Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor

Kahsai, Alem W.; Wisler, James W.; Lee, Jungmin; Ahn, Seungkirl; Cahill, Thomas J.; Dennison, S. Moses; Staus, Dean P.; Thomsen, A.; Anasti, Kara; Pani, Biswaranjan; Wingler, Laura M.; Desai, Hemant; Bompiani, Kristin M.; Strachan, Ryan T.; Qin, Xiao; Alam, S. Munir; Sullenger, Bruce A.; Lefkowitz, Robert J.

doi:10.1038/nchembio.2126

Cited by 71 publications

(57 citation statements)

References 54 publications

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“…Some are located at the intracellular face of the receptor; these are relatively large and can accommodate a wide range of compound sizes. Interestingly, most of the currently reported nonsmall-molecule β 2 AR allosteric modulators, such as nanobodies (10,22,38) and RNA aptamers (11), bind to intracellular cavities that overlap with the G-protein binding site (39). This appears to be true as well for 15 because it competes for β 2 AR binding with a nanobody (Nb60) that favors an inactive conformation and that binds to this intracellular region of the β 2 AR (22).…”

Section: Discussionmentioning

confidence: 97%

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Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Ahn

Kahsai

Pani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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139

119

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The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

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Section: Discussionmentioning

confidence: 97%

“…or RNA aptamers (11). Another approach, which has not yet been widely applied, is screening of DNA-encoded small-molecule libraries (DELs).…”

Section: Significancementioning

confidence: 99%

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Ahn

Kahsai

Pani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

139

119

View full text Add to dashboard Cite

show abstract

“…As a complementary methodology to HTS-based ligand discovery31, in vitro affinity display technologies generating peptide3233, DNA or RNA34 ligands can expose currently ‘undruggable' target space by defining new molecular topologies to inform the design of complementary ligand scaffolds in synthetic small molecules. To this end, we pursued macrocyclic polypeptide templates incorporating rigid stereochemical complexity extensively used by nature to interact across extended protein binding surfaces2935.…”

Section: Discussionmentioning

confidence: 99%

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

Dranchak

et al. 2017

Nat Commun

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Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

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“…This method is commonly used in GPCR studies where, for example, the β 2 -adrenergic receptor was incorporated into Nanodiscs and used in various binding experiments. 200–203 …”

Section: Structure and Assembly Of Nanodiscsmentioning

confidence: 99%

Nanodiscs in Membrane Biochemistry and Biophysics

2017

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Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system which provides control over size, composition and specific functional modifications on nanometer scale. In this review we attempted to combine a comprehensive list of various applications of Nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by Nanodiscs for structural and mechanistic studies of membrane proteins.

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Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor

Cited by 71 publications

References 54 publications

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

Nanodiscs in Membrane Biochemistry and Biophysics

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