BACKGROUND:Plasma accumulation of the gut microbial metabolite 4-ethylphenylsulfate (4EPS), derived from dietary amino acid, tyrosine, has been associated with cardiovascular, renal, metabolic, and neurological disorders. AngII (angiotensin II) infusion increases circulating 4EPS in mice, suggesting a potential mechanistic role. We hypothesized that 4EPS modulates AngII-regulated pathophysiology and disease progression by directly inhibiting AT1R (angiotensin II type 1 receptor).METHODS:This hypothesis was tested by combining AT1R pharmacology, cell signaling assays, ex vivo vascular studies, an AngII-induced aortic aneurysm growth model, and plasma proteomics analysis.RESULTS:in vitro, 4EPS reduced the binding of both AngII and the antagonist candesartan to AT1R and suppressed AngII-induced calcium signaling. Ex vivo, 4EPS attenuated AngII-mediated vasoconstriction. In vivo, high-fat diet-fed ApoE-null mice coinfused with AngII and 4EPS showed significant blunting of blood pressure elevation and a marked reduction in aortic aneurysm-related mortality compared with mice infused with AngII alone. Analysis of aortic remodeling revealed increased elastin preservation and decreased thickening of the intimal and medial layers in 4EPS-treated animals. Plasma proteomics indicated alterations in actin-cytoskeletal signaling pathways consistent with reduced activation of ERK (extracellular-regulated kinase) 1/2, filamin-A, and proteins involved in vascular smooth muscle cell motility.CONCLUSIONS:These findings identify 4EPS as a benign, endogenous AT1R antagonist that diminishes AngII-mediated hemodynamic and vascular pathology. By suppressing cytoskeletal signaling associated with vascular remodeling, 4EPS provides significant protection against hypertension and aortic aneurysm progression in mice, revealing a previously unrecognized protective role for a gut microbial metabolite in modulating renin-angiotensin system activity.

BACKGROUND: Plasma accumulation of the gut microbial metabolite, 4-ethylphenylsulfate (4EPS), produced from dietary protein aromatic amino acids has been observed in correlative and associative studies of cardiovascular, renal, metabolic and neurological diseases. 4EPS level increases upon AngII infusion in mice. How 4EPS alters host physiology to contribute to progression of any disease state is currently unknown. METHODS: To test the hypothesis that 4EPS interferes with angiotensin binding to AT1R, we used multiple approaches: AT1R pharmacology, cell-signaling, ex vivo vascular contraction and a mouse model of angiotensin-induced aortic aneurysm (AA) disease. ApoE-null mice were fed high-fat diet and infused with AngII, or co-infused with 4EPS and Olmesartan. BP was recorded. At the end of infusion, aortas were assessed for severity of AA, contractile response and histopathology. To evaluate signaling associated with different AA outcomes plasma proteomics analysis was done. RESULTS: In vitro, 4EPS reduced the binding of angiotensin and Candesartan to AT1R and calcium signaling. Ex vivo, 4EPS decreased vasomotor response of the aorta to AngII. In vivo, 4EPS inhibited AngII-mediated increase of BP and reduced mortality from AA. Abdominal aorta remodeling in 4EPS+AngII co-infused mice showed an increase of elastin area and reduced thickening of intimal/medial layers. Plasma proteome analysis indicated significant change in actin-cytoskeletal signaling associated with reduced ERK1/2 and Filamin-A activation, and cell motility. CONCLUSIONS: Benign antagonism of AT1R by 4EPS involves direct interaction with AT1R. Molecular mechanisms of 4EPS responsible for reduced AA associated mortality in mice are distinct from those of AT1R blocker, Olmesartan. ### Competing Interest Statement The authors have declared no competing interest.

