Angewandte
Chemie
indicate a clash of larger moieties with the peptide backbone
of the b5 subunit and thus suggest that small hydrophobic
groups are required for inhibitor stabilization.
hole, an area typically populated by active head groups of
ligands; this phenomenon has already been described in the
literature.[13,14] These two molecules, HU and MES, can be
regarded as independent fragments that can be used in future
fragment-based drug design.
Remarkably, slight changes in R2 significantly influenced
the IC50 of these HU compounds: small halogenated (tri-
fluoromethoxy in 2) and extended aliphatic R2 side chains (n-
pentoxy in 3) resulted in at least a fivefold increase in the IC50
(> 1 mm) compared to compound 1. Interestingly, a tert-
butyldimethylsiloxy moiety (in 4) in this position improved
affinity and resulted in a fivefold decrease in the IC50 (48 mm,
Ki 4.8 mm) (Table 1). The crystal structure of the latter
compound in complex with the CP at 3.2 ꢀ resolution
(Rfree = 0.228) shows strong interactions of the tert-butyldi-
methylsiloxy side chain wiwth Met45 and Ala46 in the S1
subpocket (Table ST1 and Figure S3b in the Supporting
Information). Based on these crystallographic results, molec-
ular modeling was performed to design more appropriate side
chains for R2. A 1-adamantyloxy group in R2 was identified to
give the highest docking scores (À10.7 in GlideScore)[11,12]
among a small library of 50 compounds (Figure S6 in the
Supporting Information). Surprisingly, this new HU deriva-
tive (8) displayed an IC50 of 700 nm (Ki = 0.08 mm), 320 times
lower than that of the starting compound (1) (Table 1). The
crystal structure of the CP:8 complex at 2.9 ꢀ resolution
(Rfree = 0.238, Table ST1 in the Supporting Information)
confirmed the modeling results (0.8 ꢀ rmsd between exper-
imental and modeled ligand structures; Figures S3c and S7 in
the Supporting Information) and additionally revealed the
importance of Ser118 in subunit b6, which formed a strong
hydrogen bond to the ether oxygen atom of 8 and thus further
stabilizes the position of the adamantyloxy residue in the S3
subpocket (Figure 1c).
In conclusion, HUs constitute a new class of proteasome
inhibitors that are characterized by a novel mode of ligand
binding and proteasome inhibition. The combination of
crystallographic characterization, molecular modeling, chem-
ical synthesis, and kinetic experiments identified a strong
inhibitory binding profile of these novel proteasome ligands
in defined specificity pockets that have hitherto not been
explored for proteasomal drug design. This new class of
compounds represents the smallest, reversibly and noncova-
lently bound, active-site-specific inhibitors observed to date
for the 20S proteasome that do not contain a functional
reactive head group. In addition, the hydroxyurea compounds
present many molecular properties important for pharmaco-
kinetics that may open new avenues for proteasomal drug
development.
Received: August 25, 2011
Published online: November 21, 2011
Keywords: hydroxyureas · inhibitors · lipoxygenases ·
.
proteasomes · protein crystallography
[2] P. G. Richardson, P. Sonneveld, M. W. Schuster, D. Irwin, E. A.
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Boccadoro, J. Cavenagh, W. S. Dalton, A. L. Boral, D. L.
Esseltine, J. B. Porter, D. Schenkein, K. C. Anderson, N. Engl.
These HU compounds were optimized further with
respect to the configuration generated by the R1 group. The
R enantiomer (10; IC50 = 300 nm, Ki = 34 nm) displayed a
much stronger inhibition than the S conformer (9; IC50 =
56 mm, Ki = 5.8 mm; Figure 1 and Table 1). The structural
results of CP:9 at 3.1 ꢀ resolution (Rfree = 0.231) and CP:10 at
2.8 ꢀ resolution (Rfree = 0.262, Table ST1 in the Supporting
Information) showed that only the orientation of R1 in the
S enantiomer is responsible for this decrease in binding
affinity as a result of disfavored interactions with protein main
chain atoms (Figure S8b,c). Accordingly, soaking proteasome
crystals with a racemic mixture of HU compounds yielded
CP:HU structures containing solely the R enantiomer, thus
demonstrating a strong enantioselectivity (Figure S8a in the
Supporting Information). Structural superpositions of 9 and
10 revealed that both R and the less active S enantiomer
aligned almost perfectly with regard to the N-hydroxyurea
moiety and the adamantyloxybenzene (Figure S8b in the
Supporting Information). Considering the noncovalent bind-
ing mode, this observation underlines the strength of the
binding motifs, which keep the inhibitor in place in spite of the
disfavored orientation of the Me group in R1 (9).
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[8] S. E. Wenzel, A. K. Kamada, Ann. Pharmacother. 1996, 30, 858.
[9] R. L. Bell, P. R. Young, D. Albert, C. Lanni, J. B. Summers, D. W.
[11] R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J.
Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, M. Shelley, J. K.
[12] R. A. Friesner, R. B. Murphy, M. P. Repasky, L. L. Frye, J. R.
Greenwood, T. A. Halgren, P. C. Sanschagrin, D. T. Mainz, J.
[13] M. Groll, N. Gallastegui, X. Marechal, V. Le Ravalec, N. Basse,
N. Richy, E. Genin, R. Huber, L. Moroder, J. Vidal, M. Reboud-
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In addition, an N-morpholinoethanesulfonic acid (MES)
molecule from the crystallization buffer was found in the
electron density maps of most CP:HU complex structures
(Figure S5 in the Supporting Information). The MES mole-
cule interacts with b5-Gly47N and occupies the oxyanion
Angew. Chem. Int. Ed. 2012, 51, 247 –249
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