Small-Molecule Positive Allosteric Modulators of theβ₂-Adrenoceptor Isolated from DNA-Encoded Libraries

Abstract

Conventional drug discovery efforts at the β2-adrenoceptor (β2AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β2AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β2AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β2AR compared with the closely related β1AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Multimodal small-molecule screening for human prion protein binders

“…Alternatively, purification of PrP from mammalian cells 73 and insertion into nanodiscs 74 or micelles might more faithfully recapitulate PrP's PTMs and endogenous membrane environment, potentially yielding binding sites not present on recombinant PrP. Encouragingly, DEL screening has been used successfully with nanodisc immobilized proteins 75 . We could also extend our fragment-based drug design strategy by using chemoproteomics 76 to directly assess PrP ligandability on the cell surface.…”

α1-Adrenergic Receptors in Neurotransmission, Synaptic Plasticity, and Cognition

“…There are three published reports of allosteric modulation at α 1 -ARs, and all are described as negative allosteric modulators with no known clinically-useful application as of yet ( Leppik et al., 2000 ; Sharpe et al., 2003 ; Campbell et al., 2017 ). Positive allosteric modulators have also been developed for the β 2 -AR ( Ahn et al., 2018 ; Liu et al., 2019 ). While these modulators are selective for the β 2 -AR and display cooperative signals with agonists, they are not ligand-selective nor signaling-biased.…”

Multimodal small-molecule screening for human prion protein binders

“…Purification of PrP from mammalian cells 68 and insertion into nanodiscs 69 or micelles might more faithfully recapitulate PrP's PTMs and endogenous membrane environment, potentially yielding binding sites not present on recombinant PrP. Encouragingly, DEL screening has been used successfully with nanodisc immobilized proteins 70 . We could also extend our fragment-based drug design strategy by using chemoproteomics 71 to directly assess PrP ligandability on the cell surface.…”