Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes.
The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.
Corvas International Inc., San Diego, California 92121, USA.
R
R
Krishnan
E
E
Zhang
K
K
Hakansson
R K
RK
Arni
A
A
Tulinsky
M S
MS
Lim-Wilby
O E
OE
Levy
J E
JE
Semple
T K
TK
Brunck
D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005998:Glycine / Q000378:metabolism; D006146:Guanidines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D010880:Piperidines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D000447:Aldehydes; C114376:CVS 1578; C114378:CVS 1694; C114377:CVS 1695; D006146:Guanidines; D046911:Macromolecular Substances; D010880:Piperidines; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D014357:Trypsin; D013917:Thrombin; D005998:Glycine