De novo design of self-assembling foldamers that inhibit heparin-protein interactions

Abstract

A series of self-associating foldamers have been designed as heparin reversal agents, as antidotes to prevent bleeding due to this potent antithrombotic agent. The foldamers have a repeating sequence of Lys-Sal, in which Sal is 5-amino-2-methoxy-benzoic acid. These foldamers are designed to self-associate along one face of an extended chain in a β-sheet-like interaction. The methoxy groups were included to form intramolecular hydrogen bonds that preclude the formation of very large amyloid-like aggregates, while the positively charged Lys side chains were introduced to interact electrostatically with the highly anionic heparin polymer. The prototype compound (Lys-Sal)4 carboxamide weakly associates in aqueous solution at physiological salt concentration in a monomer-dimer-hexamer equilibrium. The association is greatly enhanced at either high ionic strength or in the presence of a heparin derivative, which is bound tightly. Variants of this foldamer are active in an antithrombin III-factor Xa assay, showing their potential as heparin reversal agents.

Figure 1

Chemical structures of SalAA foldamers explored in this study. (A) Schematic (left) and crystal structure (right) of a dimeric foldamer based on the salicylamide backbone, Ac-Sal-Gly-Sal-NMe amide, in which Ac is acetyl, Sal is the indicated amino-salicylic acid methyl ether unit, Gly is glycine, and NMe amide is an N-methyl amide. (B) Chemical structures of salicylamide-α-amino acid peptides (SalAA foldamers) for C-terminal and N-terminal (C) modified series. (D) Chemical structure of compound 7, (d-Cit-Sal)₄-CONH₂; in this variant of (Lys-Sal)₄-CONH₂, compound 1, cationic lysine is replaced with a neutral citrulline amino acid.

Figure 2

Concentration-dependent CD spectra of compound 1 in the near (A) and far (B) UV regions. Extinction coefficient as a function of wavelength (C), determined from near UV–vis absorption spectra. CD mean residue ellipticity (MRE) at 333 nm and UV–vis absorption shifts in λ_max are overlaid (D) with their respective fits; K_hex equals 10^–18.4 (or 4.0 × 10^–19) M⁵ for CD MRE₃₃₃ and 10^–19.3 (or 5 × 10^–20) M⁵ for UV–vis λ_max.

Figure 3

Concentration-dependent ITC of 1. A stock solution of 1.0 mM 1 in TBS was serially diluted into the same buffer (A). The heat associated with each injection was determined by integrating the peaks. The heat generated for a given final concentration is then summed and expressed in cal/mol vs final concentration (B). The smooth line shows the best fit of a monomer-dimer-hexamer equilibrium to the data (see Methods). The weight fraction distribution for hexamer, dimer, and monomer species are plotted vs concentration (C).

Figure 4

UV–vis absorption λ_max and normalized ITC heats of dilution as a function of SalAA foldamer concentration. (A) UV–vis absorption λ_max as a function of SalAA foldamer concentration for C-terminal modifications. (B) UV–vis absorption λ_max as a function of SalAA foldamer concentration for N-terminal modifications. Normalized ITC heats of dilution (pK_(hex-mon)) values are summarized in Table 2 as a function of SalAA foldamer concentration for C-terminal (C) and N-terminal (D) modified series, and their respective fits are illustrated as lines.

Figure 5

Concentration-dependent shifts in λ_max for 1 at varying [NaCl] in 0.05 M Tris, pH 7.4. The smooth curves were generated by global fitting of the data to a monomer-hexamer equilibrium. The value of λ_max for the monomer (304 nm) and hexamer (318 nm) were globally fit, while the value of pK_(hex-mon) was allowed to vary for each curve. The fit pK_(hex-mon) values were <15 at 0 M NaCl, 17.4 at 0.05 M, 19.7 at 0.15 M, 24.1 at 0.5 M, and 26.5 at 1.0 M.

Figure 6

Near UV CD and ITC characterizations of SalAA compound 1 binding to fondaparinux. (A) Near UV CD spectra of the fondaparinux titration of compound 1, (Lys-Sal)₄-CONH₂, where increasing concentrations of fondaparinux were titrated into 25 μM compound 1. (B) Concentration-dependent ITC of fondaparinux titrated into compound 1, (Lys-Sal)₄-CONH₂. Shown are the raw data of 150 μM fondaparinux (stock solution) titrated into 50 μM compound 1. (C) Accumulated integrated heat changes as a function of fondaparinux:foldamer molar ratio for three experiments at the listed concentrations.