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PDBsum entry 1y2c
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References listed in PDB file
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Key reference
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Title
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A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-Based drug design.
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Authors
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G.L.Card,
L.Blasdel,
B.P.England,
C.Zhang,
Y.Suzuki,
S.Gillette,
D.Fong,
P.N.Ibrahim,
D.R.Artis,
G.Bollag,
M.V.Milburn,
S.H.Kim,
J.Schlessinger,
K.Y.Zhang.
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Ref.
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Nat Biotechnol, 2005,
23,
201-207.
[DOI no: ]
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PubMed id
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Abstract
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Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes
that regulate a variety of cellular processes. We describe a family of potent
PDE4 inhibitors discovered using an efficient method for scaffold-based drug
design. This method involves an iterative approach starting with low-affinity
screening of compounds followed by high-throughput cocrystallography to reveal
the molecular basis underlying the activity of the newly identified compounds.
Through detailed structural analysis of the interaction of the initially
discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray
crystallography, we identified three sites of chemical substitution and designed
small selective libraries of scaffold derivatives with modifications at these
sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved
after only two rounds of chemical synthesis and the structural analysis of seven
pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this
approach for identifying new inhibitors that can be further developed into drug
candidates.
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Figure 1.
Figure 1. Crystal structure of the pyrazole scaffold and its
derivatives in complex with PDE4B or PDE4D. (a) Crystal
structure of 3,5-dimethyl-1H-pyrazole-4-carboxylic acid ethyl
ester (pyrazole no. 2) bound to PDE4D, showing the pyrazole ring
sandwiched in the hydrophobic clamp formed by F372 and I336. The
conserved H-bond, seen in all pyrazole derivative cocrystal
structures, between the NE2 atom of the invariant glutamine and
the carboxylate group, is shown. (b) The crystal structure of
3,5-dimethyl-1-phenyl-1H-pyrazole-4-carboxylic acid ethyl ester
(pyrazole no. 8) bound to PDE4D, showing the same interactions
as its parent compound, and thus validating the dimethyl
pyrazole as a scaffold. The dimethyl pyrazole is sandwiched by
F372 and I336 and the carbonyl oxygen forms an H-bond with Q369.
The ethoxy group is tucked into the Q1 pocket. (c) Crystal
structure of
3,5-dimethyl-1-(3-nitro-phenyl)-1H-pyrazole-4-carboxylic acid
ethyl ester (pyrazole no. 21) bound to PDE4B and PDE4D. The
carbon atoms of pyrazole no. 21 bound to PDE4B and PDE4D are
shown in green and yellow respectively. The NO[2] group at the
meta-position of the phenyl ring formed H-bonds with T345, D392
in PDE4B and the two water molecules coordinating Zn2+ (omitted
for clarity). (d) Crystal structure of
1-(2-chloro-phenyl)-3,5-dimethyl-1H-pyrazole-4-carboxylic acid
ethyl ester (pyrazole no. 20) bound to PDE4B. The
Cl-substitution at the ortho-position of the phenyl ring makes
several hydrophobic contacts with residues M347, L393 and F446.
(e) Crystal structure of
1-(4-amino-phenyl)-3,5-dimethyl-1H-pyrazole-4-carboxylic acid
ethyl ester (pyrazole no. 19) bound to PDE4D. The amine group
forms three H-bonds with three water molecules, two of which are
coordinated to Mg2+. However, this amine nitrogen is also in
close proximity to the carbon atom in M273 which results in
unfavorable interactions. (f) Crystal structure of
1-(4-methoxy-phenyl)-3,5-dimethyl-1H-pyrazole-4-carboxylic acid
ethyl ester (pyrazole no. 17) bound to PDE4D. The methoxy-phenyl
group rotated 180° to point away from the di-metal ions to avoid
the repulsive interactions between the methoxy group and the
di-metal ions.
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Figure 3.
Figure 3. Pyrazole scaffold bound to PDE4B and PDE4D and the
discovery of potent pyrazole inhibitors for PDE4 in three steps.
Superposition of six different pyrazoles (nos. 2, 8, 17, 19,
20 and 21) in seven cocrystal structures with PDE4B and/or PDE4D
revealed the consistent binding mode of the scaffold moiety
(panels a -c). For clarity, only several side chains for one
PDE4B cocrystal structure are shown. The three pockets in the
active site are highlighted on the solvent accessible surface:
the metal binding pocket (M) in blue, purine-selective glutamine
and hydrophobic clamp pocket (Q) in red (which is further
divided into Q[1], Q[2] sub-pockets) and solvent-filled side
pocket (S) in green. The discovery of potent pyrazole inhibitors
for PDE4 in three steps is illustrated in panels d -f. (a) A
view looking down into the active site. The pyrazole carboxylate
scaffold fits into the narrow passage formed by the hydrophobic
clamp. (b) A view looking away from the S pocket. The pyrazole
carboxylate scaffold forms an H-bond with the invariant Q443^4B.
(c) A view looking towards the S pocket. The ethoxy group
occupies the Q[1]-pocket. The scaffold that the six different
pyrazoles share is marked by a dashed oval. (d) Scaffold
discovery. The scaffold candidate,
3,5-dimethyl-1H-pyrazole-4-carboxylic acid ethyl ester (pyrazole
no. 2), is a weak PDE4D inhibitor with IC[50] of 82  M.
(e) Scaffold validation. The derivative of the scaffold,
3,5-dimethyl-1-phenyl-1H-pyrazole-4-carboxylic acid ethyl ester
(pyrazole no. 8) has significantly increased potency towards
PDE4D with IC[50] of 0.27 M.
(f) Chemical optimization. The validated scaffold was optimized
into a potent PDE4D inhibitor,
3,5-dimethyl-1-(3-nitro-phenyl)-1H-pyrazole-4-carboxylic acid
ethyl ester (pyrazole no. 21), with IC[50] of 0.021 M.
A 4,000-fold increase in potency was achieved in two rounds of
chemical synthesis. Compounds are represented by solid surface
colored by atomic types. The active site is represented by the
blue mesh. The PDE4D is represented by cartoons where helices
are shown as cylinders and loops are shown as tubes.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Biotechnol
(2005,
23,
201-207)
copyright 2005.
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