Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain.
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10 (-4)  s (-1) , and to selectively inhibit chymase activity with an IC 50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.
F. Hoffmann-La Roche Ltd, Pharma Research & Early Development, Discovery Technologies, Basel, Switzerland.
Daniel
D
Schlatter
Simon
S
Brack
David W
DW
Banner
Sarah
S
Batey
Jörg
J
Benz
Julian
J
Bertschinger
Walter
W
Huber
Catherine
C
Joseph
Arne
A
Rufer
Anita
A
van der Klooster
Martin
M
Weber
Dragan
D
Grabulovski
Michael
M
Hennig
D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D053818:Chymases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D019151:Peptide Library; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D053818:Chymases