase, and ubiquitination analyses were carried out to probe into the mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. The lncRNA MEG3 expression was lessened in the ASCI mouse spinal cord tissues and LPS‐treated primary microglia and BV2 cells, and the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia and the neuroinflammation by regulating the NF‐κB signaling pathway. For the investigation of the potential mechanism of such, the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF‐κB axis and boosted the motor function recovery and neuroinflammation relief in the mice with SCI. The overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF‐κB axis.

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confidence: 99%

“…The amino group was then acylated through peptide coupling with various carboxylic acids to afford CHX C13-amide derivatives. Many of these amide derivatives were effective translation inhibitors, as assessed by a cellular assay for translation that measures incorporation of 2′- O -propargyl puromycin (OPP) (Figure B). , In particular, this includes benzamide 8 (IC 50 = 63 ± 4.5 nM), which is approximately 40 times more active than CHX (Figure C). Derivatives containing simple modifications to the benzamide were not as effective (Figure B).…”

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confidence: 99%

“…Motivated by these questions, we completed a total synthesis of CHX that enabled identification of analogs with increased potencies, including benzylester 2 and N-hydroxysuccinimide ester 3 (Figure 1A). 9 The relative stereochemistry of the C13methyl group was essential for the enhanced inhibitory activity of the C13-modified derivatives. We speculated that the C13modified derivatives form additional stabilizing interactions within the binding pocket similarly to lactimidomycin (LTM, 4), another E-site inhibitor that is more potent than CHX but only blocks translation at the initiating codon (Figure 1A).…”

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confidence: 99%

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Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation

Koga¹,

Hoang²,

Park³

et al. 2021

J. Am. Chem. Soc.

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Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX’s extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3′ end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

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Versatile Synthetic Route to Cycloheximide and Analogues That Potently Inhibit Translation Elongation

Cited by 5 publications

References 40 publications

Regenerating islet‐derived protein 3 gamma (Reg3g) ameliorates tacrolimus‐induced pancreatic β‐cell dysfunction in mice by restoring mitochondrial function

Regenerating islet‐derived protein 3 gamma (Reg3g) ameliorates tacrolimus‐induced pancreatic β‐cell dysfunction in mice by restoring mitochondrial function

lncRNA MEG3 restrained the M1 polarization of microglia in acute spinal cord injury through the HuR/A20/NF‐κB axis

Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation

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