Heparin Binding to an Engineered Virus-like Nanoparticle Antagonist.

The anticoagulant activity of heparin administered during medical interventions must be reversed to restore normal clotting, typically by titrating with protamine. Given the acute toxicity associated with protamine, we endeavored to generate safer heparin antagonists by engineering bacteriophage Qß virus-like particles (VLPs) to display motifs that bind heparin. A particle bearing a single amino acid change from wild-type (T18R) was identified as a promising candidate for heparin antagonism. Surface potential maps generated through molecular modeling reveal that the T18R mutation adds synergistically to adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region that is replicated 180 times over the capsid. Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle. Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin. Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP. Dynamic light scattering (DLS) experiments suggested that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent, and in vitro clotting assays confirmed the 1:1 binding stoichiometry as full antagonism of heparin is achieved. Biolayer interferometry and backscattering interferometry corroborated the strong interaction of T18R with heparin, yielding Kd ∼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists.

Bio-layer interferometry of a multivalent sulfated virus nanoparticle with heparin-like anticoagulant activity.

Heparin is a sulfated glycosaminoglycan that is routinely used as an anticoagulant. It is typically purified from bovine or porcine sources, leading to heterogeneity that poses several challenges when used clinically. We have found that the bacteriophage Qß can be selectively sulfated to yield virus-like nanoparticles (sulf-VLP) that elicit anticoagulant activity similar to heparin. In an effort to explore the binding interactions that heparin-like VLPs make with cationic targets, described herein are bio-layer interferometry studies utilizing the BLItz platform that evaluate the interaction of sulf-VLP with the cationic peptide CDK5 (50% Lys). Streptavidin biosensors modified with biotin-CDK5 were found to bind strongly to sulf-VLP and not to the underivatized nanoparticle. Titration of sulf-VLP yielded concentration-dependent sensorgrams, permitting calculation of rate and equilibrium constants: k(on) = (8 ± 3) × 10(6) s(-1) for the association phase, k(off )= (5 ± 2) × 10(-3) M s(-1) for the dissociation phase, yielding an overall dissociation constant K(D)~ 1 nM. Fitting was best achieved using an equation possessing both exponential and linear terms, suggesting a mechanism more complex than 1:1 binding. To mitigate multivalency and rebinding effects, experiments were conducted with protamine (~70% Arg) added during the dissociation phase, leading to more pronounced dissociation curves and k off values that yielded a near-linear relationship with protamine concentration.

Directed polyvalent display of sulfated ligands on virus nanoparticles elicits heparin-like anticoagulant activity.

Heparin is a sulfated glycosaminoglycan that is widely used as an anticoagulant. It is typically extracted from porcine or bovine sources to yield a heterogeneous mixture that varies both in molecular weight and in degree of sulfation. This heterogeneity, coupled with concern for contamination, has led to widespread interest in developing safer alternatives. Described herein are sulfated bacteriophage Qß virus-like particles (VLPs) that elicit heparin-like anticoagulant activity. Sulfate groups were appended to the VLP by synthesis of single- and triple-sulfated ligands that also contained azide groups. Following conversion of VLP surface lysine groups to alkynes, the sulfated ligands were attached to the VLP via copper-catalyzed azide-alkyne cycloaddition (CuAAC). MALDI-MS analysis of the intermediate alkyne VLP indicated that the majority of the coat proteins contained 5-7 of the alkyne linkers; similar analysis of the intermediate alkyne particles conjugated to a fluorescein azide suggest that nearly the same number of attachment points (3-6) are modified via CuAAC. Analysis by SDS-PAGE with fluorescent staining indicated altered migration patterns for the various constructs: compared to the wild-type nanoparticle, the modified coat proteins appeared to migrate farther toward the positive pole in the gel, with coat proteins displaying the triple-sulfated ligand migrating significantly farther. Clotting activity analyzed by activated partial thrombin time (APTT) assay showed that the sulfated particles were able to perturb coagulation, with VLPs displaying the triple-sulfated ligand approximately as effective as heparin on a per mole basis; this activity could be partially reversed by protamine. ELISA experiments to assess the response of the complement system to the VLPs indicate that sulfating the particles may reduce complement activation.