Fig 1.
Hypusination in a cell-free system.
(A) Reaction of eIF5A hypusination. (B) The hypusination reaction was performed in the presence of recombinants eIF5A (5 μg), DHPS (1 μg), DOHH (1 μg), and spermidine (100 μM) with or without one of the components. Proteins were analyzed by Western Blot with the indicated antibodies.
Fig 2.
(A) Principle of the Hyp’Assay. (B) Dose-response of the quantity of eIF5A to 0 from 6 μg with 2 μg of DHPS and DOHH and 100 μM of spermidine. (C) Dose-response of DHPS or DOHH with a fixed quantity of eIF5A (3 μg) and a fixed quantity of DOHH or DHPS (2 μg). (D) Dose-response of spermidine at two pH: 8.0 and 9.0 (E) Dose-response of DMSO in the reaction of hypusination.
Fig 3.
(A) Dose-response of GC7 to 0 from 10 μM with two concentrations of spermidine (5 and 100 μM). (B) Dose-response (0 to 1 mM) of different DHPS inhibitors; Spermine, Putrescine, or BENSPM. (C) Dose-response of different DOHH inhibitors (0 to 100 μM), CPX, DFX, EDTA, Folate, and Spermidine. (D) GC7 is added during the DHPS or the DOHH step in the reaction of hypusination with 5 μM of spermidine. (E-G) A reaction of deoxyhypusination of eIF5A is performed and revelated with Hpu98. (E) Dose-response of spermidine at two pH: 8.0 and 9.0. (F-G) GC7 (F), CPX or EDTA (G) are used in the reaction of deoxyhypusination of eIF5A with 5 μM of spermidine.
Fig 4.
(A) Model of spermidine and GC7 binding site within the DHPS active site. (B) GC7 analogs structures (structures depicted in neutral form). (C) GC7 analogs synthesis.
Fig 5.
GC7 analogs are hypusination inhibitors.
(A) Hyp’Assay: Dose-response of GC7 analogs (1, 2, 3 and 4) from 0 to 1 mM in the presence of 5 μM of spermidine. (B) MCF7 or (C) HT29 were treated with 40 μM of GC7, 1, 2, 3 or 4 for 24 h. Cells were lysed and analyzed by Western blot using the indicated antibodies. A quantification of 4 and 5 experiments is provided.