Seven transmembrane receptor (7TMR) responses are modulated by orthosteric and allosteric ligands to great therapeutic advantage. Here we introduce a unique class of negative allosteric modulator ...(NAM) – the positive allosteric modulator (PAM)-antagonist – that increases the affinity of the receptor for the agonist but concomitantly decreases agonist efficacy when cobound. Notably, the reciprocation of allosteric energy causes the orthosteric agonist to increase the affinity of the receptor for the PAM-antagonist; thus, this modulator seeks out and destroys agonist-bound receptor complexes. When contrasted with standard orthosteric and allosteric antagonists it is clear that PAM-antagonists are uniquely well suited to reversing ongoing persistent agonism and provide favorable target coverage in vivo. Specifically, the therapeutic application of PAM-antagonists to reverse pathological overactivation (e.g., endothelin vasoconstriction) is emphasized.
PAM-antagonists exhibit a completely new profile of contradictory allosteric effects that confer a unique mode of antagonism.
These molecules theoretical circumvent problems in overcoming persistent agonism and may be selectively more active in the presence of the agonist.
PAM-antagonists may have unique properties with respect to long half-times for effect and target coverage in vivo.
G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the ...transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.
Whether biomechanical force on the heart can induce exosome secretion to modulate cardiovascular function is not known. We investigated the secretion and activity of exosomes containing a key ...receptor in cardiovascular function, the angiotensin II type I receptor (AT1R).
Exosomes containing AT1Rs were isolated from the media overlying AT1R-overexpressing cells exposed to osmotic stretch and from sera of mice undergoing cardiac pressure overload. The presence of AT1Rs in exosomes was confirmed by both electron microscopy and radioligand receptor binding assays and shown to require β-arrestin2, a multifunctional adaptor protein essential for receptor trafficking. We show that functional AT1Rs are transferred via exosomes in an in vitro model of cellular stretch. Using mice with global and cardiomyocyte conditional deletion of β-arrestin2, we show that under conditions of in vivo pressure overload the cellular source of the exocytosis of exosomes containing AT1R is the cardiomyocyte. Exogenously administered AT1R-enriched exosomes target cardiomyocytes, skeletal myocytes, and mesenteric resistance vessels and are sufficient to confer blood pressure responsiveness to angiotensin II infusion in AT1R knockout mice.
AT1R-enriched exosomes are released from the heart under conditions of in vivo cellular stress to likely modulate vascular responses to neurohormonal stimulation. In the context of the whole organism, the concept of G protein-coupled receptor trafficking should consider circulating exosomes as part of the reservoir of functional AT1Rs.
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 signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. 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). 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 isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of 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.
The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has ...been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
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•κ-opioid receptor active-state crystal structure disclosed•κ-opioid receptor structure reveals features involved in biased signaling•Structure provides template for the creation of safe and effective analgesics
A crystal structure of the active κ-opioid receptor provides a guide for the development of safe and effective new analgesics.
Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly ...test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10−3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system’s name: viral evolution of genetically actuating sequences, or “VEGAS.” Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks’ time.
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•One day per round of directed molecular evolution in mammalian cells•Mutation rates of 10−3 from each round, surpassing many in vitro systems•System does not require resetting or recycling of hits•Three unique campaigns are presented, each succeeding in less than a week
The VEGAS system is a platform for directed evolution, a method for engineering DNA sequences, in mammalian cells. The system is highly mutagenic, facile, and self-contained, requiring no in vitro handling during evolution cycles. As a result, robust evolution campaigns can be run within the context of a mammalian cell signaling environment. We perform three such campaigns as a proof-of-concept: evolving a transcription factor, a G-protein coupled receptor, and llama-derived nanobodies toward specific in vivo activities.
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.
The 5-HT₂A serotonin receptor is the most abundant serotonin receptor subtype in the cortex and is predominantly expressed in pyramidal neurons. The 5-HT₂A receptor is a target of several ...hallucinogens, antipsychotics, anxiolytics, and antidepressants, and it has been associated with several psychiatric disorders, conditions that are also associated with aberrations in dendritic spine morphogenesis. However, the role of 5-HT₂A receptors in regulating dendritic spine morphogenesis in cortical neurons is unknown. Here we show that the 5-HT₂A receptor is present in a subset of spines, in addition to dendritic shafts. It colocalizes with PSD-95 and with multiple PDZ protein-1 (MUPP1) in a subset of dendritic spines of rat cortical pyramidal neurons. MUPP1 is enriched in postsynaptic density (PSD) fractions, is targeted to spines in pyramidal neurons, and enhances the localization of 5-HT₂A receptors to the cell periphery. 5-HT₂A receptor activation by the 5-HT₂ receptor agonist DOI induced a transient increase in dendritic spine size, as well as phosphorylation of p21-activated kinase (PAK) in cultured cortical neurons. PAK is a downstream target of the neuronal Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 that is important for spine remodeling. Kalirin-7 regulates dendritic spine morphogenesis in neurons but its role in neuromodulator signaling has not been investigated. We show that peptide interference that prevents the localization of kalirin-7 to the postsynaptic density disrupts DOI-induced PAK phosphorylation and spine morphogenesis. These results suggest a potential role for serotonin signaling in modulating spine morphology and kalirin-7's function at cortical synapses.
Shocks near defibrillation threshold (nDFT) strength commonly extinguish all ventricular fibrillation (VF) wavefronts, but a train of rapid, well-organized postshock activations (PAs) typically ...appears before sinus rhythm ensues. If one of the PA waves undergoes partial propagation block (wavebreak), reentry may be induced, causing VF to reinitiate and the shock to fail.
The purpose of this study was to determine whether wavebreak leading to VF reinititation following nDFT shocks occurs preferentially at the right ventricular insertion (RVI), which previous studies have identified as a key site for wavebreak.
We used panoramic optical mapping to image the ventricular epicardium of 6 isolated swine hearts during nDFT defibrillation episodes. After each experiment, the hearts were fixed and their geometry scanned with magnetic resonance imaging (MRI). The MRI and mapping datasets were spatially coregistered. For failed shocks, we identified the site of the first wavebreak of a PA wave during VF reinitiation.
We recorded 59 nDFT failures. In 31 of these, the first wavebreak event occurred within 1 cm of the RVI centerline, most commonly on the anterior side of the right ventricular insertion (aRVI) (23/31). The aRVI region occupies 16.8% ± 2.5% of the epicardial surface and would be expected to account for only 10 wavebreaks if they were uniformly distributed. By χ
analysis, aRVI wavebreaks were significantly overrepresented.
The anterior RVI is a key site in promoting nDFT failure. Targeting this site to prevent wavebreak could convert defibrillation failure to success and improve defibrillation efficacy.
In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual ...signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.
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