Young Bok Lee

Hypusine Is Essential for Eukaryotic Cell Proliferation

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)-L-lysine] is a most remarkable amino acid, occurring in all eukaryotic cells, yet occupying only a single position in one protein, eukaryotic protein synthesis initiation factor 5A (eIF-5A). The unusual structure of hypusine, its derivation from the polyamine spermidine, and its increased formation in response to growth stimulation, as well as its limited occurrence in the highly conserved amino acid sequence of eIF-5A, have aroused keen interest in the biological significance of its existence and in its relationship to eIF-5A function.

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation

SummaryThe unusual amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to itsε-amino group to form the intermediate, deoxyhypusine [Nε-(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.

Branched-chain and unsaturated 1,7-Diaminoheptane derivatives as deoxyhypusine synthase inhibitors

Deoxyhypusine synthase catalyzes the first step in the posttranslational biosynthesis of the unusual amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A (eIF-5A). eIF-5A and its single hypusine residue are essential for cell proliferation. Two series of 1,7-diaminoheptane derivatives were prepared and tested as inhibitors of human deoxyhypusine synthase. These include branched-chain saturated derivatives and both branched- and straight-chain unsaturated derivatives providing size and positional variation in branching and different torsional constraints. Of the branched-chain compounds, 7-amino-1-guanidinooctane (39) proved to be the most potent inhibitor in vitro (IC50, 34 nM), while 1,7-diamino-trans-hept-3-ene (20a) displayed the greatest inhibition (IC50, 0.7 microM) among the unsaturated compounds. Compound 39 also provided effective inhibition of hypusine production in Chinese hamster ovary cells in culture. Considerations of the in vitro inhibition data reported here, along with earlier findings, allowed some speculation concerning the conformation of the substrate spermidine during its productive interaction at the active site of deoxyhypusine synthase.

Synthesis and anticancer activity of new 1-[(5 or 6-substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives

A series of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives were synthesized and evaluated as an anticancer agent. From screening of quinoxalinyl-piperazine compound library, we identified that many compounds inhibited proliferation of various human cancer cells at nanomolar concentrations. Among them, one of the fluoro quinoxalinyl-piperazine derivatives showed its IC(50) values ranging from 11 to 21nΜ in the growth inhibition of cancer cells. This compound also displayed a more potent effect than paclitaxel against paclitaxel resistant HCT-15 colorectal carcinoma cells. The potency of this novel compound was further confirmed with the synergistic cytotoxic effect with several known cancer drugs such as paclitaxel, doxorubicin, cisplatin, gemcitabine or 5-fluorouracil in cancer cells. This strong cell killing effect was derived from the induction of apoptosis. Mechanistic studies have shown that this quinoxalinyl-piperazine compound is a G2/M-specific cell cycle inhibitor and inhibits anti-apoptotic Bcl-2 protein with p21 induction. Thus the results suggest that our compound has potential use in the growth inhibition of drug resistant cancer cells and the combination therapy with other clinically approved anticancer agents as well.

Enzyme-substrate intermediate at a specific lysine residue is required for deoxyhypusine synthesis. The role of Lys329 in human deoxyhypusine synthase.

Deoxyhypusine synthase catalyzes the first step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A. We recently reported biochemical evidence for a covalent enzyme-substrate intermediate involving a specific lysine residue (Lys329) in human deoxyhypusine synthase (Wolff, E. C., Folk, J. E., and Park, M. H. (1997) J. Biol. Chem. 272, 15865–15871). In an effort to evaluate the role of this enzyme-substrate intermediate in catalysis, we carried out site-directed mutagenesis (Lys to Arg and/or Ala) of the conserved lysine residues in human deoxyhypusine synthase. A drastic reduction in enzyme intermediate formation and enzymatic activities was observed with mutant proteins with substitution at Lys287 but not with those with mutations at residues 141, 156, 205, 212, 226, 251, or 338. Lys to Ala or Lys to Arg substitution at Lys329 totally abolished covalent enzyme-substrate intermediate formation and deoxyhypusine synthesis activity, indicating that Lys329 is the unique site for the enzyme intermediate and that it is absolutely required for deoxyhypusine synthesis in the eukaryotic translation initiation factor 5A precursor. The K329A mutant showed spermidine cleavage activity (∼6% of the wild type enzyme) suggesting that in contrast to deoxyhypusine synthesis, spermidine cleavage can occur without enzyme intermediate formation.

