Development of an Allostery Responsive Chromatographic Method for Screening Potential Allosteric Modulator of Beta2-adrenoceptor from a Natural Product-Derived DNA-Encoded Chemical Library

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.