Molecular Pharmacology最新文献

Biased signaling and ligand bias, often termed functional selectivity or selective nuclear receptor modulation, have been reported for nuclear receptor partial agonists over the past 20 years. Whether signaling differences produced by partial agonists result from less intense modulation, off-target effects, or biased signaling remains unclear. A commonly postulated mechanism for biased signaling is coactivator favoritism, where agonists induce different coactivator recruitment profiles. We find that both GW1929 (full agonist) and MRL24 (partial agonist) favor recruitment of 100 to 300 residue regions from S-motif coactivators compared with a reference full agonist (rosiglitazone), yielding 95% bias value confidence intervals of 0.05-0.17 and 0.29-0.38, respectively. Calculations based on these data indicate that GW1929 and MRL24 would induce 30% to 60% higher S-motif coactivator occupancy at the receptor compared with rosiglitazone. We compare the transcriptional effects of these same three ligands on human adipocytes using RNA sequencing and exploratory Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Only 50% (rosiglitazone) and 77% (GW1929) of all gene expression changes are shared between these full agonists after 3 hours of exposure. After 24 hours of exposure, 13/98 KEGG pathways appear more intensely modulated by rosiglitazone than GW1929 (e.g., 95% confidence interval of bias in the regulation of lipolysis in adipocytes pathway is 0.03-0.09), despite similar signaling for the remaining 85 affected pathways. Similarly, rosiglitazone has an unusually large effect on several lipid metabolism-related pathways compared with the partial agonist MRL24. These data indicate that nuclear receptor full and partial agonists can induce biased signaling, likely through differences in coactivator recruitment. SIGNIFICANCE STATEMENT: Many nuclear receptor partial agonists cause fewer adverse effects and similar efficacy compared with full agonists, potentially by inducing biased agonism. Our data support the idea that partial agonists, and a full agonist, of the nuclear receptor Peroxisome proliferator-activated receptor gamma (PPARγ) are biased agonists, causing different signaling by inducing PPARγ to favor different coactivators. These data indicate that biased agonism can occur in nuclear receptors and should be considered in efforts to develop improved nuclear receptor-targeted drugs.

Nav1.8 sodium channels (Nav1.8) are an attractive therapeutic target for pain because they are prominent in primary pain-sensing neurons with little expression in most other kinds of neurons. Recently, two Nav1.8-targeted compounds, VX-150 and VX-548, have shown efficacy in clinical trials for reducing pain. We examined the characteristics of Nav1.8 inhibition by these compounds. The active metabolite form of VX-150 (VX-150m) inhibited human Nav1.8 channels with an IC₅₀ of 15 nM. VX-548 (suzetrigine) was even more potent (IC₅₀ 0.27 nM). Both VX-150m and VX-548 had the unusual property of "reverse use-dependence," whereby inhibition could be relieved by repetitive depolarizations, a property seen before with another Nav1.8 inhibitor, A-887826. The relief of VX-548 inhibition by large depolarizations occurred with a time constant of ∼40 milliseconds that was not concentration-dependent. Reinhibition at negative voltages occurred with a rate that was nearly proportional to drug concentration, consistent with the idea that relief of inhibition reflects dissociation of drug from the channel and reinhibition reflects rebinding. The relief of inhibition by depolarization suggests a remarkably strong and unusual state-dependence for both VX-150m and VX-548, with very weak binding to channels with fully activated voltage sensors despite very tight binding to channels with voltage sensors in the resting state. SIGNIFICANCE STATEMENT: The Nav1.8 sodium channel (Nav1.8) is a current target for new drugs for pain. This work describes the potency, selectivity, and state-dependent characteristics of inhibition of Nav1.8 channels by VX-150 and VX-548, compounds that have recently shown efficacy for relief of pain in clinical trials but whose mechanism of interaction with channels has not been described. The results show that the compounds share an unusual property whereby inhibition is relieved by depolarization, demonstrating a state-dependence different from most sodium channel inhibitors.

