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The mode of binding of four active-site directed inhibitors to human thrombin
has been determined by x-ray crystallographic analysis. The inhibitors studied
are benzamidine, PPACK, NAPAP, and MD-805, of which the last three are compounds
evolved specifically to inhibit thrombin. Crystal structures were determined in
the presence of both the inhibitor and the undecapeptide [des-amino
Asp55]hirudin(55-65) which binds distant from the active site. Despite having
significantly different chemical structures, NAPAP and MD-805 bind to thrombin
in a very similar "inhibitor binding mode" which is not that expected by direct
analogy with the binding of substrate. Both inhibitors bind to thrombin in a
similar way as to trypsin, but thrombin has an extra loop, the "Tyr-Pro-Pro-Trp
loop," not present in trypsin, which gives further binding interactions and is
seen to move somewhat to accommodate binding of the different inhibitors. The
fact that NAPAP and MD-805 require different stereochemistry for potent
inhibition is demonstrated, and its structural basis clarified. The wealth of
data on analogs and variants of these lead compounds is shown to be compatible
with this inhibitor binding mode.
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