Allosteric
ligands are promising drugs owing to their remote regulations
of the orthosteric ligand signaling pathway. There are few allosteric
ligands due to the lack of handy and efficacious method for the screening.
Herein, we developed an affinity chromatographic method for allosteric
ligand screening by immobilizing purified beta2 adrenoceptor (β2-AR) onto macroporous silica gel by a two-point tethering
method. The method relies on the occupation of the orthosteric site
by an antagonist and the chelation of N-terminal His-tag of the receptor
and Ni2+ coated on the gel. The immobilized β2-AR demonstrated the greatest allosteric responsive feature
when Cmpd-15 (0.25 μM) was included in the mobile phase. Under
the same conditions, the association constants of three agonists (salbutamol,
terbutaline, and tulobuterol) reduced to 47%, 19%, and 27% compared
with the data without the inclusion of Cmpd-15 in the mobile phase.
APF was screened as a potential allosteric modulator of β2-AR by applying the immobilized receptor in a natural product-derived
DNA-encoded chemical library (DEL). Relying on these results, we reasoned
that the current method has potential in screening allosteric ligands
of the receptor. We expect that it is applicable for the discovery
of new allosteric binding sites of a target protein and screening
allosteric modulators of the other receptors from complex samples.
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