β-arrestins are multifaceted adaptor proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and signaling. It is emerging that receptor-specific determinants specify these divergent functions at GPCRs, yet this remains poorly understood. Here, we set out to identify the receptor determinants responsible for β-arrestin-mediated regulation of the chemokine receptor C-X-C motif chemokine receptor 5 (CXCR5). Using bioluminescence resonance energy transfer, we show that β-arrestin1 and β-arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand C-X-C motif chemokine ligand 13 (CXCL13). The carboxy-terminal tail of CXCR5 contains several serine/threonine residues that can be divided into three discrete phospho-site clusters based on their position relative to transmembrane domain 7. Mutagenesis experiments revealed that the distal and medial phospho-site clusters, but not the proximal, are required for agonist-stimulated β-arrestin1 or β-arrestin2 recruitment to CXCR5. Consistent with this, we provide evidence that the distal and medial, but not proximal, phospho-site clusters are required for receptor desensitization. Surprisingly, the individual phospho-site clusters are not required for agonist-stimulated internalization of CXCR5. Further, we show that CXCL13-stimulated CXCR5 internalization and ERK1/2 phosphorylation, but not desensitization, remain intact in human embryonic kidney 293 cells lacking β-arrestin1 and β-arrestin2. Our study provides evidence that β-arrestins are recruited to CXCR5 and are required for desensitization but are dispensable for internalization or signaling, suggesting that discrete receptor determinants specify the divergent functions of β-arrestins. SIGNIFICANCE STATEMENT: C-X-C motif ligand 13 (CXCL13) and C-X-C motif chemokine receptor 5 (CXCR5) are important in the immune system and are linked to diseases, yet regulation of CXCR5 signaling remains poorly understood. We provide evidence that a phospho-site cluster located at the extreme distal carboxyl-terminal tail of the receptor is responsible for β-arrestin recruitment and receptor desensitization. β-arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that β-arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of β-arrestins.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca²⁺-permeable component of excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurologic, and neurodegenerative disorders. We recently described the thieno[2,3-day]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, human embryonic kidney cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 μM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances coagonist potency, and reduces single-channel conductance. We also show that EU1622-240 facilitates NMDAR activation when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg²⁺ sensitivity, in contrast to channels arising from activation of NMDARs with both coagonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. SIGNIFICANCE STATEMENT: NMDA receptors are expressed throughout the central nervous system and are permeable to calcium. EU1622-240 increases open probability and agonist potency while reducing single-channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one coagonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, ABCB1 and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by ABCB1, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, that are expressed at the zebrafish BBB. We evaluated the luminescence and transporter substrate status of 16 NanoLuc substrates. We identified eight substrates that were efficiently pumped out by ABCB1, six by Abcb4, seven by ABCG2, and seven by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. SIGNIFICANCE STATEMENT: The ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. This study screened 16 NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relationship for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both inhibition of [³H]creatine uptake and transport associated currents allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds - i.e., compound 1 (2-(N-benzylcarbamimidamido)acetic acid), MIPA572 (=carbamimidoylphenylalanine), and MIPA573 (=carbamimidoyltryptophane) that blocked CRT-1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIPA574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). SIGNIFICANCE STATEMENT: The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1.

Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by GPCR kinases (GRKs) from the GRK2/3 subfamily. While these relatively 'downstream' steps in the agonist-selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished 'upstream' by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs, and that agonist-selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved PH domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist-selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist-selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and utilizes the NTD as an agonist-selective sensor. Significance Statement This study investigates how GPCR kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist-selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N-terminus is a key determinant of agonist discrimination.

NMDA-type ionotropic glutamate receptors are critically involved in excitatory neurotransmission and their dysfunction is implicated in many brain disorders. Allosteric modulators with selectivity for specific NMDA receptor subtypes are therefore attractive as therapeutic agents, and sustained drug discovery efforts have resulted in a wide range of new allosteric modulators. However, evaluation of allosteric NMDA receptor modulators is limited by the lack of operational ligand-receptor models to describe modulator binding dissociation constants (K_B) and effects on agonist binding affinity (α) and efficacy (β). Here, we describe a pharmacological equilibrium model that encapsulates activation and modulation of NMDA receptors, and we apply this model to afford deeper understanding of GluN2A-selective negative allosteric modulators (NAMs), TCN-201, MPX-004, and MPX-007. We exploit slow NAM unbinding to examine receptors at hemi-equilibrium when fully occupied by agonists and modulators to demonstrate that TCN-201 display weaker binding and negative modulation of glycine binding affinity (K_B = 42 nM, α = 0.0032) compared to MPX-004 (K_B = 9.3 nM, α = 0.0018) and MPX-007 (K_B = 1.1 nM, α = 0.00053). MPX-004 increases agonist efficacy (β = 1.19), whereas TCN-201 (β = 0.76) and MPX-007 (β = 0.82) reduce agonist efficacy. These values describing allosteric modulation of diheteromeric GluN1/2A receptors with two modulator binding sites are unchanged in triheteromeric GluN1/2A/2B receptors with a single binding site. This evaluation of NMDA receptor modulation reveals differences between ligand analogs that shape their utility as pharmacological tool compounds and facilitates the design of new modulators with therapeutic potential. Significance Statement Detailed understanding of allosteric NMDA receptor modulation requires pharmacological methods to quantify modulator binding affinity and the strengths of modulation of agonist binding and efficacy. We describe a generic ligand-receptor model for allosteric NMDA receptor modulation and use this model for the characterization of GluN2A-selective NAMs. The model enables quantitative evaluation of a broad range of NMDA receptor modulators and provides opportunities to optimize these modulators by embellishing the interpretation of their structure-activity relationships.

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The beta 2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. But the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remains incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in HEK293 cells using FLAG co-immunoprecipitation coupled to SILAC labeling and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and six out of seven isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately three-fold, and increased both miniGs and arrestin recruitment to B2AR more than two fold each, without noticeably changing EC50 with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the amount of signaling-competent receptors at the plasma membrane. Significance Statement This study presents a new interactome of the agonist-stimulated beta 2 adrenergic receptor (B2AR), a paradigmatic GPCR that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting B2AR access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.