Receptor Assay Research Articles

Dual agonism of FP and EP3 receptors by ricinoleic acid: Plausible therapeutic in glaucoma

Aims/Purpose: Prostaglandin analogues used in glaucoma therapy act on prostaglandin F (FP) receptor to enhance the uveoscleral outflow, however they do not have affinity for prostaglandin E3 (EP3) receptors which are extensively present in iris‐ciliary body‐trabecular meshwork. Ricinoleic acid (RA) derived from the castor oil used as labor inducer as it causes EP3 receptor agonism and release of prostaglandins. This property of dual agonism, makes it an ideal candidate for ocular hypotensive therapy. Therefore, this study was designed to study the interaction RA with FP and EP3 receptor.Methods: The three‐dimensional structure modeling and docking was performed for FP and E3 receptor with ricinoleic acid and their respective positive controls. To study interaction of RA with FP and EP3 receptor, calcium binding and cAMP release assay were performed in ovarian, kidney, corneal epithelial and trabecular meshwork cell lines. The variable concentration of RA were compared to the positive and negative controls. The ovarian and trabecular meshwork cells supernatant were assessed for the levels of PGF2alpha using ELISA.Results: The in‐silico study showed RA higher binding affinity for EP3 receptor as compared to its natural (PGE2) and synthetic ligand (Misoprostol). The RA had comparatively less affinity for FP receptor compared to controls. In calcium release and cAMP assay, RA variable doses (10‐1000nM) were able to increase the release of calcium and decrease the cAMP levels in dose dependent manner. The dose response of RA for the EP3 agonism was significantly higher than latanoprost, and travoprost. The EP3 agonism by RA caused the release of PGF2alpha at lower levels, which plateaued after the 200nM dose.Conclusions: The study shows that at lower doses RA had higher interaction with EP3 but comparable affinity for FP receptors. The release of prostaglandin by EP3 agonism might be responsible for its dual agonism. The compound can be tested further for its hypotensive properties in glaucoma.

Evaluation of a new soluble transferrin receptor assay and comparison to three measurement procedures.

Soluble transferrin receptor (sTfR) is a useful marker in the differentiation of anemia. Clinical utility is limited by lack of standardization between measurement procedures and interpretative recommendations. Our objective was to evaluate the analytical performance of a research sTfR immunoturbidimetric assay (Alinity c, Abbott Diagnostics) and compare it to three established measurement procedures. Assay imprecision was assessed with 7 panels across the analytical measuring interval. 159 patient samples were measured across four instrument systems (Alinity c [Abbott Diagnostics], Tina-quant c502 [Roche Diagnostics], Quantex Biokit [Werfen], and ACCESS [Beckman Coulter]). Ferritin was also measured to calculate an sTfR/Log Ferritin ratio. Sera from 100 reference individuals were assayed for sTfR and ferritin (Alinity) for reference interval (RI) verification (sTfR) or establishment (sTfR index). Assay imprecision met defined goals. Method comparison between Alinity c and ACCESS sTfR assays showed good agreement (slope: 1.06, intercept: -0.12, r: 0.989). Comparisons across other assays demonstrated significant proportional bias with slopes ranging from 0.44 (Tina-quant c502, mean bias: -2.52mg/L) to 1.24 (Quantex Biokit, mean bias: 0.60mg/L). A proportional bias was observed between other instruments. While the sTfR RI was verified on the Alinity assay, agreement in interpretation (within vs outside RI) between Alinity and other platforms ranged from 74.2 to 80.5%. We report the first characterization of the performance of a research sTfR immunoturbidimetric assay (Alinity c, Abbott Diagnostics). Our findings emphasize the lack of harmonization between measurement procedures and result interpretation for sTfR and sTfR index, necessitating standardization efforts and clinical studies.

Comparison of Histopathological Type in Breast Carcinoma Between Premenopausal & Postmenopausal Women

