NIH Research Festival
FARE Award Winner
Bioactive compounds possess unfavorable properties for drug development due to on-target, off-tissue mediated side effects. One alternative is to use fragments that can react to form a bioactive product upon exposure to a certain stimulus or chemical. We sought to apply this approach for activation of the parathyroid hormone receptor-1 (PTHR1), which regulates skeletal development, and mineral ion homeostasis. Two fragments (PTH1-11 and PTH12-34) together comprise the prototypical peptide agonist of PTHR1 (PTH1 34). Here we describe methodology for the in-situ synthesis of conjugates that resemble PTH1 34 through peptide template-induced dimerization and click (azide-alkyne) reactions between the fragment peptides PTH1-11 and PTH12-34.
Templated dimerization was achieved in presence of a heterodimeric peptide (HDP), fusing PTH fragments containing click chemistry handles to one of two Nbs that bind to short peptide epitopes. HDP was used to bring fusions comprised of Nbs and PTH fragments with complementary click handles, into proximity.
Gel electrophoresis analysis of the kinetics of the reaction between azide- and alkyne-functionalized Nbs showed that HDP accelerated product formation. We also used a fluorescence resonance energy transfer assay and fluorophore-labeled Nbs to corroborate the finding that HDP induces proximity between Nbs. During the cell-based assay, HDP facilitated click chemistry-induced dimerization. Whereas PTH fragment-click handle-Nb conjugates were weakly active alone or when added in tandem, the addition of HDP to the tandem mixture resulted in strong activation of the PTHR1.
Altogether, this approach entails a new strategy for in situ synthesis of a bioactive agonist from its inactive fragment peptides.
Scientific Focus Area: Chemical Biology
This page was last updated on Monday, September 25, 2023