New Synthetic Thrombin Inhibitors: Molecular Design and Experimental Verification

BACKGROUND: The development of new anticoagulants is an important goal for the improvement of thromboses treatments. OBJECTIVES: The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. METHODS: Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. RESULTS: New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. CONCLUSIONS: The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor

Preclinical evaluation of drugs in animals helps researchers to select potentially informative clinical laboratory markers for human trials. To assess the utility of animal thrombin generation (TG) assay, we studied the sensitivity of animal plasmas to triggers of TG, human Tissue Factor (TF), and Activated Factor XI (FXIa). Pooled human, mouse, rat, guinea pig, rabbit, bovine, sheep, and goat plasmas were used in this study. TF- or FXIa-triggered TG and clotting were measured via fluorescence and optical density, respectively. Thrombin peak height (TPH) and time (TPT), clot time (CT), and fibrin clot density (FCD) were all analyzed. The trigger low and high sensitivity borders (LSB and HSB) for each assay parameter were defined as TF and FXIa concentrations, providing 20 and 80% of the maximal parameter value, unless the baseline (no trigger) value exceeded 20% of the maximal, in which case, LSB was derived from 120% of baseline value. Normal human samples demonstrated lower TPH HSB than most of the animal samples for both TF and FXIa. Animal samples, except mice, demonstrated lower TPT LSB for FXIa versus humans. Most rodent and rabbit samples produced baseline TG in the absence of TG triggers that were consistent with the pre-activation of blood coagulation. FCD was not sensitive to both TF and FXIa in either of the plasmas. Animal plasmas have widely variable sensitivities to human TF and FXIa, which suggests that optimization of trigger concentration is required prior to test use, and this complicates the extrapolation of animal model results to humans

New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics

Design of mutations in the reactive site region of Inf4.

<p><b>(A)</b> A web-logo representation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144940#pone.0144940.ref041" target="_blank">41</a>] of the P²–P^5’ binding loop sequences of 89 proteinase inhibitors from the Kazal family [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144940#pone.0144940.ref039" target="_blank">39</a>]. The amino acid positions are numbered by the wt-Inf4 sequence Phe9–Val15. Sequences of the corresponding reactive site fragments from wt-Inf4 and its mutants are shown below. <b>(B)</b> The MD simulation indicated a π-cation interaction (yellow dashed line) in wt-Inf4 between the phenyl group of Phe9 and the guanidinium group of Arg10. The nitrogen atoms are shown in blue, oxygen in red, and hydrogen in white. The torsion angles ψ, ϕ, χ₁, and χ₂ that describe the scissile bond conformation and rotations of the Arg10 side chain are drawn. <b>(C</b>) Superposition (main chain atoms) of wt-Inf4 (the most abundant conformation over the MD trajectory) with the structures of Inf4 mutants whose reactive loop solely adopted the canonical conformation. A close-up view of the reactive site region is indicated with a rectangle (wt-Inf4 –cyan, Mutant A–yellow, Mutant B–orange, Mutant C–green, Mutant 15 –magenta; this coloring is also used in the following Figs). The scissile bond of wt-Inf4 is indicated with a wavy line; a star indicates the Arg10 carbonyl oxygen. <b>(D)</b> A Coomassie-stained 12.5% SDS-PAGE showing the one-step purification procedure for one of the Inf4 versions expressed in <i>E</i>. <i>coli</i> BL21(DE3) cells (Trx-Mutant B is shown as an example). The protein samples applied to the gel are as follows: <u>M</u>–molecular weight marker (kDa); <u>Induction 0 h</u> and <u>4 h</u>–cell lysates before 1 mM IPTG induction and 4 h later, respectively; <u>Debr</u> and <u>Sol</u>–insoluble and soluble fractions, respectively; <u>Fl-thr</u>–flow-through of the <u>Sol</u> fraction applied to the column; <u>Wash</u>–washing the column with 50 mM Tris, pH 7.5, 500 mM NaCl, 100 mM imidazole; <u>Resin</u>–Chelating Sepharose with bound and washed Trx-Mutant B (26 kDa); <u>Elt</u>–Chelating Sepharose after Trx-Mutant B elution with 50 mM Tris-HCl, pH 7.5, 500 mM imidazole; thereafter, the protein sample was applied on the SOURCE 30Q column. <b>(E)</b> Following the two-step purification, the Trx-fused protein was cleaved with bovine thrombin. <u>Thrombin (mU)</u>–indicated amounts of bovine thrombin (0, 1, 2, 3, 4, 6, and 10 milliunits) were added to 10 μg of Trx-Mutant B resulting in two separate bands of Mutant B (13.1 kDa) and Trx (12.9 kDa). <b>(F)</b><u>Mutant B</u>–indicated quantities of the Mutant B protein (<u>10</u>, <u>1</u>, and <u>0.5</u> μg loaded into gel) that was purified after the cleavage reaction, as described in the sub-section “Expression of the thioredoxin-fused infestin-4 and its mutants”.</p

