Site-directed targeting of plasminogen activator inhibitor-1 as an example for a novel approach in rational drug design.

As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition of PAI-1. Since monoclonal antibodies are in most cases not ideal for therapeutic use, the question of whether smaller molecules exert comparable effects is a hot issue. To answer this question, Cys residues were introduced in PAI-1 at positions previously identified as determining the epitope of a PAI-1-inhibiting antibody, MA-8H9D4, resulting in PAI-1-R300C, PAI-1-Q303C, and PAI-1-D305C. Subsequently, low molecular mass sulfhydryl-specific reagents (i.e. BODIPY 530/550 IA (molecular mass 626 Da) and BODIPY FL C(1)-IA (molecular mass 417 Da)) were allowed to react covalently with the cysteine. The functional distribution (inhibitory versus substrate) toward tissue-type plasminogen activator was determined for the labeled and the unlabeled samples. Labeling at position 300 leads to a 1.7- and 2.2-fold increase in SI value for BODIPY 530/550 IA and BODIPY FL C(1)-IA, respectively. Labeling at position 303 results in a 3.3- and 1.9-fold increase of the SI value for the large and the small label, respectively. At position 305, the SI values are 3.1-fold increased for both labels. The effect (on SI and on serpin activity) of the manipulations at these positions is in good agreement with the effect exerted by MA-8H9D4. In conclusion, our study provides proof of concept for the proposed approach in evaluating whether targeting a functional epitope with a small synthetic compound may be a feasible strategy in rational drug design.