S03

Heparin

Heparin-binding aryl amide

Fig. 8.14 Major repeat of heparin and a heparin binding aryl amide. Backbone rigidifying H-bonds are shown as dashed lines.

inhibitory effect on cholesterol uptake in either model. On the other hand, while none of the previously tested a-peptide inhibitors induced effects in whole CaCO-2 cells, a b-peptide nonamer decreased the absorption rate of radiolabeled cholesterol to background levels (indicated by an increase in T1//2 of cholesterol absorption rate from 102 min to 2.6 h). This suggested that the b-peptide nonamer was not susceptible to proteolytic degradation, unlike its a-peptide counterparts.

8.2.2.8 Heparin-inhibiting Aryl Amides

Heparin is a linear, highly sulfated polysaccharide composed of repeating l-iduronic acid and d-glucosamine units [176]. Heparin demonstrates anticoagulant activity and has therefore been utilized therapeutically as a treatment for thrombotic diseases [176, 177]. Due to the presence of multiple sulfate functionalities, heparin carries an overall negative charge, and heparin is therefore recognized by cationic peptides (such as antithrombin) bearing either consensus sequence XBBXBX or XBBBXXBX, where B is a basic amino acid residue and X is any other amino acid residue [178]. Furthermore, it has been suggested that the basic residues of heparin-binding peptides are localized on one face of either an a-helix or b-sheet. While several potent heparin-binding peptides have been reported [179-182], these still suffer the drawbacks of proteolytic instability and limited bioavailability.

Oligomeric 1,3-disubstituted aryl amides have been designed to strongly interact with heparin [183]. Aryl amides were designed to display appropriately spaced cationic groups to bind the negative charges on heparin (Fig. 8.14). Molecular dynamic simulations showed that binding was mostly attributed to ionic interactions and that increasing positive charge increased heparin binding [183]. Chromagenic assay studies revealed the ability of aryl amides to compete with an-tithrombin to bind full-length heparin and low molecular weight heparin analogs (IC50 = 22.5 mM). Schild plot analysis revealed the designed aryl amide could inhibit heparin-antithrombin complex formation with dissociation constants in the micromolar range (1.8-6.7 mM). Furthermore, the aryl amide designs could inhibit heparin induced clotting in activated partial thromboplastin time clotting assays with comparable efficacy to the clinically used heparin neutralization agent protamine. Importantly, the aryl amides were determined to be nonhemolytic at concentrations as high as 1 mM.

Fig. 8.15 Aryl amide inhibitor of calmodulin. 8.2.2.9 Calmodulin-inhibiting Aryl Amides

Calmodulin is an attractive therapeutic target due to its implications in multiple biochemical pathways such as metabolism, apoptosis, inflammatory responses, muscle contraction, intracellular movement, short-term and long-term memory, nerve growth and the immune response [184, 185]. Three residues at the i, i + 3, and i + 7 positions of the smMLCK peptide are critical for CaM-binding [186]. Recapitulation of similar functional groups onto the appropriate scaffold may therefore yield potent and highly selective CaM-binding peptidomimetics.

Molecular modeling studies revealed that the arrangement of tert-butyl groups on a thioether substituted aryl amide scaffold closely matched the arrangement of hydrophobic residues presented by the helical CaM-binding peptide smMLCK (Fig. 8.15) [187]. Modeling also showed that d-phenylalanine residues presented on both ends of the aryl amide were adequately accommodated by the hydrophobic binding pockets of CaM globular domains [187]. Inhibitory concentrations (Ki) of the aryl amide inhibitors were determined through fluorescence polarization competition assays by titrating aryl amides against 1:1 complexes of CaM and the high-affinity CaM-binding peptide mastoparin X (MaX). Addition of inhibitors resulted in a shift of the maximum fluorescence emission from 327 to 341 nm, indicating release of MaX from CaM. Aryl amides demonstrated inhibitory concentration in the nanomolar range with the most potent inhibitor having a Ki = 7.10 nM [187]. Experiments with 2-D(1H15N)-HSQC NMR also suggested that the aryl amides bound CaM similarly to CaM-binding peptides smMLCK and MARCKS, demonstrating that the designed aryl amides were suitable pepti-domimetics though with limited solubility [187].

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