Table 1 lists a number of putative GPCRs identified by NC-IUPHAR [197], for which preliminary evidence for an endogenous ligand has been published, or for which there exists a potential link to a disease, or disorder. These GPCRs have recently been reviewed in detail [153]. The GPCRs in Table 1 are all Class A, rhodopsin-like GPCRs. Class A orphan GPCRs not listed in Table 1 are putative GPCRs with as-yet unidentified endogenous ligands.Table 1: Class A orphan GPCRs with putative endogenous ligands GPR3GPR4GPR6GPR12GPR15GPR17GPR20 GPR22GPR26GPR31GPR34GPR35GPR37GPR39 GPR50GPR63GPR65GPR68GPR75GPR84GPR87 GPR88GPR132GPR149GPR161GPR183LGR4LGR5 LGR6MAS1MRGPRDMRGPRX1MRGPRX2P2RY10TAAR2 In addition the orphan receptors GPR18, GPR55 and GPR119 which are reported to respond to endogenous agents analogous to the endogenous cannabinoid ligands have been grouped together (GPR18, GPR55 and GPR119).

Angiotensin II receptors, Type 1 (AT1R) and Type 2 (AT2R) are 7TM receptors that play critical roles in both the physiological and pathophysiological regulation of the cardiovascular system. While AT1R blockers (ARBs) have proven beneficial in managing cardiac, vascular and renal maladies they cannot completely halt and reverse the progression of pathologies. Numerous experimental and animal studies have demonstrated that β-arrestin biased AT1R-ligands (such as SII-AngII, S1I8, TRV023, and TRV027) offer cardiovascular benefits by blocking the G protein signaling while retaining the β-arrestin signaling. However, these ligands failed to show improvement in heart-failure outcome over the placebo in a phase IIb clinical trial. One major limitation of current β-arrestin biased AT1R-ligands is that they are peptides with short half-lives, limiting their long-term efficacy in patients. Additionally, β-arrestin biased AT1R-ligand peptides, may inadvertently block AT2R, a promiscuous receptor, potentially negating its beneficial effects in post-myocardial infarction (MI) patients. Therefore, developing a small molecule β-arrestin biased AT1R-ligand with a longer half-life and specificity to AT1R could be more effective in treating heart failure. This approach has the potential to revolutionize the treatment of cardiovascular diseases by offering more sustained and targeted therapeutic effects.

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop (ECL) 2 of β1-adrenergic receptor (β1AR) is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. We have previously shown that IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blocker compared to non-IgG3(+) β1AR AAbs. Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, it is critical to determine the mechanistic basis of the unique beneficial signaling pathway that may have therapeutic potential. HEK 293 cells stably expressing human β1AR (HEK-β1AR) was generated and treated with β1AR AAbs. Minimal changes in phosphorylation of β1AR was observed following IgG3(+) β1AR AAbs, while significant phosphorylation of β1ARs was observed with non-IgG3(+) β1AR AAbs. To dissect the diverse signaling responses mediated by IgG3(+) and non-IgG(+) β1AR AAbs, HEK-β1AR cells were treated with IgG3(+) or non-IgG3(+) β1AR AAbs. β1ARs were immunoprecipitated and subjected to mass-spectrometry analysis to identify unique co-immunoprecipitating proteins that could complex with receptor in presence of IgG3(+) or non-IgG3(+) β1AR AAbs. One of the proteins that was differentially recruited to the β1AR complex was Insulin Receptor Substrate 4 (IRS4). Traditionally, IRS4 is a signaling hub downstream of insulin/insulin growth factor receptors mediating glucose uptake. The differential interaction of IRS4 with β1AR in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs reflects that IRS4 could be a key signaling regulator of uniquely biased signal transduction pathway in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs. Mechanisms underlying this unique IRS4 mediated signaling modulation will be discussed.

Background: The clinical data suggest an association of autoantibodies (AAb) targeting angiotensin II type1 receptor (AT1R) and outcomes in heart failure (HF) and preeclampsia. Notably, patient’s risk increases 2 to 4-fold for heart diseases and hypertension. Although AT1R blockers (ARBs) have emerged as a key component in the therapeutic arsenal against CVD, we believe that rather than completely blocking AT1R signaling, maintaining a balanced signaling is crucial for cardiovascular homeostasis. Therefore, we posit that reducing AT1R overactivation and blocking AAb-mediated activation could benefit cardiac health and attenuate the progression of CVD. In our previous study (PMID:36440576), we identified small molecules capable of inhibiting AAb-induced AT1R signaling. Leveraging these observations, we have developed a novel AT1R allosteric ligand that inhibits autoantibody binding to AT1R while still retaining normal AT1R signaling. Methods: Using computational and medicinal chemistry approaches, we designed a series of novel AT1R ligands and obtained compounds with >98% purity through chemical synthesis. Monoclonal antibodies mimicking AT1R-AAb have been generated. Serum samples from dilated cardiomyopathy (DCM) and preeclampsia (PE) patient cohorts were collected and presence of AT1R-AAb was determined using ELISA and epitope-peptide competition studies. Pharmacological characterization of compounds was performed using calcium mobilization, 125I -AngII binding, ex vivo vasoconstriction, and ELISA assays. Results: We identified an AT1R allosteric compound that reduced AngII-mediated calcium signaling with a 5-fold shift in EC 50 . The IC 50 was determined to be 18 µM. It reduces the AngII-mediated vasoconstriction while blocking both monoclonal and patient-derived autoantibody binding with an IC 50 of 5 µM. Conclusion: We have identified a novel AT1R allosteric ligand with negative allosteric modulator (NAM) properties, which has the potential to block autoantibodies and could serve as a new generation of ARBs. Uniquely, it retains beneficial AT1R signaling without competing with AngII, and its allosteric ligand property can be tailored to achieve optimal AT1R signaling.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing β1- versus β2-adrenergic receptors (β1AR and β2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of β2AR, but not β1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on β2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical β2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR. Allosteric modulation is crucial in metabolic regulation but unexplored in gut microbehost interactions. Here the authors show gut microbe-derived phenylacetylglutamine acts as a negative allosteric modulator of β2-adrenergic receptors, impacting heart function.

Introduction Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop 2 (ECL2) of β1AR is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. Hypothesis IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blockers. Methods Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, in silico studies were conducted to evaluate the interaction between the IgG3(+) antibody and the known structure of β1AR. In silico epitope prediction suggested that the extracellular loop 1 (ECL1) could be a potential epitope, wherein binding by IgG3(+) AAbs at ECL1 could facilitate unique biased signaling in response to β-blockers. Peptides representing human β1AR ECL 1 was synthesized and antibodies targeting the human β1AR ECL 1 was generated by immunizing the mice. HEK 293 cells expressing human β1AR were pre-treated with serum containing antibodies against ECL 1 of β1AR followed by either β1AR selective agonist dobutamine (DOB) or β-blocker metoprolol stimulation. Downstream β1AR signaling was measured by cAMP generation. To test for specificity of ECL1 in the modulating β1AR responses, the cells were incubated with ECL 1 peptides along with antibodies against ECL1 of human β1AR as competitors and cAMP generation assessed following metoprolol. Results Treatment of human β1AR expressing HEK 293 cells with serum from mouse immunized with ECL1 of human β1AR showed no baseline changes in the cAMP generation. However, pretreatment of these cells with ECL1 antibodies followed by stimulation with DOB surprisingly showed marked reduction in cAMP generation in contrast to adjuvant controls which showed significant increase in cAMP. While, treatment of these ECL1 antibody pretreated cells with metoprolol unexpectedly resulted in cAMP generation which is blocked in the adjuvant controls. This shows that antibodies against the ECL1 of human β1AR binds to the β1AR and is able to allosterically modulate downstream signaling mediated by agonist (DOB) or β-blocker (metoprolol) as at baseline it does not alter β1AR function. To validate that antibodies against the ECL1 of human β1AR allosterically modulates metoprolol signaling, the cells were also pre-treated with competing doses of ECL1 peptide followed by metoprolol treatment. Consistent with our hypothesis, incubation with ECL1 peptide remarkably blocked cAMP generation in response to metoprolol. This shows that antibody binding to the ECL1 of human β1AR mediates this unique signaling in response to metoprolol wherein, a traditionally cAMP blocking β-blocker metoprolol now mediates cAMP generation. Conclusions These observations suggest that the allosteric binding of β1AR ECL1 by the antibodies mediates unexpected downstream signal that could underlie the benefits observed in the patients harboring IgG3(+) AAbs. Mechanisms underlying this unique allosteric modulation will be discussed in our presentation.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG–MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG–MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.

Abstract The actions of angiotensin II (Ang II) are mediated by AT 1 and AT 2 receptors ( nomenclature as agreed by nomenclature as agreed by the the NC-IUPHARNC-IUPHAR Subcommittee on Angiotensin receptors [ Subcommittee on Angiotensin receptors [6363, , 155155]] ), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, olmesartan,

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at . G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The actions of angiotensin II (Ang II) are mediated by AT1 and AT2 receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Angiotensin receptors [63, 155]), which have around 30% sequence similarity. The decapeptide angiotensin I, the octapeptide angiotensin II and the heptapeptide angiotensin III are endogenous ligands. losartan, candesartan, olmesartan, telmisartan, etc. are clinically used AT1 receptor blockers.

Manganese complex of (N'1E,N'3E)-N'1,N'3-bis(2-hydroxybenzylidene)isophthalo-hydrazide [H4L] was designed, spectroscopically analyzed, and confirmed via GC-MS, FTIR, CHNS, UV-VIS, magnetic susceptibility measurements, and molar electric conductivity. The data confirmed the formation of ligand H4L and [Mn2(H2L)Cl]Cl.2H2O complex. Ligand H4L acts as a bi-negative hexadentate and tetra-negative hexadentate coordinating through two amide carbonyl, two azomethine, and two deprotonated OH groups. The magnetic and spectral data proposed a square-planar and a tetrahedral structure. The TGA data confirmed the final metal oxide form for the Mn ligand complex. Additionally, an in-vitro assessment of the ligand H4L confirmed its ability to control cancer cell proliferation in both breast (MCF7) and lung (A549) cancer cell lines while the Mn ligand complex possessed an anticancer effect in both cancer cells at the nano-molar level. Further, the in silico data showed four hydrogen bond interactions of the ligand with G-quadruplex DNA. This strengthens our hypothesis that the hydrazone complex acts as an anticancer agent by targeting the G-quadruplex DNA.

Background: Aortic aneurysm (AA) is a “silent killer” human disease with no effective treatment. Although the therapeutic potential of various pharmacological agents have been evaluated, there are no reports of β-arrestin–biased AT1R (angiotensin-II type-1 receptor) agonist (TRV027) used to prevent the progression of AA. Methods: We tested the hypothesis that TRV027 infusion in AngII (angiotensin II)-induced mouse model of AA prevents AA. High–fat-diet–fed ApoE (apolipoprotein E gene)-null mice were infused with AngII to induce AA and co-infused with TRV027 and a clinically used AT1R blocker Olmesartan to prevent AA. Aortas explanted from different ligand infusion groups were compared with assess different grades of AA or lack of AA. Results: AngII produced AA in ≈67% male mice with significant mortality associated with AA rupture. We observed ≈13% mortality due to aortic arch dissection without aneurysm in male mice. AngII-induced AA and mortality was prevented by co-infusion of TRV027 or Olmesartan, but through different mechanisms. In TRV027 co-infused mice aortic wall thickness, elastin content, new DNA, and protein synthesis were higher than untreated and Olmesartan co-infused mice. Co-infusion with both TRV027 and Olmesartan prevented endoplasmic reticulum stress, fibrosis, and vasomotor hyper responsiveness. Conclusions: TRV027-engaged AT1R prevented AA and associated mortality by distinct molecular mechanisms compared with the AT1R blocker, Olmesartan. Developing novel β-arrestin–biased AT1R ligands may yield promising drugs to combat AA.

Thiosemicarbazone compound has pronounced anticancer activity, which increases by metal ions pres-ence. New Ni(II), Cu(II), Pd(II), and Pt(II) complexes synthesized from 2-(2,3-dihydroxy benzylidene)-N-ethylhydrazine-1-carbothioamide ligand H3L and had been characterized via NMR, GC-MS, FTIR, CHNS, UV-vis, TGA, magnetic susceptibility measurements, and molar electric conductivity. X-ray crystallo-graphic data and the refinement structure of thiosemicarbazide ligand indicated a clear light colorless block with the formula C10H13N3O2S, which belongs to a monoclinic system with P1 21/ c1 space group and adopts orthorhombic crystal with a = 5.1509(3) A; b = 10.2856(6) A; c = 11.3261(6) A. The ligand and the metal complexes were also tested for antiproliferation activity against the MCF7 breast and A549 lung cancer cell lines. The data showed that Ni(II), Cu(II), Pd(II), and Pt(II) complexes had potent an-tiproliferation activity at the micromolar level. Pt(II) complex with square planar geometry exhibits the highest antiproliferation activity, while Ni(II) complex with distorted octahedral geometry showed the lowest antiproliferation activity in both cell lines.(c) 2022 Elsevier B.V. All rights reserved.

Purpose Development of pediatric left ventricular assist devices (LVADs) has severely lagged behind that of adult LVADs, primarily due to the size and hemocompatibility constraints of pediatric anatomy. To quantify sources of blood trauma within a device, we proposed a hemocompatibility assessment platform (HAP) that can evaluate the hemocompatibility of individual components of LVADs. To eliminate the hemolysis induced by the HAP itself, we incorporated passive magnetic bearings to suspend the rotor radially and an active magnetic bearing (AMB) to control the axial position. In this study, we evaluated AMB forces of 2 geometries and validated the model by comparing its predictions with experimental results. Methods The AMB comprises a stator with an electromagnetic coil and a rotor with a permanent magnet (PM) ring (1 mm and 2 mm thickness). The AMB numerical model was established in COMSOL 5.5 using the finite-element method. The magnetic forces generated by the AMB were evaluated by increasing the rotor-stator gap from 0.1 mm to 0.5 mm with a 0.1 mm increment and by varying the coil current from -2 A to 2 A with a 1 A increment. AMB force data was collected experimentally by a precision force transducer on an XYZ-micrometer-driven stage. Results The average error of the numerical models was 8.8% and 7.0% for 1 mm and 2 mm thick PM rings, respectively. Higher errors were seen at small (<0.2 mm) rotor-stator gaps. The change in magnetic force due to electrical current, or magnetic stiffness, is greater in the thicker PM ring. For both PM ring sizes, the AMB exhibits high magnetic stiffness from -1 A to 1 A, though it saturates for currents of -2 A and 2 A. This region of high current stiffness was identified as the optimal control region. Conclusion AMB force generation was characterized as a function of current and gap, both experimentally and numerically. In future work, this function will be used to tune a control algorithm to modulate current supplied to the AMB, ultimately stabilizing the rotor axially. Additionally, if design specifications such as PM ring thickness or material change, the numerical model can be used to quickly generate a new function for the AMB control algorithm.

Table 1 lists a number of putative GPCRs identified by NC-IUPHAR [161], for which preliminary evidence for an endogenous ligand has been published, or for which there exists a potential link to a disease, or disorder. These GPCRs have recently been reviewed in detail [121]. The GPCRs in Table 1 are all Class A, rhodopsin-like GPCRs. Class A orphan GPCRs not listed in Table 1 are putative GPCRs with as-yet unidentified endogenous ligands.Table 1: Class A orphan GPCRs with putative endogenous ligands GPR3GPR4GPR6GPR12GPR15GPR17GPR20 GPR22GPR26GPR31GPR34GPR35GPR37GPR39 GPR50GPR63GPR65GPR68GPR75GPR84GPR87 GPR88GPR132GPR149GPR161GPR183LGR4LGR5 LGR6MAS1MRGPRDMRGPRX1MRGPRX2P2RY10TAAR2 In addition the orphan receptors GPR18, GPR55 and GPR119 which are reported to respond to endogenous agents analogous to the endogenous cannabinoid ligands have been grouped together (GPR18, GPR55 and GPR119).