High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases

Hong, Jun Young; Malgapo, Martin Ian P.; Liu, Yinong; Yang, Min; Zhu, Chengliang; Zhang, Xiaoyu; Tolbert, Patricia L.; Linder, Maurine E.; Lin, Hening

doi:10.1021/acschembio.1c00258

Cited by 10 publications

(12 citation statements)

References 22 publications

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“…Because the importance of DHHC7 in the positive feedback loop, DHHC7 could be the ideal target for the HCC treatment. Although, as a known palmitoyl acyltransferase inhibitor, 2-BP is reported to have an antitumor effect ( 38 ), it is weak and nonspecific (IC 50 is around 16 μM for DHHC7 in vitro) ( 24 , 25 ). A potential synthetic inhibitor, MY-D-4, based on the acylation-coupled lipophilic induction of the polarization method, has an IC 50 of around 1 μM for DHHC7 ( 25 ).…”

Section: Discussionmentioning

confidence: 99%

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

Jiang,

Xu,

Zhu

et al. 2023

Sci. Signal.

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Constitutive activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) contributes to the malignancy of many cancers such as hepatocellular carcinoma (HCC) and is associated with poor prognosis. STAT3 activity is increased by the reversible palmitoylation of Cys 108 by the palmitoyltransferase DHHC7 (encoded by ZDHHC7 ). Here, we investigated the consequences of S-palmitoylation of STAT3 in HCC. Increased ZDHHC7 abundance in HCC cases was associated with poor prognosis, as revealed by bioinformatics analysis of patient data. In HepG2 cells in vitro, DHHC7-mediated palmitoylation enhanced the expression of STAT3 target genes, including HIF1A , which encodes the hypoxia-inducible transcription factor HIF1α. Inhibiting DHHC7 decreased the S-palmitoylation of STAT3 and decreased HIF1α abundance. Furthermore, stabilization of HIF1α by cyclin-dependent kinase 5 (CDK5) enabled it to promote the expression of ZDHHC7 , which generated a positive feedback loop between DHHC7, STAT3, and HIF1α. Perturbing this loop reduced the growth of HCC cells in vivo. Moreover, DHHC7, STAT3, and HIF1α were all abundant in human HCC tissues. Our study identifies a pathway connecting these proteins that is initiated by S-palmitoylation, which may be broadly applicable to understanding the role of this modification in cancer.

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Section: Discussionmentioning

confidence: 99%

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

Jiang,

Xu,

Zhu

et al. 2023

Sci. Signal.

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“…A full HTS was thereafter performed to identify inhibitors of the activity of the N -palmitoyltransferase Hhat and will be reported in due course according to the authors. Acyl-cLIP was also tested for the detection of zDHHC3, zDHHC7, and zDHHC20 activities, showing that this assay can be adapted for the screening of zDHHC enzyme modulators ( 33 , 39 , 57 ).…”

Section: Discussionmentioning

confidence: 99%

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Salaün¹,

Takizawa²,

Galindo³

et al. 2022

Journal of Biological Chemistry

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“…A full HTS was thereafter performed to identify inhibitors of the activity of the N-palmitoyltransferase Hhat and will be reported in due course according to the authors. Acyl-cLIP was also tested for the detection of zDHHC3, 7 and 20 activities, showing that this assay can be adapted for the screening of zDHHC enzyme modulators (33,39,49).…”

Section: Discussionmentioning

confidence: 99%

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Salaün

Takizawa

Galindo

et al. 2022

Preprint

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Protein S-acylation is a reversible post-translational modification that modulates the localisation and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC domain-containing proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyse S-acylation in a two-step process involving auto-acylation of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. In this study, we developed and implemented a novel FRET-based high throughput assay for the discovery of compounds that interfere with auto-acylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. A screen of >350,000 compounds identified two related tetrazole containing compounds (TTZ-1 and -2) that inhibited both zDHHC2 auto-acylation and substrate S-acylation in cell-free systems. Furthermore, these compounds were also active in HEK293T cells, where they inhibited substrate S-acylation mediated by different zDHHC enzymes, with some apparent isoform selectivity. Resynthesis of the hit compounds confirmed their activity, providing sufficient quantities of material for further investigations. The assays developed herein provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating the cellular activities of S-acylation and zDHHC enzymes.

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High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases

Cited by 10 publications

References 22 publications

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

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