Background: Breast carcinoma is a significant health concern affecting women worldwide, with variationsin histopathological characteristics based on menopausal status. Understanding these differences is crucialfor guiding clinical management and improving patient outcomes. Objective: This study aimed to comparethe histopathological types of breast carcinoma between premenopausal and postmenopausal women.Methods: A cross-sectional comparative study was conducted at the Department of Surgery, RajshahiMedical College, Rajshahi, from September 2021 to August 2022. Fifty female patients with breastcarcinoma were included, with 25 participants in each group: premenopausal (Group A) and postmenopausal(Group B). Specimens were obtained after modified radical mastectomy and subjected to histopathologicalexamination and immunohistochemistry for receptor assays in two reputable laboratories. Results: Amongthe 50 patients, premenopausal women (n=16, 64%) were mostly aged 31-40 years, while postmenopausalwomen (n=12, 48%) were aged 51-60 years. Invasive Ductal Carcinoma (IDC) was the most common type,present in 15 premenopausal women (60%) and 18 postmenopausal women (72%). Estrogen Receptor(ER) positivity was higher in premenopausal women (n=17, 68%) compared to postmenopausal women(n=13, 52%). Progesterone Receptor (PR) positivity was more frequent in premenopausal women (n=16,64%) than in postmenopausal women (n=11, 44%). HER2 positivity was observed in 5 premenopausal(20%) and 7 postmenopausal women (30%). Postmenopausal women had larger tumors, higher histologicalgrades, and more lymph node involvement. Conclusions: This study highlights distinct histopathologicalcharacteristics of breast carcinoma between premenopausal and postmenopausal women. Premenopausalpatients tended to present with a higher frequency of IDC and ER-positive tumors, whereas postmenopausalpatients showed a higher prevalence of IDC and variability in receptor expression. These findings underscorethe importance of considering menopausal status in the diagnosis and management of breast carcinoma.

Characterization of novel nitazene recreational drugs: Insights into their risk potential from in vitro µ-opioid receptor assays and in vivo behavioral studies in mice

2-Benzylbenzimidazole derivatives or ‘nitazenes’ are increasingly present on the recreational drug market. Here, we report the synthesis and pharmacological characterization of 15 structurally diverse nitazenes that might be predicted to emerge or grow in popularity. This work expands the existing knowledge about 2-benzylbenzimidazole structure-activity relationships (SARs), while also helping stakeholders (e.g., forensic toxicologists, clinicians, policymakers) in their risk assessment and preparedness for the potential next generation of nitazenes. In vitro µ-opioid receptor (MOR) affinity was determined via competition radioligand (3[H]DAMGO) binding assays in rat brain tissue. MOR activation (potency and efficacy) was studied by means of a cell-based β-arrestin 2 recruitment assay. For seven nitazenes, including etonitazene, opioid-like pharmacodynamic effects (antinociception, locomotor activity, body temperature changes) were evaluated after subcutaneous administration in male C57BL/6 J mice. The results showed that all nitazenes bound to MOR with nanomolar affinities, and the functional potency of several of them was comparable to or exceeded that of fentanyl. In vivo, dose-dependent effects were observed for antinociception, locomotor activity, and body temperature changes in mice. SAR insights included the high opioid-like activity of methionitazene, iso-butonitazene, sec-butonitazene, and the etonitazene analogues 1-ethyl-pyrrolidinylmethyl N-desalkyl etonitazene and ethylene etonitazene. The most potent analogue of the panel across all functional assays was α’-methyl etonitazene. Taken together, through critical pharmacological evaluation, this work provides a framework for strengthened preparedness and risk assessments of current and future nitazenes that have the potential to cause harm to users.

Establishing a Relationship between In Vitro Potency in Cell-Based Assays and Clinical Efficacious Concentrations for Approved GLP-1 Receptor Agonists.

Background: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) play an important role in the treatment of type 2 diabetes (T2D) and obesity. The relationship between efficacy and dosing regimen has been studied extensively for this class of molecules. However, a comprehensive analysis of the translation of in vitro data to in vivo efficacious exposure is still lacking. Methods: We collected clinical pharmacokinetics for five approved GLP-1RAs to enable the simulation of exposure profiles and compared published clinical efficacy endpoints (HbA1c and body weight) with in-house in vitro potency values generated in different cell-based assays. Additionally, we investigated the correlation with target coverage, expressed as a ratio between the steady state drug exposure and unbound potency, body weight, or HbA1c reduction in patients with T2D. Results: We found that the best correlation with in vivo efficacy was seen for in vitro potency data generated in cellular assays performed in the absence of any serum albumin or using ovalbumin. Residual variability was larger using in vitro potency data generated in endogenous cell lines or in the presence of human serum albumin. For the human receptor assay with no albumin, exposures above 100-fold in vitro EC50 resulted in >1.5% point HbA1c reduction, while a 5% BW reduction was related to approximately 3× higher exposures. A similar relationship was seen in the ovalbumin assay. Conclusions: Overall, the relationship established for in vitro potency and in vivo efficacy will help to increase confidence in human dose prediction and trial design for new GLP-1RAs in the discovery and early clinical phases.

