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

Abstract

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.

Surface-enhanced Raman spectroscopy as a powerful method for the analysis of Chinese herbal medicines

“…In order to improve specificity in SERS detection, functionalized probes are usually developed and used to modify the substrate. [61][62][63] Although the affinity screening method based on immobilized proteins has demonstrated dramatic advances in screening bioactive compounds from natural products, [64][65][66] it is still challenged by long screening time, binder loss and low contents in samples. To address the urgent needs, Zhao et al have employed a dual-functional SERS probe to screen and detect bioactive ingredients binding to the target protein from Gardenia jasminoides extract.…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…Below, we will briefly discuss some synthetic small molecular weight allosteric regulators since they, like small endogenous regulators (cholesterol, metal ions, etc. ), are described in detail in a number of reviews and experimental works [ 417 , 418 , 419 , 420 , 421 , 422 ], and we will pay more attention to autoantibodies and pepducins.…”

“…Cmpd-15 is able of entering the cell and binding specifically to the intracellular allosteric site formed by the cytoplasmic ends of TM1, TM2, TM3, and TM7, as well as the ICL1 and the H8 helix of β 2 -AR [ 68 ]. The result of this is the stabilization of the receptor in an inactive state, a decrease in the affinity of β 2 -AR for orthosteric agonists, and inhibition of its binding to G proteins and β-arrestins [ 68 , 421 ]. As shown for the polyethylene glycol-carboxylic acid derivative Cmpd-15PA, which has the same pharmacological characteristics as Cmpd-15, the polar groups of this derivative bind to the allosteric site through interactions with polar amino acid residues (Thr274 6.36 , Ser329 8.47 , Asp331 8.49 , Asn69 2.40 , and Arg63 ICL1 ) while the cyclohexyl and phenyl rings of Cmpd-15PA interacted with the hydrophobic package formed by the residues Val54 1.53 and Ile58 1.57 at the end of TM1, Leu64 ICL1 (ICL1), Ile72 2.43 (TM2), Leu275 6.37 (TM6), as well as Tyr326 7.53 from the conserved NPxxY sequence located in TM7 and Phe332 8.50 located in the H8 helix [ 68 ].…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…Below, we will briefly discuss some synthetic small molecular weight allosteric regulators, since they, like small endogenous regulators (cholesterol metal inos, etc. ), are described in detail in a number of reviews and experimental works [416][417][418][419][420][421], and we will pay more attention to autoantibodies and pepducins.…”

“…Cmpd-15 is able to enter the cell and bind specifically to the intracellular allosteric site formed by the cytoplasmic ends of TM1, TM2, TM3 and TM7, as well as the ICL1 and the H8 helix of β2-AR [62]. The result of this is the stabilization of the receptor in an inactive state, a decrease in the affinity of β2-AR for orthosteric agonists and inhibition of its binding to G-proteins and β-arrestins [62,420]. As shown for the polyethylene glycol-carboxylic acid derivative Cmpd-15PA, which has the same pharmacological characteristics as Cmpd-15, the polar groups of this derivative bind to the allosteric site through interactions with polar amino acid residues (Thr274 6.36 , Ser329 8.47 , Asp331 8.49 , Asn69 2.40 , Arg63 ICL1 ), while the cyclohexyl and phenyl rings of Cmpd-15PA interacted with the hydrophobic package formed by the residues Val54 1.53 and Ile58 1.57 at the end of TM1, Leu64 ICL1 (ICL1), Ile72 2.43 (TM2), Leu275 6.37 (TM6), as well as Tyr326 7.53 from the conserved NPxxY sequence located in TM7, and Phe332 8.50 located in the H8 helix [62].…”