Synthesis, anticancer activity and pharmacokinetic analysis of 1-[(substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives

Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives published in Bioorg. Med. Chem.2010, 18, 7966, we further explored the synthesis of 7 or 8-substituted quinoxalinyl piperazine derivatives. From in vitro studies of the newly synthesized compounds using human cancer cell lines, we identified some of the 8-substituted compounds, for example 6p, 6q and 6r, which inhibited the proliferation of various human cancer cells at nanomolar concentrations. Compound 6r, in particular, showed the lowest IC(50) values, ranging from 6.1 to 17nM, in inhibition of the growth of cancer cells, which is better than compound 6k (compound 25 in the reference cited above). In order to select and develop a leading compound among the quinoxaline compounds with substitutions on positions 5, 6, 7 or 8, the compounds comparable to compound 6k in in vitro cancer cell growth inhibition were chosen and their pharmacokinetic properties were evaluated in rats. In these studies, compound 6k showed the highest oral bioavailability of 83.4%, and compounds 6j and 6q followed, with 77.8% and 57.6%, respectively. From the results of in vitro growth inhibitory activities and the pharmacokinetic study, compound 6k is suggested for further development as an orally deliverable anticancer drug.

A Novel Anti‐Cancer Agent, 1‐(3,5‐Dimethoxyphenyl)‐4‐[(6‐Fluoro‐2‐Methoxyquinoxalin‐3‐yl)Aminocarbonyl] Piperazine (RX‐5902), Interferes With β‐Catenin Function Through Y593 Phospho‐p68 RNA Helicase

1‐(3,5‐Dimethoxyphenyl)‐4‐[(6‐fluoro‐2‐methoxyquinoxalin‐3‐yl)aminocarbonyl] piperazine (RX‐5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX‐5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of 3H‐labeled RX‐5902 to Y593 phospho‐p68 RNA helicase. We further demonstrated RX‐5902 inhibited the β‐catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX‐5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the β‐catenin pathway, such as c‐Myc, cyclin D1 and p‐c‐Jun. Therefore, our study indicates that the inhibition of Y593 phospho‐p68 helicase ‐ β‐catenin interaction by direct binding of RX‐5902 to Y593 phospho‐p68 RNA helicase may contribute to the anti‐cancer activity of this compound. J. Cell. Biochem. 116: 1595–1601, 2015.

Abstract 1790: Evaluation of UCK2 protein expression as a potential predictive biomarker of RX-3117

Background: A novel, orally bioavailable nucleoside analogue, RX-3117, is a prodrug activated intracellularly by Uridine Cytidine Kinase 2 (UCK2) that is thought to be expressed predominantly in tumor tissue. RX-3117 is currently being evaluated in a Phase Ib/IIa multi-center, open-label clinical study in patients with advanced pancreatic and bladder cancer. In this study, we aimed to determine the relation between UCK2 tissue protein expression and the efficacy of RX-3117 in mice xenograft models and also UCK2 protein expression in a panel of human cancer tissues relative to normal tissue. Methods: The UCK2 protein expression in tumor tissues was analyzed by immunoblotting using clone 22-1 rabbit monoclonal antibody. The validated procedure for the immunohistochemistry (IHC) of UCK2 with clone 22-1 was performed in a panel of human formalin-fixed paraffin-embedded (FFPE) cancer and normal tissues. Results: The immunoblotting protein level of UCK2 normalized to β-actin and corresponding tumor growth inhibition (oral RX-3117 dose of 500mg/kg, TIWK) were 57 and 67% in MiaPaCa2, 30 and -5% in BxPC3, 199 and 92% in Colo-205, 21 and 90% in Caki-1, 2 and 39% in A549, and 146 and 79% in H460, respectively. This data indicates an anti-tumor efficacy trend in a UCK2-dependent manner. The IHC of UCK2 showed that positive staining of UCK2 in cancer tissues was observed in 20/20 bladder cancer tissues (100% frequency), 19/20 CRC tissues (95% frequency), 18/20 NSCLC tissues (90% frequency), and 19/20 pancreatic cancer tissues (95% frequency). Average H-Scores of UCK2 in cancer tissues vs. normal tissues were 104 vs. 9 in lung, 97 vs. 20 in bladder, 67 vs. 41 in pancreas and 39 vs. 21 in colon, respectively. Conclusions: The current data showed a correlation trend between UCK2 protein expression level and degree of antitumor activity of RX-3117 in xenograft models. It also supports a higher UCK2 protein expression level in human cancer tissues compared to their normal tissues. This suggests that RX-3117 activity may be specific to tumor tissue, and quantification of UCK2 expression in human cancer tissues may be useful as a predictive biomarker to select patients for their sensitivity to RX-3117 in future clinical studies. Citation Format: Young Bok B. Lee, Deog J. Kim, Christina George, Chang-ho Ahn, Julie Frank, Reza Mazhari, Lisa M. Dauffenbach, Eric P. Olsen. Evaluation of UCK2 protein expression as a potential predictive biomarker of RX-3117 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1790. doi:10.1158/1538-7445.AM2017-1790

