PDBsum entry 1y2e

Hydrolase

PDB id: 1y2e

Contents

Protein chains

326 a.a. *

Ligands

5DE ×2

EDO ×14

Metals

_ZN ×2

_MG ×2

Waters ×271

* Residue conservation analysis

PDB id:

1y2e

Name:

Hydrolase

Title:

Catalytic domain of human phosphodiesterase 4d in complex with 1-(4- amino-phenyl)-3,5-dimethyl-1h-pyrazole-4-carboxylic acid ethyl ester

Structure:

Camp-specific 3',5'-cyclic phosphodiesterase 4d. Chain: a, b. Fragment: catalytic domain of human phosphodiesterase 4d. Synonym: dpde3, pde43. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: pde4d. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.190 R-free: 0.218

Authors:

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.J.Zhang

Key ref:

G.L.Card et al. (2005). A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design. Nat Biotechnol, 23, 201-207. PubMed id: 15685167 DOI: 10.1038/nbt1059

Date:

22-Nov-04 Release date: 01-Mar-05

Protein chains

Q08499  (PDE4D_HUMAN) -  3',5'-cyclic-AMP phosphodiesterase 4D from Homo sapiens

Seq: 809 a.a.

Struc: 326 a.a.

Enzyme reactions

• Enzyme class: E.C.3.1.4.53 - 3',5'-cyclic-AMP phosphodiesterase.
• Reaction: 3',5'-cyclic AMP + H2O = AMP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1038/nbt1059

Nat Biotechnol 23:201-207 (2005)

PubMed id: 15685167

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design.

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.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Biotechnol (2005, 23, 201-207) copyright 2005.

Figures were selected by the author.