164 Combining in silico, in vitro, and in vivo approaches for studying taste perception and preferences of domestic cats and dogs

Abstract Developing food products for pet cats and dogs is an important and challenging area of the food industry. The diet offered must be both balanced and palatable to ensure the amount of food eaten meets their nutritional requirements. The taste perception of pets is different from humans in many cases (Li et al., 2005) and therefore species-specific research is necessary. Hence, understanding the flavor preferences of cats and dogs is an important area of research and requires multiple disciplines and scientific approaches. We have developed a range of complimentary methods for studying taste perception and preferences of domestic cats and dogs In silico: homology models of the pet taste receptors have been developed. These models are used to identify candidate compounds and elucidate the mechanisms of taste perception of pets. In vitro: cell-based high throughput screening assays of pet taste receptors have also been developed. These assays are used to identify taste-active compounds and confirm response to those identified via the in silico method to refine the model. In vivo: finally, a two-bottle choice test for cats has been developed. This test is used to confirm hedonic responses and determine optimum concentrations of single compounds and mixtures identified by the in silico and/or in vitro methods. Using this novel approach, we now have a deeper understanding of the taste perception and preferences of pets, with specific examples including Umami (McGrane et al., 2023) and Kokumi (Laffitte et al., 2021) taste perception of cats and bitter taste perception of dogs (Gibbs et al., 2022). This approach has also enabled us to reduce the number of in vivo tests required, so implementing the 3Rs, by prioritizing the most promising tastants using the in silico and in vitro methods. Gibbs, M., Winnig, M., Riva, I., Dunlop, N., Waller, D., Klebansky, B., Logan, D.W., Briddon, S.J., Holliday, N.D., McGrane, S.J. 2022. Bitter taste sensitivity in domestic dogs (Canis familiaris) and its relevance to bitter deterrents of ingestion. PLoS One. 17:e0277607. Laffitte, A., Gibbs, M., Hernangomez de Alvaro, C., Addison, J., Lonsdale, Z.N., Giribaldi, M.G., Rossignoli, A., Vennegeerts, T., Winnig, M., Klebansky, B., Skiles, J., Logan, D.W., McGrane, S.J. 2021. Kokumi taste perception is functional in a model carnivore, the domestic cat (Felis catus). Sci Reports 2021 111. 11:1–17. Li, X., Li, W., Wang, H., Cao, J., Maehashi, K., Huang, L., Bachmanov, A.A., Reed, D.R., Legrand-Defretin, V., Beauchamp, G.K., Brand, J.G. 2005. Pseudogenization of a sweet-receptor gene accounts for cats’ indifference toward sugar. PLoS Genet. 1:0027–0035.McGrane, S.J., Gibbs, M., Hernangomez De Alvaro, C., Dunlop, N., Winnig, M., Klebansky, B., Waller, D. 2023. Umami taste perception and preferences of the domestic cat (Felis catus), an obligate carnivore. Chem Senses. 48.

Improving the Sensitivity of Protein Quantification by Immunoaffinity Liquid Chromatography─Triple Quadrupole Mass Spectrometry Using an Iterative Transition Summing Technique.

The desire to reach ever-diminishing lower limits of quantification (LLOQ) to probe changes in low abundance protein targets has led to enormous progress in sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumentation. To maximize signal and reduce noise, many approaches have been employed, including specific immunoaffinity (IA) enrichment and reducing the LC flow to the nanoflow (nLC) level; however, additional sensitivity gains may still be required. Recently, a technique termed "echo summing" has been described for small-molecular-weight analytes on a triple quadrupole (QqQ) MS where multiple iterations of the same, single selected reaction monitoring (SRM) transition are collected, summed, and integrated, yielding significant analyte dependent signal-to-noise (S/N) improvements. Herein, the direct applicability of echo summing to protein quantification by sequential IA combined with nLC-MS/MS (IA-nLC-MS/MS) is described for a beta nerve growth factor (NGF) and a soluble asialoglycoprotein receptor (sASGPR) assay from human serum. Five iterations of echo summing outperformed traditional collection in relative average accuracy (-1.5 ± 7.7 vs -41.7 ± 10.7% bias) and precision (7.8 vs 18.4% coefficient of variation (CV)) of the low-end quality control (QC) sample (N = 4) for NGF and improved functional sensitivity of serially diluted serum QC samples (N = 5 each population) approximately 2-fold (1.96 and 2.00-fold) for two peptides of sASGPR. Echo summing also extended the minimum quantifiable QC level for sASGPR 4-fold lower. Similar gains are believed to be achievable for most protein IA-nLC-MS/MS assays.

