3062
A. Blommaert et al. / Bioorg. Med. Chem. 12 (2004) 3055–3062
9. Anne, C.; Turcaud, S.; Quancard, J.; Teffo, F.; Meudal,
ꢀ
4H), 2.75–3.10(m, 4H), 3.10–3.30(m, 4H), 3.4 and 3.55
(t, 2H), 4.1–4.3 (m, 4H), 4.6–4.9 (m, 4H), 6.9–7.4 (m,
44H), 7.85 (m, 4H), 8.25–8.65 (m, 6H). MS (ESI)
(M+Na)þ m=z ¼ 1443.4.
H.; Fournie-Zaluski, M. C.; Roques, B. P. Developement
of potent inhibitors of Botulinum neurotoxin type B.
J. Med. Chem. 2003, 46, 4648–4656.
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synaptobrevin-II bound to botulinum neurotoxin type B
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of the catalytic and binding sites of Clostridium botulinum
neurotoxin B. Nat. Struct. Biol. 2000, 7, 693–699.
12. Anne, C.; Cornille, F.; Lenoir, C.; Roques, B. P. High-
throughput assay determination of botulinum type B
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253–261.
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4.7. Enzyme assay
The inhibitory potencies of all compounds were mea-
sured by using the light chain of BoNT/B as previously
described12 with some slight modifications. Briefly,
BoNT/B LC (0.35 ng) was preincubated for 30 min at
37 ꢁC in 90 lL of 20mM Hepes, pH 7.4 with increasing
concentrations (10ꢀ10 to 10ꢀ5 M) of inhibitor. Com-
pounds containing a free thiol group were tested in the
presence of 0.1 mM DTT in order to avoid oxidation. All
other compounds were tested in the absence of DTT. The
fluorescent substrate Syb 60-94 [Pya74-Nop77]
(Km ¼ 47 M) was then incubated for 30min and the
reaction stopped by addition of 0.2 N HCl at 4 ꢁC. Ver-
ification through HPLC analysis showed no reduction in
situ of compounds containing disulfide bridges (data not
shown) except for compound 25 when tested in the
presence of 10equiv of DTT added prior to incubation in
order to evaluate the affinity of its thiol counterpart 26.
The percentage of degradation was measured directly in
a 96 wells plate. The IC50 values were determined from
logarithmic dose–degradation curves and the values of
the inhibitory constant (Ki of compounds 1–12 were
calculated according to the equation of Cheng and
Prusoff24 and are expressed as the mean SEM of three
separate experiments each in triplicate.
14. Chauvel, E. N.; Llorens-Cortes, C.; Coric, P.; Wilk, S.;
ꢀ
Roques, B. P.; Fournie-Zaluski, M. C. Differential inhi-
bition of aminopeptidase A and aminopeptidase N by
new-amino-thiols. J. Med. Chem. 1994, 37, 2950–2957.
15. Bischoff, L.; David, C.; Martin, L.; Meudal, H.; Roques,
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B. P.; Fournie-Zaluski, M.-C. 2,4-Dinitrophenyl 4-meth-
oxybenzyldisulfide: a new efficient reagent for electrophilic
sulfenylation of b-amino ester enolates. J. Org. Chem.
1997, 62, 4848–4850.
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asymmetric synthesis. Pure Appl. Chem. 1990, 62, 1241–1250.
17. Josien, H.; Martin, A.; Chassaing, G. Asymmetric syn-
thesis of L-pyrenylalanine and L-9-fluorenylglycine via
room temperature alkylations of a sultam-derived glycine
imine. Tetrahedron Lett. 1991, 32, 6547–6550.
18. Schiavo, G.; Santucci, A.; Dasgupta, B. R.; Mehta, P. P.;
Jontes, J.; Benfenati, F.; Wilson, M. C.; Montecucco, C.
Botulinum neurotoxins serotypes A and E cleave SNAP-
25 at distinct COOH-terminal peptide bonds. FEBS Lett.
1993, 335, 99–103.
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