Aleksandra Zivkovic

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase α

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo−) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.

A Class of 5-Benzylidene-2-phenylthiazolinones with High Potency as Direct 5-Lipoxygenase Inhibitors

A novel class of potent direct 5-lipoxygenase (5-LO) inhibitors bearing a thiazolinone-scaffold identified by virtual screening is presented. A range of substitutions and the importance of the 2-phenyl moiety were evaluated. This series is characterized by high potency in intact polymorphonuclear leukocytes and a cell-free system, exemplified by (Z)-2-(4-chlorophenyl)-5-(4-methoxybenzylidene)-5H-thiazol-4-one (18, IC(50) = 0.28 and 0.09 μM). These disubstituted thiazolinones may possess potential for intervention with inflammatory and allergic diseases and certain cancer types.

Determinants of the Unexpected Stability of RNA Fluorobenzene Self Pairs

Fluorine-substituted base analogues have proven invaluable as “nonpolar nucleoside isosteres” to probe the physical forces that govern the stabilities of nucleic acids. When paired against natural bases, fluorinated analogues destabilize DNA and RNA helices and exhibit little binding sequence specificity. 5] These observations make Watson–Crick base pairing involving hydrogen bonds to fluorine unlikely. When paired opposite one another, however, a considerable degree of stability is regained, and a selective pairing of fluorinated bases in the context of nucleic acids is observed. Weak C F···H C dipolar interactions have been implicated as acting as stabilizing forces in this case. Apparently, the role of fluorine in molecular recognition strongly depends on the surrounding molecular environment. Similar effects have been observed in the fields of medicinal chemistry and protein design, in which the fluorophilicity/fluorophobicity of the protein environment affects the affinity of fluorine-substituted ligands or the stabilizing influence of fluorine-containing artificial amino acids. With the goal of addressing the influence of the environment on the molecular recognition thermodynamics of organic fluorine, we have undertaken a combined experimental/computational study of fluo ACHTUNGTRENNUNGrobenzene self-pairing in the context of duplex RNA. We report here the first systematic study of the determinants of the surprising stability of fluorobenzene-based self-pairs with increasing fluorine-substitution. Motivated by preliminary modeling results, we synthesized novel ribonucleoside analogues in which the nucleobases are replaced by benzene or fluorine-substituted benzenes, respectively 13] (Scheme 1 and in the Supporting Information). The modified nucleosides were tested in a defined 12-mer RNA duplex (5’-CUU UUC XUU CUU paired with 3’-GAA AAG YAA GAA). The nucleoside analogues were introduced at positions X and Y, respectively, to form a base pair in the duplex. We anticipated that this supramolecular system should be particularly apt for investigation of the molecular recognition properties of organic fluorine. Here we focus on results obtained for homo-self-pairs (that is, positions X and Y were occupied by the same nucleotide) of 1–5. The 2,4,6-trifluorobenzene-substituted nucleoside analogue and the pentafluorinated species were omitted, as steric effects due to bis-ortho substitution result in large destabilization. Likewise, we restrict ourselves to the homologous set of benzene derivatives 1–5 instead of also considering, for example, indoleor benzimidazole-based base analogues. That way we can minimize any influence due to variation in shape or size of the base analogues or stacking interactions (see also below) on the observed stabilities. The CD spectra of the RNA duplexes with the modified bases follow the typical curves for an A-type helix (Figure S2 in the Supporting Information). Thus, the structure of the duplex RNA is not disturbed by incorporation of our modified nucleosides, in agreement with previous findings. The thermodynamic stabilities of the modified RNA duplexes were determined by thermal denaturation as monitored by UV absorbance in a phosphate buffer (20 mm, pH 7.0) containing NaCl (140 mm). The thermodynamic data were extracted from the melting curves by means of a two-state model for the transition from duplex to single strand. Not unexpectedly, our measurements demonstrate that the pairing preference of fluorinated bases is higher in selfpairs (Figure 1; Table 1) than in pairs with natural bases. In both cases, the stability increases incrementally with the number of fluorine substituents in the base analogue, with the largest gain in stability observed in the first two fluorination steps (1!2 : DDG=1.7 kcalmol , 2!3 : 1.4 kcalmol ). Surprisingly, this leads to RNA duplex stabilities with self-paired bases 3, 4, and 5 (11.6, 11.8 and 12.2 kcalmol , respectively) that are similar to or exceed that of the natural AU base pair (11.9 kcalmol ). In stark contrast, in the case of a 12-mer DNA double helix, the presence of two self-pairs of 5 bases resulted in an overall destabilization of the duplex by 4.6 kcalmol 1 compared to the natural AT base pairs, and the stability increase observed on going from two self-pairs of 1 bases to two self-pairs of 5 bases is much less pronounced (DDG=0.6 kcalmol ). What is the molecular origin of the stepwise stability increase and the unexpected overall stability in the RNA case? To address this question, we performed 10 ns molecular dynamics (MD) simulations and free energy calculations together with a structural component analysis for RNA duplexes containing homo-self-pairs of 1–5, including solvent and consider[a] H. Kopitz, Prof. Dr. H. Gohlke Pharmazeutisches Institut Christian-Albrechts-Universit t zu Kiel Gutenbergstr. 76, 24118 Kiel (Germany) Fax: (+49)431-880-1352 E-mail : gohlke@pharmazie.uni-kiel.de [b] Dr. A. Živkovic, Prof. Dr. J. W. Engels Fachbereich Biochemie, Chemie und Pharmazie, Goethe-Universit t Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Structures of the base analogues that form self pairs. R is always the ribosephosphate moiety. 1=benzene; 2=4-fluorobenzene; 3=2,4-difluorobenzene; 4=2,4,5-trifluorobenzene; 5=2,3,4,5-tetrafluorobenzene.