Analysis of fXIIa binding by Inf4 variants.

<p><b>(A</b>) The <i>CT</i>_<i>3</i> values (μM) measured in the aPTT assay in normal plasma are plotted against the <i>K</i>_<i>i</i> values for the Inf4 variants and the inhibitors of fXIIa from other families (CTI, CMTI-III, LCTI-III). The mean values ± SD are presented (n = 3). (<b>B</b>) Lineweaver-Burk plot (1/V, nM^-1*sec, versus 1/S, μM^-1) of fXIIa inhibition by 0, 0.5 and 1 nM of Mutant B at various concentrations (100, 200, 300, and 400 μM) of S-2302 substrate (mean + SD values, n ≥ 3). The data fitting with linear functions is shown with dots. (<b>C, D)</b> Complex of fXIIa (light violet) docked with wt-Inf4 (<b>C</b>) and Mutant B (<b>D</b>). The P¹ Arg10 residue of these inhibitors formed a salt bridge with S¹ Asp185 of fXIIa and the scissile bond was close (3.2–3.9 Å) to the catalytic residues Ser191 and His40. <b>(E)</b> A diagram representing the mean values of ln(1/<i>K</i>_<i>i</i>) (left axis) (n = 3; SD was approx. 0.2, i.e., 1%), where <i>K</i>_<i>i</i> is the inhibitory constant of the Inf4 variants for fXIIa; this value is proportional to the energy term. The values of the energy-like scoring function <i>SF</i> (right axis) were calculated with the ClusPro server.</p

Inf4 mutants did not inhibit FXa.

<p><b>(A)</b> Residual amidolytic activity of fXa at various concentrations of the Inf4 variants – 100% activity of fXa corresponds to a <i>k</i>_<i>cat</i> value of 240 s^-1. The mean ± SD values are shown (n = 3). Data fitting with a hyperbola is shown with dots. <b>(B, C)</b> Complexes of either wt-Inf4 <b>(B</b>; cyan) or Mutant B <b>(C</b>; orange) docked with fXa (secondary structure ribbons and C atoms are beige). Hydrogen bonds and salt bridges between charged functional groups are shown as yellow dashed lines. Red dashed lines show distances between the atoms involved in the cleavage reaction: 1) direction of the nucleophilic attack of Ser195 O^γ on the Arg10 carbonyl carbon and 2) direction of the proton transfer from His57 N^ε2 to the Asn11 carbonyl nitrogen (during the formation of the tetrahedral intermediate, a proton is acquired by His57 from Ser195, then a proton is transferred to the scissile bond to effect the cleavage). The scissile bond (shown as wavy line) of wt-Inf4 <b>(B)</b> was located in the fXa pocket properly for formation of the intermediate. P¹ Arg10 was bound to S¹ Asp189 and Gly218 by salt bridges. In addition, the Asn11 side chain of Mutant B <b>(C)</b> interfered with the catalytic Ser195 O^γ and His57 N^ε2 atoms.</p

Mutant B selectively inhibits the contact pathway of coagulation.

