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

The present study reports the discovery of a small-molecule negative allosteric modulator for the β2-adrenergic receptor (β2AR) via in vitro affinity-based iterative selection of highly diverse DNA-encoded small-molecule libraries. Characterization of the compound demonstrates its selectivity for the β2AR and that it negatively modulates a wide range of receptor functions. More importantly, our findings establish a generally applicable, proof-of-concept strategy for screening DNA-encoded small-molecule libraries against purified G-protein–coupled receptors (GPCRs), which holds great potential for discovering therapeutic molecules

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states

Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation

G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states

Helminth induced suppression of macrophage activation is correlated with inhibition of calcium channel activity.

Helminth parasites cause persistent infections in humans and yet many infected individuals are asymptomatic. Neurocysticercosis (NCC), a disease of the central nervous system (CNS) caused by the cestode Taenia solium, has a long asymptomatic phase correlated with an absence of brain inflammation. However, the mechanisms of immune suppression remain poorly understood. Here we report that murine NCC displays a lack of cell surface maturation markers in infiltrating myeloid cells. Furthermore, soluble parasite ligands (PL) failed to induce maturation of macrophages, and inhibited TLR-induced inflammatory cytokine production. Importantly, PL treatment abolished both LPS and thapsigargin-induced store operated Ca2+ entry (SOCE). Moreover, electrophysiological recordings demonstrated PL-mediated inhibition of LPS or Tg-induced currents that were TRPC1-dependent. Concomitantly STIM1-TRPC1 complex was also impaired that was essential for SOCE and sustained Ca2+ entry. Likewise loss of SOCE due to PL further inhibited NFkB activation. Overall, our results indicate that the negative regulation of agonist induced Ca2+ signaling pathway by parasite ligands may be a novel immune suppressive mechanism to block the initiation of the inflammatory response associated with helminth infections

Sortase ligation enables homogeneous GPCR phosphorylation to reveal diversity in β-arrestin coupling

The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and β-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer's mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in β-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and β-arrestins with GPCRs' transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the β2-adrenergic receptor (β2AR) allows stabilization of a high-affinity receptor active state by β-arrestin1, permitting us to define elements in the β2AR and β-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the β2AR, the muscarinic acetylcholine receptor 2 and the μ-opioid receptor reveals that the ability of β-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of β-arrestin1 correlates with its ability to attenuate, or "desensitize," G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor-transducer interactions

Helminth antigens impairment of TRPC1-STIM1 channel assembly.

<p>Co-immunoprecipitation of TRPC1 and STIM1 from BMDMs in resting, LPS-, and Tg-stimulations. A) Cells were pulsed with medium alone, PL at 25 ug/ml, LPS at 10 ng/ml, or PL before the addition of LPS to the medium. B) Cells were pulsed with medium alone, PL at 25 ug/ml, Tg at 2 µM, or PL before the addition of Tg to the medium. Immunoprecipitation of TRPC1-STIM1 complex from cell was prepared by using Anti-STIM1 Ab. Anti-TRPC1 was used for western blot detection in both Fig. 8 A and B. The average pixel intensity of the respective bands from three independent experiments was measured. This was done using the imaging software Quality One-4.6.7 (Bio-Rad).</p

Helminth antigens inhibit Tg induced Ca²⁺ release and Ca²⁺ entry.

<p>Fura 2 fluorescence measurements in J774 cells. Cells were pulsed with medium alone, Tg at 1/ml for 20 mins before the addition of LPS to the medium. Analog plots of the fluorescence ratio (340/380) from an average of 0- 50 cells are shown in (A) and (B). (C) The bar graph indicates the average data on calcium release (first peak) and calcium entry (second peak) under these conditions. ** indicate significance (p = <0.01). (D) Inward currents were induced upon store depletion at −80 mV in control and PL treated cells Respectively IV curves under these conditions are shown in (E). Average (8–10 recordings current intensity at −80 mV are shown in (F). * indicate significance (p = <0.05).</p

Infiltrating myeloid cells display reduced expression of surface maturation markers during murine NCC.

<p><i>In-situ</i> IF staining was performed on frozen sections of HBSS infected (mock) and parasite infected brains of mice at 1 wk and 2 wk p.i. Brain cryosections were analyzed for expression of maturation markers MHC II, CD80, and CD86 using fluorochrome conjugated antibodies (red-PE) and macrophages specific marker CD11b (green-Alexa 488). Nuclear staining DAPI is depicted in blue. Staining for these maturation markers MHC-II (A1), CD80 (B1), and CD86 (C1) was undetected/barely detected in mock infected mice. Parasite infected mice brains displayed large number of infiltrating myeloid cells stained positive for CD11b (A2–A3, B2–B3, and C2–C3). MHC-II was undetected in many of the CD11b+ accumulated cells in the CNS of NCC mice at both 1 wk (A2) and 2 wk p.i. (A3) (arrow). CD80 (B2–B3) and Cd86 (C2–C3) were undetected/barely detected in the CD11b+ myeloid cells around the parasite (P) in the CNS of NCC mice both at 1 wk and 2 wk p.i. Results are from one representative experiment of atleast three independent experiments (n = 4 per each time point).</p

Helminth antigens downregulated surface maturation markers on BMDMs.

<p>BMDMs were pulsed with medium alone, PL at 25/ml, LPS at 10 ng/ml, or PL at 25 ug/ml twenty min prior to the addition of LPS to a final concentration of 10 ng/ml in the medium (PL+LPS) and cultured for a total of 20–24 h period. BMDMs were harvested and FACS analysis was performed to measure surface expression of MHC-II, CD80, and CD86. Dotted line in the histogram shows control staining with isotype/fluorochrome control Ab, and filled line shows signal with specific Ab.</p