Immobilization of neurotensin receptor through immunologic recognition between ALFA tag and nanobody for...

Fabrication of bioactive interface by immobilizing proteins with pharmacological importance onto a solid surface is instrumental for interrogating drug actions and discovery of new drug molecules. In current work, we have developed a hybrid method for the interface fabrication by using Halo-tagged nanobody as a bidirectional adaptor for simultaneous capture of ALFA-tagged proteins onto solid surface and capturing proteins of the interest with a fused short epitope sequence. To this end, we engineered G protein-coupled receptors with a fused ALFA tag comprised of 13 amino acids and an ALFA-specific nanobody (Nb^ALFA) fused to a Halo tag for the covalent immobilization onto a silica surface chemically modified with the Halo substrate. This strategy was applied to incorporate β₂-adrenergic receptor (β₂AR) into a silica sphere-based chromatographic stationary phase. Both salbutamol and methoxyphenamine showed specific retention profiles in the β₂AR column with measured dissociation constant (K_d) as 58.50 μM and 115.20 μM respectively. We employed this strategy for human neurotensin receptor 1 (NTR1), a class A GPCR member involved in schizophrenia, diabetes, and cancer but with scarce verified ligands. The proved peptide ligands of NTR1 show retention time of 4.64 ± 0.04 min and 1.83 ± 0.05 min with K_d of 40.00 μM and 49.13 μM respectively in the NTR1 column. Extracts of Ziziphi Spinosae Semen (ZSS) were further applied to the NTR1 column and a natural product, spinosin, was discovered with estimated K_d of 5.16 μM and further identified by LC-MS. Molecular docking reveals that spinosin binds to orthosteric pocket of NTR1. Taken together, we developed a hybrid method for immobilizing GPCRs and fabricating the bioactive chromatographic column for measuring drug interactions and identified a new ligand binding to NTR1.