Ceramide synthesis regulates T cell activity and GVHD development

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for a variety of hematologic malignances, yet its efficacy is impeded by the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production, and migration of alloreactive donor T cells. Hence, strategies to limit GVHD are highly desirable. Ceramides are known to contribute to inflammation and autoimmunity. However, their involvement in T-cell responses to alloantigens is undefined. In the current study, we specifically characterized the role of ceramide synthase 6 (CerS6) after allo-HCT using genetic and pharmacologic approaches. We found that CerS6 was required for optimal T cell activation, proliferation, and cytokine production in response to alloantigen and for subsequent induction of GVHD. However, CerS6 was partially dispensable for the T cell-mediated antileukemia effect. At the molecular level, CerS6 was required for efficient TCR signal transduction, including tyrosine phosphorylation, ZAP-70 activation, and PKCθ/TCR colocalization. Impaired generation of C16-ceramide was responsible for diminished allogeneic T cell responses. Furthermore, targeting CerS6 using a specific inhibitor significantly reduced T cell activation in mouse and human T cells in vitro. Our study provides a rationale for targeting CerS6 to control GVHD, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments.

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P3 and phosphoinositide 3-kinase

Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors.

Abstract FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.

Synthesis of Modified RNA-Oligonucleotides for Structural Investigations

Abstract RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) and 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U- rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.

RNA RECOGNITION BY FLUOR-AROMATIC SUBSTITUTED

RNA exhibits a higher structural diversity than DNA and is an important molecule in the biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops, etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing, and hydrogen bond is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-phenyl-β-d-ribofuranose (B), 1′-deoxy-1′-(4-fluorophenyl)-β-d-ribofuranose (4 FB), 1′-deoxy-1′-(2,4-difluorophenyl)-β-d-ribofuranose (2,4 DFB), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-β-d-ribofuranose (2,4,5 TFB), 1′-deoxy-1′-(2,4,6-trifluorophenyl)-β-d-ribofuranose, 1′-deoxy-1′-(pentafluorophenyl)-β-d-ribofuranose (PFB), 1′-deoxy-1′-(benzimidazol-1-yl)-β-d-ribofuranose (BI), 1′-deoxy-1′-(4-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 FBI), 1′-deoxy-1′-(6-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 FBI), 1′-deoxy-1′-(4,6-difluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4,6 DFBI), 1′-deoxy-1′-(4-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 TFM), 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (5 TFM), and 1′-deoxy-1′-(6-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 TFM). These amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5′). Only one position was modified, marked as X and Y, respectively. UV melting profiles of those oligonucleotides were measured.