A strategy to profile prime and non-prime proteolytic substrate specificity.
A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4-P1 non-prime site substrate specificity. The data was used to synthesize biased donor-quencher positional scanning libraries to profile the P1'-P4' prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1'-P4' positional scanning libraries. From these libraries focused donor-quencher libraries incorporating P4-P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4-P4' substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.
Petrassi
H Michael
HM
Genomics Institute of the Novartis Research Foundation, Drug Discovery, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Williams
Jennifer A
JA
Li
Jun
J
Tumanut
Christine
C
Ek
Jared
J
Nakai
Takashi
T
Masick
Brian
B
Backes
Bradley J
BJ
Harris
Jennifer L
JL
D000595:Amino Acid Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques; D003198:Computer Simulation; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D003374:Coumarins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D013917:Thrombin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases