13 research outputs found
Medium-chain fatty acids suppress lipotoxicity-induced hepatic fibrosis via the immunomodulating receptor GPR84
食事性肥満から肝炎発症に関わる制御因子の同定 --中鎖脂肪酸油による予防・GPR84標的NASH治療薬の可能性--. 京都大学プレスリリース. 2023-01-18.Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein–coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake–induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH
Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels.
Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding
Synthesis and Biological Evaluation of Lysophosphatidic Acid Analogues Using Conformational Restriction and Bioisosteric Replacement Strategies
Lysophosphatidic
acid (LPA) is a key player in many physiological
and pathophysiological processes. The biological activities of LPA
are mediated through interactions withat leastsix
subtypes of G-protein-coupled receptors (GPCRs) named LPA1–6. Developing a pharmacological tool molecule that activates LPA subtype
receptors selectively will allow a better understanding of their specific
physiological roles. Here, we designed and synthesized conformationally
restricted 25 1-oleoyl LPA analogues MZN-001 to MZN-025 by incorporating its glycerol linker into dihydropyran,
tetrahydropyran, and pyrrolidine rings and variating the lipophilic
chain. The agonistic activities of these compounds were evaluated
using the TGFα shedding assay. Overall, the synthesized analogues
exhibited significantly reduced agonistic activities toward LPA1, LPA2, and LPA6, while demonstrating
potent activities toward LPA3, LPA4, and LPA5 compared to the parent LPA. Specifically, MZN-010 showed more than 10 times greater potency (EC50 = 4.9
nM) than the standard 1-oleoyl LPA (EC50 = 78 nM) toward
LPA5 while exhibiting significantly lower activity on LPA1, LPA2, and LPA6 and comparable potency
toward LPA3 and LPA4. Based on the MZN-010 scaffold, we synthesized additional analogues with improved selectivity
and potency toward LPA5. Compound MZN-021,
which contains a saturated lipophilic chain, exhibited 50 times more
potent activity (EC50 = 1.2 nM) than the natural LPA against
LPA5 with over a 45-fold higher selectivity when compared
to those of other LPA receptors. Thus, MZN-021 was found
to be a potent and selective LPA5 agonist. The findings
of this study could contribute to broadening the current knowledge
about the stereochemical and three-dimensional arrangement of LPA
pharmacophore components inside LPA receptors and paving the way toward
synthesizing other subtype-selective pharmacological probes
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Illuminating G-Protein-Coupling Selectivity of GPCRs
Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling
Structural basis for lysophosphatidylserine recognition by GPR34
Abstract GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34
Probing the Hydrophobic Binding Pocket of G‑Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS<sub>1</sub> by Docking-Aided Structure–Activity Analysis
The
ligands of certain G-protein-coupled receptors (GPCRs) have
been identified as endogenous lipids, such as lysophosphatidylserine
(LysoPS). Here, we analyzed the molecular basis of the structure–activity
relationship of ligands of GPR34, one of the LysoPS receptor subtypes,
focusing on recognition of the long-chain fatty acid moiety by the
hydrophobic pocket. By introducing benzene ring(s) into the fatty
acid moiety of 2-<i>deoxy</i>-LysoPS, we explored the binding
site’s preference for the hydrophobic shape. A tribenzene-containing
fatty acid surrogate with modifications of the terminal aromatic moiety
showed potent agonistic activity toward GPR34. Computational docking
of these derivatives with a homology modeling/molecular dynamics-based
virtual binding site of GPR34 indicated that a kink in the benzene-based
lipid surrogates matches the L-shaped hydrophobic pocket of GPR34.
A tetrabenzene-based lipid analogue bearing a bulky <i>tert</i>-butyl group at the 4-position of the terminal benzene ring exhibited
potent GPR34 agonistic activity, validating the present hydrophobic
binding pocket model
Conformational Constraint of the Glycerol Moiety of Lysophosphatidylserine Affords Compounds with Receptor Subtype Selectivity
Lysophosphatidylserine (LysoPS) is
an endogenous lipid mediator
that specifically activates membrane proteins of the P2Y and its related
families of G protein-coupled receptors (GPCR), GPR34 (LPS<sub>1</sub>), P2Y10 (LPS<sub>2</sub>), and GPR174 (LPS<sub>3</sub>). Here, in
order to increase potency and receptor selectivity, we designed and
synthesized LysoPS analogues containing the conformational constraints
of the glycerol moiety. These reduced structural flexibility by fixation
of the glycerol framework of LysoPS using a 2-hydroxymethyl-3-hydroxytetrahydropyran
skeleton, and related structures identified compounds which exhibited
high potency and selectivity for activation of GPR34 or P2Y10. Morphing
of the structural shape of the 2-hydroxymethyl-3-hydroxytetrahydropyran
skeleton into a planar benzene ring enhanced the P2Y10 activation
potentcy rather than the GPR34 activation
Probing the Hydrophobic Binding Pocket of G‑Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS<sub>1</sub> by Docking-Aided Structure–Activity Analysis
The
ligands of certain G-protein-coupled receptors (GPCRs) have
been identified as endogenous lipids, such as lysophosphatidylserine
(LysoPS). Here, we analyzed the molecular basis of the structure–activity
relationship of ligands of GPR34, one of the LysoPS receptor subtypes,
focusing on recognition of the long-chain fatty acid moiety by the
hydrophobic pocket. By introducing benzene ring(s) into the fatty
acid moiety of 2-<i>deoxy</i>-LysoPS, we explored the binding
site’s preference for the hydrophobic shape. A tribenzene-containing
fatty acid surrogate with modifications of the terminal aromatic moiety
showed potent agonistic activity toward GPR34. Computational docking
of these derivatives with a homology modeling/molecular dynamics-based
virtual binding site of GPR34 indicated that a kink in the benzene-based
lipid surrogates matches the L-shaped hydrophobic pocket of GPR34.
A tetrabenzene-based lipid analogue bearing a bulky <i>tert</i>-butyl group at the 4-position of the terminal benzene ring exhibited
potent GPR34 agonistic activity, validating the present hydrophobic
binding pocket model