Potent bivalent thrombin inhibitors: Replacement of the scissile peptide bond at P-1-P-1 ' with arginyl ketomethylene isosteres

We have designed highly potent synthetic bivalent thrombin inhibitors, whic h consist of an active site blocking segment, a fibrinogen recognition exos ite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite s imultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethyle ne isosteres Arg Psi-[CO-CH2-X]P-1' were incorporated into the bivalent inh ibitors as P-1-P-1' segment to eliminate the scissile bond. The P-1' residu e is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thr ombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-Arg Psi[CO-CH2-S]Gly-(Gly)(4 )-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH showed t he lowest K-i value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K-i = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successful ly reduced the size of the inhibitor from similar to 7 kDa of recombinant h irudin to similar to 2 kDa without losing the affinity.