The crystal structures of four active site-directed
thrombin inhibitors, 1–4, in a complex with human
α-thrombin have been determined and refined at up to
2.0 Å resolution using X-ray crystallography. These
compounds belong to a structurally novel family of inhibitors
based on a 2,3-disubstituted benzo[b]thiophene
structure. Compared to traditional active-site directed
inhibitors, the X-ray crystal structures of these complexes
reveal a novel binding mode. Unexpectedly, the lipophilic
benzo[b]thiophene nucleus of the inhibitor
appears to bind in the S1 specificity pocket.
At the same time, the basic amine of the C-3 side chain
of the inhibitor interacts with the mostly hydrophobic
proximal, S2, and distal, S3, binding
sites. The second, basic amine side chain at C-2 was found
to point away from the active site, occupying a location
between the S1 and S′1 sites.
Together, the aromatic rings of the C-2 and C-3 side chains
sandwich the indole ring of Trp60D contained in the thrombin
S2 insertion loop defined by the sequence
“Tyr-Pro-Pro-Trp.” [The thrombin residue
numbering used in this study is equivalent to that reported for
chymotrypsinogen (Hartley BS, Shotton DM, 1971, The
enzymes, vol. 3. New York: Academic Press. pp
323–373).] In contrast to the binding mode of
more classical thrombin inhibitors (d-Phe-Pro-Arg-H,
NAPAP, Argatroban), this novel class of
benzo[b]thiophene derivatives
does not engage in hydrogen bond formation with Gly216
of the thrombin active site. A detailed analysis of the
three-dimensional structures not only provides a clearer
understanding of the interaction of these agents with thrombin,
but forms a foundation for rational structure-based drug
design. The use of the data from this study has led to
the design of derivatives that are up to 2,900-fold more
potent than the screening hit 1.