Abstract P5-03-13: The anticancer effects of Supinoxin® (RX-5902) in triple-negative breast cancer MDA-MB-231 through phosphorylated p68 on Tyr593

Several studies have indicated that the DEAD box RNA helicase DDX5/p68 plays several important roles in cancer (1, 2). In particular, p68 that is phosphorylated on Tyr593 has been shown to be associated with cell transformaton, epithelial mesenchymal transition (EMT) and cell migration (3). Therefore, phosphorylated p68 may be a promising target for novel anti-cancer therapeutics. We previously reported that 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl) aminocarbonyl] piperazine (RX-5902, Supinoxin®) inhibits the growth of cancer cells at low nanomolar concentrations by interacting with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway (4). In this study, we sought to determine whether phosphorylated p68 on Tyr593 plays a key role in RX-59029s ability to inhibit cancer cell growth by knocking down p68. p68-siRNA efficiently down-regulated the expression of phosphorylated p68 on Tyr593 as well as p68 in the triple-negative (TN) breast cancer cell line, MDA-MB-231. Exposure of p68-siRNA-transfected cells to the IC50 concentration of RX-5902 protected MDA-MB-231 cells from the cytotoxic effects of RX-5902, indicating the phosphorylated p68 on Tyr593 is a key molecule for RX-5902 cytotoxic effects. We also examined the tumor growth inhibition (TGI) of RX-5902 in the human TN-breast tumor (MDA-MB-231) xenograft mouse model. Not only did RX-5902 demonstrate potent efficacy in this model but also oral administration with RX-5902 resulted in dose-dependent TGI and extended the overall survival of these animals. Oral administration of 160, 320 and 600 mg/kg of RX-5902 showed 54.4%, 84.4% and 100% TGI, respectively whereas 5 mg/kg of Abraxane (iv) showed only 48.2% TGI at day 29. Further studies demonstrated the inhibitory effects of RX-5902 on cellular motility in MDA-MB-231 in wound healing assays, suggesting the potential function of phosphorylated p68 on Tyr593 in cell migration (5). These data support the potential therapeutic activity of RX-5902 in triple negative breast cancers. A Phase 1 study of RX-5902 on relapse/refractory solid tumors is ongoing. References 1. Fuller-Pace, FV, RNA Biology 10, 121–132 (2013) 2. Dai et al. Journal of Experimental & Clinical Cancer Research, 33, 64-71 (2014 3. Yang et al., Cell, 127, 139–155 (2006) 4. Kost et al., Journal of Cellular Biochemistry ;online: 3 FEB 2015 05:14PM EST | DOI: 10.1002/jcb.25113) (2015). 5. Remenyi et al, presented at 2015 AACR (2015). Citation Format: Kim DJ, Yang MY, Lee YB, Remenyi J, Fuller-Pace F. The anticancer effects of Supinoxin® (RX-5902) in triple-negative breast cancer MDA-MB-231 through phosphorylated p68 on Tyr593. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-13.