Genotype and X-chromosome inactivation are associated with disease severity in females with X-linked Alport syndrome.

Male patients with X-linked Alport syndrome (XLAS) generally develop end-stage kidney disease in early or middle adulthood and show distinct genotype-phenotype correlations. Female patients, however, show various phenotypes ranging from asymptomatic to severe with no genotype-phenotype correlations. However, the factors affecting the severity of XLAS in female patients are unclear. Since X-chromosome inactivation (XCI) affects the severity of certain female X-linked diseases, we investigated whether genotype and XCI were associated with XLAS severity in female patients in a large Japanese cohort. Among 139 female patients with genetically diagnosed XLAS at our institution, we conducted XCI analysis on peripheral blood leukocytes using the human androgen receptor assay method and analyzed two cohorts. In 74 adult female patients, we evaluated the correlation between kidney function (creatinine-estimated glomerular filtration rate [Cr-eGFR] optimized for Japanese individuals) and genotype/XCI using multivariable linear regression analysis, and in 65 pediatric female patients, we evaluated the correlation between kidney function (Cr-eGFR optimized for Japanese individuals) and genotype/XCI using multivariable linear regression analysis. We also investigated the correlation between the development of proteinuria (urine protein-to-creatinine ratio above normal for the patient's age) and genotype/XCI using multivariable Cox proportional hazard analysis. In adult female patients, XCI pattern was significantly associated with Cr-eGFR (regression coefficient estimate = -0.53, P=0.004), whereas genotype was not (P=0.892). In pediatric female patients, both genotype and XCI pattern were significant independent risk factors for the development of proteinuria (hazard ratio [HR], 3.702; 95% confidence interval [CI], 1.681-8.150; P=0.001 and HR, 1.043; 95% CI, 1.061-1.070; P=0.001, respectively), whereas both genotype and XCI pattern were not associated with Cr-eGFR (P=0.20, P=0.67, respectively). Genotype and XCI are factors associated with the severity in females with XLAS.

Comparison of Receptor Status in Breast Carcinoma Between Premenopausal and Postmenopausal Women

Background: Breast cancer is the most prevalent cancer affecting women worldwide. The immunohistochemical status of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) plays a crucial role in determining breast cancer treatment options and prognosis. These receptor statuses are known to be influenced by menopausal transition. Objective: This cross-sectional comparative study aimed to assess the frequency of ER, PR, and HER2/Neu positivity or negativity and their association with menopausal status among female breast carcinoma patients. Methodology: The study was conducted over one year, from September 2021 to August 2022, at the Department of Surgery, Rajshahi Medical College, Rajshahi. A total of 50 female patients with breast carcinoma were included in the study, divided into two groups: Group A (Premenopausal women) and Group B (Postmenopausal women). Specimens were collected after modified radical mastectomy and sent to renowned laboratories for histopathological and immunohistochemical receptor assays. Data were collected through a predesigned semi-structured questionnaire and analyzed using SPSS (version 24.0). Results: The study found no significant relationship between receptor status and menopausal status or increasing age. However, a significant histopathological association was observed with receptor status, with ER and PR positivity associated with grade II tumors (p-value <0.011). The most common histopathological type was ductal cell carcinoma, and its incidence increased with parity (p-value <0.009). Conclusions: This study revealed no significant difference in hormone receptor and HER2 Neu expression between premenopausal and postmenopausal breast carcinoma patients. Further research with a larger sample size is recommended for a more comprehensive understanding of this relationship.

Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.

New synthetic opioids: Advances of receptor assays as tools for pharmacological characterization and analytical screening

New synthetic opioids: Advances of receptor assays as tools for pharmacological characterization and analytical screening

Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.

CARD11 regulates the thymic Treg development in an NF-κB-independent manner.

CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner. We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO–K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

Porcine low-density lipoprotein receptor plays an important role in classical swine fever virus infection

ABSTRACT Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

Functional characterization of the kinin receptor in the Chagas disease vector Rhodnius prolixus; activity of native kinins and potent biostable Aib-containing insect kinin analogs

The causative agent for Chagas disease, Trypanosoma cruzi, is transmitted to a human host in the urine/feces of the kissing bug, Rhodnius prolixus, following blood feeding. Kinins are important chemical messengers in the overall control of blood feeding physiology in R. prolixus, including hindgut contractions and excretion. Thus, disruption in kinin signaling would have damaging consequences to the insect but also interfere with the transmission of Chagas Disease. Here, a heterologous functional receptor assay was used to confirm the validity of the previously cloned putative kinin G-protein-coupled receptor, RhoprKR, in Rhodnius prolixus. Three native R. prolixus kinins were chosen for analysis; two possessing the typical kinin WGamide C-terminal motif and one that possesses an atypical C-terminal WAamide. All three are potent (EC50 values in the nM range), with high efficacy, on CHO-K1-aeq cells expressing the RhoprKR, thereby confirming ligand binding. Members of three other R. prolixus peptide families, which are also myotropins (tachykinins, pyrokinins and sulfakinins) elicited little or no response. In addition, this heterologous receptor assay was used to test characteristics of kinin mimetics previously tested on tick and mosquito kinin receptors. Five α-aminoisobutyric acid (Aib) containing analogs were tested, and four found to have considerably higher potencies than the native kinins, with EC50 values in the pM range. Interestingly, adding Aib to the atypical WAamide kinin improves its EC50 value from 2 nM to 39 pM. Biostable kinin analogs may prove useful leads for novel pest control strategies. Since T. cruzi is transmitted to a human host in the urine/feces after blood feeding, disruption in kinin signaling would also interfere with the transmission of Chagas Disease.

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.

Abstract 12379: AT1-AA (Angiotensin II Type 1 Receptor Autoantibody) Aggravates Abdominal Aortic Aneurysm Through Activating AT1R/Gq Allosterically

Background: Angiotensin II type 1 receptor (AT1R) is a key mediator of the rennin-angiotensin-aldosterone system (RAAS). Autoantibodies against AT1R (AT1-AA), as an activator of AT1R, was found in abdominal aortic aneurysm (AAA) related diseases, such as hypertension and atherosclerosis. However, the role of AT1-AA in AAA is still unclear. Methods: We used angiotensin II (AngII)-infused male ApoE -/- mice model of AAA. Ultrasonography measurement, histological assessment, western blot, qRT-PCR and RNA sequencing were used to assess the severity of AAA. Nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay, radio assay of receptors and homogeneous time resolved fluorescence (HTRF) were used to measure the activation feature of AT1R and the affinity of AT1R with AT1-AA or AngII. Results: AT1-AA increased the incidence of AAA (vehicle vs. AT1-AA: 47% vs. 81%; P<0.05), which can be attenuated by AT1R blocker Candesartan. Furthermore, AT1-AA greatly increased severity of AAA, due to augmenting the diameter of abdominal aorta and the degradation of elastin by enhancing the expression of MMP2/9 (matrix metalloproteinase) and the accumulation of inflammatory cells. And AT1-AA could induce phenotypic switch of smooth muscle cells. But AT1-AA does not increase the plasma level of AngII. Mechanically, AT1-AA enhanced AngII-induced recruitment of Gq, but not β-arrestin1/2. AT1-AA potentiated the AngII-mediated Gq-dependent inositol phosphate (EC50 shift=3.829 nM) and the activation of PKC and ERK1/2. The responses were abolished by AT1R blocker and Gq inhibitor. The affinity of AT1-AA for the AT1R in cells was calculated to be 75.49 ± 4.7 nM. AT1-AA cannot compete for the binding of AngII to AT1R. However, AT1-AA significantly potentiated the binding of AngII with AT1R allosterically (mean K D ± SEM: 34.11 ± 0.9765 nM at 0 nM AT1-AA vs. 25.63 ± 1.826 nM at 100 nM AT1-AA; p<0.05), without altering the maximum binding. Conclusion: AT1-AA, as an allosteric activator of AT1R, promotes AAA through increasing the affinity of AngII with AT1R and activating Gq biasedly.

A sweeter future: Using protein language models for exploring sweeter brazzein homologs.

With growing concerns over the health impact of sugar, brazzein offers a viable alternative due to its sweetness, thermostability, and low risk profile. Here, we demonstrated the ability of protein language models to design new brazzein homologs with improved thermostability and potentially higher sweetness, resulting in new diverse optimized amino acid sequences that improve structural and functional features beyond what conventional methods could achieve. This innovative approach resulted in the identification of unexpected mutations, thereby generating new possibilities for protein engineering. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.