Immobilization of neurotensin receptor through immunologic recognition between ALFA tag and nanobody for affinity-based ligand discovery.

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 (NbALFA) 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 (Kd) 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 Kd 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 Kd 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.