title: Enantiomers of 3-methylspermidine selectively modulate deoxyhypusine synthesis and reveal important determinants for spermidine transport.
abstract: Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conserved-hypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (S)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (R)- over the (S)-enantiomer as a substrate. (R)-3-MeSpd competed with [(14)C]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (Ki = 141 μM vs 19 μM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (S)-3-MeSpd, but not DHS substrate (R)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis.
authors.affiliation: †School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Mervi T
authors.initials: MT
authors.lastname: Hyvönen
authors.affiliation: ‡Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.
authors.forename: Maxim
authors.initials: M
authors.lastname: Khomutov
authors.affiliation: §Université Paris Diderot - Paris, U.F.R. Sciences du Vivant, 35 rue Hélène Brion, 75205 Paris CEDEX 13, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Petit
authors.affiliation: †School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Janne
authors.initials: J
authors.lastname: Weisell
authors.affiliation: ‡Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Kochetkov
authors.affiliation: †School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Leena
authors.initials: L
authors.lastname: Alhonen
authors.affiliation: †School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Jouko
authors.initials: J
authors.lastname: Vepsäläinen
authors.affiliation: ‡Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.
authors.forename: Alex R
authors.initials: AR
authors.lastname: Khomutov
authors.affiliation: †School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Tuomo A
authors.initials: TA
authors.lastname: Keinänen
mesh_terms: D001692:Biological Transport; D045744:Cell Line, Tumor; D000518:Eflornithine / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D008297:Male; D065108:Ornithine Decarboxylase Inhibitors / Q000494:pharmacology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D000518:Eflornithine; D008239:Lysine; D065108:Ornithine Decarboxylase Inhibitors; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D013095:Spermidine; D000097574:Eukaryotic Translation Initiation Factor 5A; C560108:1,8-diamino-3-methyl-4-azaoctane; C100028:hypusine; C034924:deoxyhypusine; C063507:deoxyhypusine synthase