Computational protocol: Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

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Protocol publication

[…] The compounds composing the focused library were identified using computational chemistry methods. Briefly, the eMolecules catalogue of commercially available compounds was filtered for acceptable drug-like molecular properties. After filtering, compounds were computationally scored (using both 2D and 3D methods) for their potential to occupy portions of the D-amino acid and FAD-binding pockets within hDAAO. The 1016 best scoring compounds were purchased from eMolecules for screening. Please see Supplementary Online Data (at http://www.bioscirep.org/bsr/034/bsr034e133add.htm) for details on library assembly and screening.Compound 1 (4H-furo[3,2-b]pyrrole-5-carboxylic acid) was synthesized as described previously []. Compound 2 [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid] was purchased from eMolecules as an original compound from the focused library screen. Compound 3 [4-hydroxy-6-(2-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)ethyl)pyridazin-3(2H)-one], Compound 5 (6-(2,4-dihydroxyphenethyl)-4-hydroxypyridazin-3(2H)-one) and Compound 6 (6-(2,4-dimethoxyphenethyl)-4-hydroxypyridazin-3(2H)-one) were synthesized and characterized by NMR and MS. Synthesis and analytical details are described in the Supplementary Online Data. ADP disodium salt was purchased from Sigma-Aldrich and Compound 4 was purchased from eMolecules. [...] Full-length, untagged hDAAO protein was prepared as described previously []. The hDAAO preparation at 2 mg/ml was dialysed overnight at 4°C against 50 mM sodium phosphate, pH 6.6 and 10 μM FAD prior to crystallography. hDAAO samples were incubated overnight with 250 μM compound 3 or 1 mM compound 2 and then subjected to crystallization trials. Crystals of the compound 3 complex were grown in sitting drops containing 0.4 μl hDAAO+0.4 μl crystallant consisting of 30% (w/v) PEG2000MME, 0.1 M potassium thiocynate. Crystals of the compound 2 complex were grown in sitting drops containing 0.4 μl hDAAO+0.4 μl crystallant consisting of 13.64% (w/v) PEG3350, 0.1 M Tris pH 7.4, 0.15 M potassium citrate tribasic. Crystals were harvested in 20% (v/v) ethylene glycol and flash frozen in liquid nitrogen. X-ray data for the hDAAO:FAD:compound 2 complex crystals were collected at SSRL beam line 7-1 and reduced with the XDS/XSCALE package to 2.85 Å. The CCP4 program Phaser was used for Molecular Replacement using chain A from the 3CUK structure as the search model. The model was iteratively extended in real space using Coot and refined in reciprocal space using Refmac5. The final model had R/Rfree=0.24/0.29. X-ray data for the hDAAO:FAD:compound 3 complex crystals were collected at APS beam line 21-idf and reduced with the XDS/XSCALE package to 2.4 Å. The CCP4 program Molrep was used for Molecular Replacement using chain A from the 3CUK structure as the search model. The model was iteratively extended in real space using Coot and refined in reciprocal space using Refmac5. The final model had R/Rfree=0.18/0.23. […]

Pipeline specifications

Software tools eMolecules, XDS, CCP4, Coot, REFMAC5, Molrep
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Nervous System Malformations, Anti-N-Methyl-D-Aspartate Receptor Encephalitis
Chemicals Benzoic Acid