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. 2019 Mar 18;9(1):4737.
doi: 10.1038/s41598-019-41187-0.

Anti-alcohol Abuse Drug Disulfiram Inhibits Human PHGDH via Disruption of Its Active Tetrameric Form Through a Specific Cysteine Oxidation

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Free PMC article

Anti-alcohol Abuse Drug Disulfiram Inhibits Human PHGDH via Disruption of Its Active Tetrameric Form Through a Specific Cysteine Oxidation

Quentin Spillier et al. Sci Rep. .
Free PMC article

Abstract

Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of PHGDH inhibition by DSF. Residual activity percentage of PHGDH (A) upon incubation with DSF (50 µM) for the indicated times and (B) after the rapid dilution assay experiment with DSF (50 µM). All experiments values were performed in triplicates at each compound dilution and error bars show the standard deviation. Data were collected at 37 °C with a PHGDH concentration of 12 ng/µL in 50 mM Tris and 1 mM EDTA at pH 8.5.
Figure 2
Figure 2
(A) Dose-response curve of CuET on WT PHGDH. (B) PHGDH inhibition (IC50) of CuETa. All experiments to determine IC50 values were performed in triplicates at each compound dilution.Under bracket: 95% confidence interval. Data were collected at 37 °C with a PHGDH concentration of 12 ng/µL in 50 mM Tris and 1 mM EDTA at pH 8.5.
Figure 3
Figure 3
Proposed Mechanism of Interaction between PHGDH and (A) Disulfiram (B) 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (DSF and AMS reacts with sulfhydryl groups of free (reduced) cysteine residues forming a mixed disulfide). Masses after coupling are given for only one reactive cysteine.
Figure 4
Figure 4
Western-blot of the different conditions. All samples were incubated with DSF and/or AMS during 1 h at room temperature before running on a 12% SDS-Tris-Glycine Page gel. A PHGDH. B PHGDH with 2 mM AMS. C PHGDH with 100 µM DSF. D PHGDH with 100 µM DSF and 2 mM AMS. Original uncropped Western-blot is available in the Supplementary Information File Fig. S3.
Figure 5
Figure 5
Percentage of the three oxidized cysteine residues (C111, C116 and C281) at tested concentrations. Determined from the peptide spectrum matches (PSMs) of their precursors after trypsinization and analysis by nanoUHPLC/MS. DSF at various concentrations was incubated with PHGDH in 50 mM Tris and 1 mM EDTA at pH 8.5.
Figure 6
Figure 6
(A) Overview of the PHGDH cysteine residues (111, 116 and 281) (PDB code 2G76). (B) Zoomed-in region highlighting the targeted Cys116 on the two monomers.
Figure 7
Figure 7
Cross-linking experiment of PHGDH with BS3 at various DSF concentrations A. MW marker. B 0 µM. C 1 µM. D 5 µM. E 10 µM. F 50 µM. G 100 µM. H 250 µM. I 500 µM.). PHGDH was incubated with DSF during 30′ before cross-linking. Lane B was used as control without DSF. Lane A (MW marker) was used to deduce the oligomerization state of PHGDH. Original exposure of the uncropped gel is available in the Supplementary Information File (Fig. S4).
Figure 8
Figure 8
(A) Representative immunoblot for PHGDH on UM-UC-3 cancer cells; (B) Dose-response curves of DSF. (C) UM-UC-3-PHGDH− and UM-UC-3-PHGDH+ cell proliferation inhibition by DSF. All experiments to determine IC50 values were performed in n = 6 at each compound dilution, and all IC50 values were averaged on two or more independent experiments. Under bracket: 95% confidence interval.

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