Design of a linker for trivalent thrombin inhibitors: interaction of the main chain of the linker with thrombin.
N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990). The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds. Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 1.7-3.4 nM. The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker. The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition. Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 0.51 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length. Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM). Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
Szewczuk
Z
Z
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Gibbs
B F
BF
Yue
S Y
SY
Purisima
E
E
Zdanov
A
A
Cygler
M
M
Konishi
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry
D006629:Hirudins; D011480:Protease Inhibitors; D013917:Thrombin