Development of New Cyclic Plasmin Inhibitors with Excellent Potency and Selectivity

Abstract

The trypsin-like serine protease plasmin is a target for the development of antifibrinolytic drugs for use in cardiac surgery with cardiopulmonary bypass or organ transplantations to reduce excessive blood loss. The optimization of our recently described substrate-analogue plasmin inhibitors, which were cyclized between their P3 and P2 side chains, provided a new series with improved efficacy and excellent selectivity. The most potent inhibitor 8 binds to plasmin with an inhibition constant of 0.2 nM, whereas K(i) values >1 μM were determined for nearly all other tested trypsin-like serine proteases, with the exception of trypsin, which is also inhibited in the nanomolar range. Docking studies revealed a potential binding mode in the widely open active site of plasmin that explains the strong potency and selectivity profile of these inhibitors. The dialkylated piperazine-linker segment contributes to an excellent solubility of all analogues. Based on their overall profile the presented inhibitors are well suited for further development as injectable antifibrinolytic drugs.

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

“…Aprotinin, a plasmin inhibitor in limited use in some countries, also suffers from a lack of specificity [1,5,6]. Several plasmin inhibitors being developed as antifibrinolytics include KDI-L17R [15] textilinin-1 [16] and cyclic peptidomimetics [13,14]. Yet, these are active site inhibitors and their selectivity for plasmin is challenging because of the similarity of active site geometries of a number of trypsin-like proteases.…”

“…These approaches include substrate-based design of linear and cyclic peptidomimetics [10][11][12][13][14], mutagenesis of key residues to engineer Kunitz-and Kazal-type protein/peptide inhibitors [15][16][17], covalent inhibition through a reactive nitrile or aldehyde warhead [18,19], and structure-based computational inhibitor design [20,21]. Each of these approaches typically targets the enzyme's active site.…”

“…The monomeric scaffolds included chalcones (compounds 1-10), flavonoids (11)(12)(13)(14)(15)(16) [30][31][32], sucrose octasulfate (17) [40], quinazolinones (18 and 19) [37], and tetrahydro-isoquinolines (20)(21)(22)(23)(24)(25)(26)(27) [36], whereas the dimeric scaffolds comprised flavonoid-quinazolinone heterodimers (28)(29)(30)(31)(32)(33)(34) [37], bis-quinazolinones homodimers (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) [37], and bis-flavonoid homodimers (48-55). In addition to the inherent diversity of the scaffolds in this library, NSGMs also differed in the number (1 to 8) and orientation of the sulfate groups.…”

Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines

“…A relatively strong trypsin inhibition was still found for the phenylsulfonyl and cyclohexylsulfamoyl analogues 29 and 31, with K i values of 62 and 21 nM, respectively, which is in the same range as found for the reference compounds 2 and 3 ( Table 2). 25,27 The strong subnanomolar inhibitory potency of compounds 3, 29, and 31 suggested that plasmin should tolerate some P1 modifications, which lead to a generally reduced affinity against all trypsin-like serine proteases. Therefore, additional inhibitors with the longer piperazine-dipropionyl linker have been prepared, which provided slightly more potent inhibitors than the shorter piperazine-diacetyl linker.…”

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

“…In contrast, no cleavage could be observed after the P2 lysine, because the reference 2,4-dichlorophenylacetyl-Lys-Lys-OH is more hydrophilic and elutes at 25.95 min. Moreover, under identical conditions approximately 42% of the known chromogenic trypsin substrate methylsulfonyl-D-cyclohyexylalanine-Gly-Arg-pnitroanilide 84 was hydrolyzed after the P1-Arg residue.…”