Structure of 3,4-dichloroisocoumarin-inhibited factor D.
Factor D (D) is a serine protease essential in the activation of the alternative complement pathway. Only a few of the common serine protease inhibitors inhibit D, binding covalently to the serine hydroxyl of the catalytic triad. 3,4-Dichloroisocoumarin (DCI) is a mechanism-based inhibitor which inhibits most serine proteases and many esterases, including D. The structure of the enzyme:inhibitor covalent adduct of D with DCI, DCI:D, to a resolution of 1.8 A is described, which represents the first structural analysis of D with a mechanism-based inhibitor. The side chain of the ring-opened DCI moiety of the protein adduct undergoes chemical modification in the buffered solution, resulting in the formation of an alpha-hydroxy acid moiety through the nucleophilic substitution of both Cl atoms. The inhibited enzyme is similar in overall structure to the native enzyme, as well as to a variety of isocoumarin-inhibited trypsin and porcine pancreatic elastase (PPE) structures, yet notable differences are observed in the active site and binding mode of these small-molecule inhibitors. One region of the active site (residues 189-195) is relatively conserved between factor D, trypsin, and elastase with respect to amino-acid sequence and to conformation. Another region (residues 214-220) reflects the amino-acid substitutions and conformational flexibility between these enzymes. The carbonyl O atom of the DCI moiety was found to be oriented away from the oxyanion hole, which greatly contributes to the stability of the DCI:D adduct. The comparisons of the active sites between native factor D, DCI-inhibited factor D, and various inhibited trypsin and elastase (PPE) molecules are providing the chemical bases directing our design of novel, small-molecule pharmaceutical agents capable of modulating the alternative complement pathway.
Cole
L B
LB
BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive, Birmingham, AL 35244-2812, USA.
Kilpatrick
J M
JM
Chu
N
N
Babu
Y S
YS
D000818:Animals; D048588:Benzoxazines; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism; D011416:Complement Factor D / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003170:Complement Pathway, Alternative; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D049934:Isocoumarins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism; D010196:Pancreatic Elastase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013552:Swine
D048588:Benzoxazines; D002219:Carbamates; D003374:Coumarins; D049934:Isocoumarins; D010078:Oxazines; D015842:Serine Proteinase Inhibitors; C054779:(1-(5-chloro-4-oxo-4H-3,1-benzoxazin-2-yl)-2-methylpropyl)carbamic acid 1,1-dimethylethyl ester; C046235:3,4-dichloroisocoumarin; D010196:Pancreatic Elastase; C493488:CFD protein, human; D011416:Complement Factor D