<p><b>(A–D</b>) Thrombin generation in normal platelet-poor plasma was triggered with a mixture of calcium chloride and either kaolin (<b>A</b>) or TF (5 pM final concentration) <b>(B)</b> and was monitored for 90 min. Frozen-thawed plasma was preincubated at 37°C with various concentrations of Mutant B as indicated in the legend (0.2, 0.5, 1, 2, and 5 μM), CTI (0.5 and 5 μM) or vehicle (control). The assay parameters including the time to the thrombin peak <b>(C)</b> and the peak amplitude <b>(D)</b> were calculated for the experiments with the activation by kaolin (black bars) or 5 pM TF (gray bars). The mean values ± SD are shown; each experiment was performed in duplicate and repeated twice. <b>(E, F)</b> The thromboelastography assay was carried out in plasma preincubated with Mutant B (1, 2, 5, 10, and 20 μM), CTI (5 and 15 μM), or vehicle (control). <b>(E)</b> Thromboelastogram representing clotting in frozen-thawed platelet-poor plasma from healthy volunteers, drawn into a flask without any activator (coagulation was triggered by the contact pathway from flask walls). <b>(F)</b> Diagram showing the mean R-time values (n = 2) for thromboelastograms, obtained as a result of the assay either in normal platelet-poor plasma without activator (black bars), or in fXII-depleted plasma activated with 0.6 pM TF (gray bars). <b>(G)</b> Prolongation of the whole blood clotting time by Mutant B. The time to the visually detected clotting was measured in whole blood collected into tubes, which were prefilled with either vehicle (final concentration in blood 30 mM Hepes pH 7.4, empty squares) or Mutant B (10 μM, filled squares), without any other anticoagulant. Individual data from 5 donors and the median values are presented; for each donor the experiment was performed in duplicate. P-value (* < 0.005) was estimated using the Wilcoxon rank test.</p

Wt-Inf4 and CTI are the most potent inhibitors of fXIIa and have some off-target activities.

<p><b>(A)</b> The residual amidolytic activity (%) of fXIIa upon incubation with various concentrations of wt-Inf4 (red), CTI (black), LCTI-III (cyan), or CMTI-III (magenta). <b>(B, C)</b> The residual amidolytic activity (%) of the coagulation-related proteases plasma kallikrein (purple pentagon), plasmin (cyan), fXIa (blue), fIXa (purple sphere), fXa (red), thrombin (black), fVIIa (magenta), and aPC (orange) upon incubation with various concentrations (logarithmic scale) of wt-Inf4 <b>(B)</b> and CTI <b>(C)</b>.</p

Application of Mutant B in the thrombodynamics assay.

<p>The thrombodynamics assay in platelet free plasma. <b>(A)</b> A representative dark-pole image of the assay cuvette filled with recalcified normal plasma (dark background) 30 min after the start of the thrombodynamics assay. TF was localized on the surface of a rectangular insert at the upper side of the cuvette (“Surface-immobilized TF”) and triggered a frontal growth of the bright fibrin clot (“TF-initiated clot”) in the downwards direction. Additional TF-independent artifact clots appeared far from the activator. <b>(B)</b> A percentage of the cuvette area occupied by fXIIa-initiated, TF-independent artifact clots in frozen-thawed normal plasma versus time (min). If no fXIIa inhibitor was added, then these clots appeared (occupied 5% of the cuvette area) 10 min after the assay was initiated and occupied the entire cuvette (100% of the cuvette area) 30 min later. Plasma samples were pre-incubated at 37°C with a vehicle (empty squares) or Mutant B (filled squares) and CTI (filled triangles) at their <i>CT</i>_<i>3</i> concentrations: 20 μM and 10 μM, respectively. The mean values are presented for n = 10. <b>(C)</b> The size of the TF-initiated fibrin clot in a downward direction (μm) versus time (min) in fXII-depleted plasma. The plasma was preincubated at 37°C with Mutant B (20 μM, filled triangles) or vehicle (empty squares; mean values for n = 3 are shown). <b>(D)</b> A percentage of the cuvette area occupied by fXIa-initiated, TF-independent clots in frozen-thawed normal plasma versus time (min). These clots appeared approximately 10–12 min after the assay was initiated and occupied the entire cuvette 20 min later. The plasma samples were preincubated with a vehicle, Mutant B (20 μM) or CTI (10 μM). The mean values are presented for n = 10. <b>(E)</b> Images of the assay cuvettes 30 min after the assay was initiated, filled with normal frozen-thawed plasma that was spiked with 20 pM fXIa (“hyper”) or vehicle (“normal”) and preincubated with Mutant B (“MutB”) or vehicle (“no inhibitor“).</p