| Autor: |
Cheong HY; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States., Groner M; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States., Hong K; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States., Lynch B; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States., Hollingsworth WR; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States., Polonskaya Z; Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States., Rhee JK; Department of Food Science and Engineering, Ewha Womans University , Seou 03760, Korea., Baksh MM; School of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Finn MG; School of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States., Gale AJ; Department of Molecular and Experimental Medicine, The Scripps Research Institute , La Jolla, California 92037, United States., Udit AK; Department of Chemistry, Occidental College , Los Angeles, California 90041, United States. |
| Abstrakt: |
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 Qβ 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 K d ∼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists. |
