Virus-Based Heparin Reversal Agents
Synergistic polyvalent interactions allow biology to exploit low affinity binding events to affect physiological responses. Exploiting this, bacteriophage Q-beta was used as a multivalent platform for the display of motifs that antagonize heparin. Our prior work generated a series of particles synthetically and genetically. While several of these constructs showed good activity in aPTT clotting assays as heparin antagonists, we have focused on mutant T18R as a promising candidate for heparin antagonism that can be readily generated reproducibly and in large quantities. Utilizing mutant K16M as the negative control, these two mutants differ in surface charge by 360 charge units taking into account the 180 coat proteins per capsid. Surface potential maps generated through molecular modeling reveal that the T18R mutation complements adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region. Fluorescence spectroscopy using fluorescein-labeled heparin (HepFL) (Invitrogen, ~12 kDa) was performed to evaluate dissociation constants and binding stoichiometry. Titration of HepFL with T18R resulted in a concentration-dependent change in emission intensity that appears to approach a limiting value. Binding stoichiometries were estimated at 67?7 HepFL per VLP, or 2.7?0.3 coat protein subunits per HepFL. Analogous experiments with K16M did not yield a consistent change in emission intensity, suggesting no significant interaction between the VLP and saccharide. Hill plots were generated for each HepFL concentration and the resulting dissociation constants were averaged to yield Kd = (2.9?4.5) x 10-10M. These biophysical measurements confirm the activity of T18R, and open the door for designing additional antagonists with greater activity and specificity for heparin.
Cheong, Ho Yong, " Virus-Based Heparin Reversal Agents" (2011). URC Student Scholarship.
John Stauffer Summer Research Fellowship in Chemistry/Biochemistry