F. Esra Önen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3628–3631
3631
Figure 3. ThyX activity was measured by monitoring decrease in A340 as the function of time (see Materials and methods). Molecules 14b and 16h were used at 20 lM. Where
indicated, BSA (240 lg/ml) or Triton X-100 (0.1% v/v) were included in reaction mixtures.
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15. Assay reactions, with a final volume of 200 ll, consisted of 200 lM NADPH,
Figure 4. NADPH oxidation activity (nmol/min) of PBCV-1 ThyX as the function of
the added dUMP. Experiment was performed in the absence and presence of inhi-
bitors (20 lM) 14b (class I) and 16h (class II).
5 lM CH2H4 folate, 5 lM dUMP, 1 mM MgCl2, 1% glycerol, 62.5 lM FAD and
12.5 lM of viral PBCV-1 ThyX. Note that active site configurations of this viral
enzyme is very similar to that of ThyX proteins from pathogenic species (not
shown). The reactions were initiated by injection of NADPH to each well of the
we detected a benzoyl and a triazole derivative that demonstrated
inhibition of the catalytic activity. Optimization of these leads by
further structure–activity relationship (SAR) analyses and screen-
ing of the library against additional methyltransferases are under
investigation and will be the subject of future communications.
Microtiter 96-well clear flat-bottom plate, followed by rapid shaking of the
microplate. ThyX activity was determined by following the decrease of A340
(due to oxidation of NADPH) that was measured with a reading interval of 30 s
for duration of 15 min. All assays used
a kinetic mode of a multilabel
microplate reader [CHAMELEON II (Hidex, Finland)] with an injector. All
screening reactions were performed in triplicates. The primary screen was
performed with the small molecules dissolved in DMSO, including DMSO alone
as low-activity control. The concentration of screened compounds was 20 lM
unless otherwise indicated. Dose–response curves of the chosen compounds
were determined using a wide range of compounds.
Acknowledgments
E. Onen thank the French Embassy in Turkey/ARC and the
French Ministry of Education, respectively, for fellowships. We also
thank J. Ulmer for helpful comments on the manuscript. The
authors are grateful to CNRS, INSERM, and Ministére de l’Education
Nationale, de l’Ensei-gnement Supérieur et de la Recherche ACI
‘Molecules & Cibles Thérapeutiques’ (02L0524) and Region Ile-de-
Franee (CPER, SESAME) for financial support. H.M. also acknowl-
edges the support from Fondation Bettencourt-Schueller.
16. 2-(N-{[(4R)-3-benzoyl-2-phenyl-1,3-thiazolidine-4-yl]methyl}hexanamido)acetic
acid (14b). 1H NMR (CDCl3) d 0.87–1.60 (m, 7H, H-19 à H-21), 1.68–2.25 (m, 4H,
H-17+H-18), 3.91–4.27 (m, 4H, H-6+H-8), 4.69–4.98 (m, 3H, H-14+H-7) 6.05
(sl, 1H, H-5), 7.38–7.81 (m, 10H, H-arom).13C NMR (CDCl3) d 14.0 (C-21), 22.4
(C-20), 26.0 (C-19), 31.5 (C-20), 31.8 (C-17), 36.2 (C-6), 50.6 (C-14), 53.3 (C-16),
58.8 (C-7), 65.8 (C-5), 127.0, 128.3, 128.8, 129.5, 134.2, 138.3 (C-arom). LC–MS
Calcd for C25H30N2O4S 454.59À m/z 472 (M+Na)+. Purity by ELSD 99%.
17. Ethyl (4R)-2-phenyl-3-({(1-[3-2,2,2-trifluoroacetamido)propyl]-1H-1,2,3- triazol-
4-yl}carbonyl)-1,3-thiazolidine-4-carboxylate (16h). 1H NMR (CDCl3) d 1.27–1.37
(m, 3H, H-1), 1.93–2.20 (m, 2H, H-17), 3.14–3.56 ( m, 4H, H-5+H-18), 4.21–4.43
(m, 4H, H-2+H-16), 5.04–5.16 (m,1H, H-4), 6.04–6.18 (m, 0.6H, H-6), 6.49 (s,
0.4H, H-6), 7.12–8.31 (m, 6H, H-arom+H-15). 13C NMR (CDCl3) d 14.1 (C-1),
29.1 (C-17), 31.3 (C-5), 34.8 (C-5), 36.9 (C-18), 47.7 (C-16), 61.9 (C-2), 65.0 (C-
4), 65.7 (C-4), 67.1 (C-6), 68.6 (C-6), 127.1, 128.3, 129.4, 130.0 (C-arom). LC–
MS: m/z 486 (M+H)+, 508 (M+Na)+. Purity by ELSD 99%.
References and notes
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