Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH2. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH2, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a Ki of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′

Hydrogen bond distances between the catalytic His 57 NE2 and Ser195 OG in thrombin complexes.

<p>N.P. - not present.</p

Hydrogen bond distances between thrombin Gly193 N and Ser195 N and bound inhibitors.

<p>N.P. - not present.</p

Inhibition of factor Xa and trypsin by tetrapeptide inhibitors.

<p>Inhibition of factor Xa and trypsin by tetrapeptide inhibitors.</p

Inhibition of bovine thrombin-induced cleavage of the chromogenic substrate (S2238) by the peptide inhibitors.

<p>Inhibition of bovine thrombin-induced cleavage of the chromogenic substrate (S2238) by the peptide inhibitors.</p

Distance between the catalytic Ser195 residue of thrombin and the P1 residue carbonyl carbon in thrombin-inhibitor complexes.

<p>Distance between the catalytic Ser195 residue of thrombin and the P1 residue carbonyl carbon in thrombin-inhibitor complexes.</p

Stereo view of the active-site region of unliganded human α-thrombin.

<p>Thrombin residues are represented as thin lines (carbon depicted in cyan, oxygen in red, nitrogen in blue and sulfur in yellow). The side chains of Lys60F and Asp189 (carbon represented in pink, other elements as above), and of Asp102, His57 and Ser195 (carbon represented in yellow, other elements as above) are shown as stick models.</p

Structure of the peptide inhibitors characterized in complex with human α-thrombin.

<p>fPrI (d-Phe-Pro-d-Arg-Ile-CONH₂), fPrC (d-Phe-Pro-d-Arg-Cys-CONH₂), fPrt (d-Phe-Pro-d-Arg-d-Thr-CONH₂).</p

Prolongation of thrombin time by peptide inhibitors.

<p>Human plasma thrombin times were measured in the absence of inhibitor (NI) and in the presence of 0.10 mM (green bar) 0.25 mM (white bars), 0.5 mM (blue bars) or 1 mM (red bars) of the indicated tetrapeptide.</p

Inhibition of amidolytic activity of α-thrombin by peptide inhibitors.

<p>Cleavage of a chromogenic substrate (S2238) by bovine α-thrombin in the absence (•) and in the presence (○ - 5 µM; ▪ - 15 µM) of the tetrapeptides fPrI (A), fPrC (B) and fPrt (C). Data correspond to a representative set of peptide concentrations of at least three independent experiments. The derived K_M (3.65±0.3 µM) and V_max (15.17±0.18 µM/min) values for the reaction of bovine α-thrombin towards the S2238 substrate are in good agreement with the previously published kinetics parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034354#pone.0034354-Wagner1" target="_blank">[35]</a>.</p