Human Receptor Research Articles

R150S mutation in the human oxytocin receptor: gain-of-function effects and implication in autism spectrum disorder

This study investigates the rs547238576 (R150S) missense variant in the oxytocin receptor (OXTR) gene, previously observed through screening of rare variants in Japanese individuals with autism spectrum disorders (ASD). Contrary to the anticipated loss-of-function, R150S exhibits gain-of-function effects, enhancing oxytocin (OXT) sensitivity, ligand-binding affinity, and OXT-induced Ca2+ mobilization in vitro. This suggests R150S may alter OXT signaling, potentially contributing to the excitatory/inhibitory imbalance seen in ASD and other psychiatric disorders. Our findings underscore the significance of genetic variations in OXTR on functional activity and highlight the necessity for population-specific genetic study and in vitro analysis to elucidate genetic susceptibilities to neuropsychiatric conditions.

PHARMACOLOGICAL INHIBITION REVEALS PARTICIPATION OF THE ENDOCYTIC COMPARTMENT IN POSITIVE FEEDBACK IL-6 SECRETION IN HUMAN SKELETAL MYOTUBES

IL-6 is an important cytokine involved in metabolic, immunological, and cell-fate responses. It is released upon stimulation by skeletal muscle cells through partially characterized mechanisms. In some cell types, IL-6 has been reported to activate a positive feedback loop involving endocytic vesicles, but evidence is mostly based on transcription and signal transduction mechanisms and is very scarce in muscle cells. Our aim was to directly demonstrate the presence of positive feedback in the ATP-induced release of IL-6 into the supernatant of human skeletal muscle cultures. The total release (production) of IL-6 was reduced for higher volumes of supernatant, when the secreted IL-6 molecules are more diluted, and enhanced when the supernatant volume was lower. In addition, secretion was impaired both by tocilizumab, a blocker of human IL-6 receptors, and by the soluble form of the receptor. The secretion in response to ATP was also inhibited by treatment with the endocytosis inhibitor dynasore, and by disruption of the acidic gradient of the endocytic compartment using different methods (chloroquine, NH4Cl or monensin). IL-6 secretion was also impaired by NED-19, a specific inhibitor of the two pore channels receptor mediating Ca2+ release from the endolysosomal compartment. IL-6 and ATP increased IL-6 mRNA levels, an effect blocked by tocilizumab. Altogether, our results demonstrate that ATP-secreted IL-6 activates a positive loop based on IL-6 receptors, endocytosis, two pore channels and IL-6 transcription. Given the importance of muscle IL-6 as a systemic regulator and as an inflammatory mediator, our study can help to understand muscle pathophysiology.

Polypurine Reverse Hoogsteen hairpins as a therapeutic tool for SARS-CoV-2 infection

Although COVID-19 pandemic was declared no longer a global emergency by the World Health Organization in May 2023, SARS-CoV-2 is still infecting people across the world. Many therapeutic oligonucleotides such as ASOs, siRNAs or CRISPR-based systems emerged as promising antiviral strategies for the treatment of SARS-CoV-2. In this work we explored the inhibitory potential on SARS-CoV-2 replication of Polypurine Reverse Hoogsteen Hairpins (PPRHs), CC1-PPRH and CC3-PPRH, targeting specific polypyrimidine sequences within the replicase and Spike regions, respectively, and previously validated for COVID-19 diagnosis. Both PPRHs bound to their target sequences in the viral genome with high affinity in the order of nM. In vitro, both PPRHs reduced viral replication by more than 92% when transfected into VERO-E6 cells 24 hours prior infection with SARS-CoV-2. In vivo intranasal administration of CC1-PPRH in K18-hACE2 mice expressing the human ACE receptor protected all the animals from SARS-CoV-2 infection. The properties of PPRHs position them as promising candidates for the development of novel therapeutics against SARS-CoV-2 and other viral infections.

Structural Studies of the Human α1 Glycine Receptor via Site-Specific Chemical Crosslinking Coupled with Mass Spectrometry

By identifying distance constraints, chemical crosslinking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical crosslinking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors (pLGICs) that function in fast neurotransmission. A single chemically-reactive cysteine was re-introduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional crosslinker, was site-specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocrosslinking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intra-subunit and inter-subunit sites of α1 GlyR crosslinking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical crosslinks within full-length integral membrane protein assemblies in a native-like lipid environment.

Biomineralized Nuclear Receptor Protein-Selection Mass Spectrometry for the Discovery of Drugs and Toxics.

Protein-selection mass spectrometry is cost-effective for the discovery of drugs and toxics. Nuclear receptors (NRs) are major targets for pharmaceuticals and endocrine-disrupting chemicals and are, thus, widely used as "bait" proteins. However, their application is limited due to the tendency to lose protein activity during cold storage. To address this problem, we introduced a novel biomineralization-based approach to preserve activity in NRs, exemplified by human retinoic acid receptor alpha (hRARα), a target for cancer and leucocythemia therapy. Since information on the coordination chemistry of metal ion and NR protein complexes is almost unavailable, we applied peptide mapping analysis for the first time for the rational design of his-hRARα-Co phosphate nanobiomaterial with high bioactivity. This nanobiomaterial successfully captured hRARα bioactive chemicals from a Chinese herb and environmental water and discovered an unsaturated fatty acid, (±)-(9Z,11E)-13-hydroxy-9,11-octadecadienoic acid ((±)13-HODE), which exhibited strong hRARα antagonistic activity.

Mono-palmitoyl-N-alkylurea ligands as specific activators of human Toll-like receptor 2/6 heterodimer.

Ligands for Toll-like-receptor 2 (TLR2) have demonstrated significant potential as immune-stimulating components in synthetic vaccines. Activation of TLR2 relies on the formation of dimeric complexes with either TLR1 or TLR6 and the nature of these dimers can impact therapeutic outcomes. The lipopeptide-based TLR2 ligands Pam3CysSK4 and Pam2CysSK4 have been extensively studied, and their recognition by different TLR-receptor heterodimers, TLR2/TLR1 and TLR2/TLR6, respectively, has been established. However, the high lipophilicity of these ligands, containing multiple palmitoyl residues, can result in solubility issues when used as vaccine adjuvants. To address this, we previously synthesized a less lipophilic ligand containing a single palmitoyl chain called mini-UPam, which effectively stimulates human moDC maturation. We here probe the receptor-dimer specificity of several mini-Upam derivatives and reveal that these mini-UPam are hTLR2/TLR6 selective ligands and that the introduction of longer urea alkyl chains does not shift the binding specificity to hTLR2/TLR1 heterodimers, in contrast to their Pam2CysSK4 and Pam3CysSK4 counterparts, pointing to a different binding mode of the UPam ligands.

A novel bioinformatics pipeline for the identification of immune inhibitory receptors as potential therapeutic targets.

Despite major successes with inhibitory receptor blockade in cancer, the identification of novel inhibitory receptors as putative drug targets is needed due to lack of durable responses, therapy resistance, and side effects. Most inhibitory receptors signal via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and previous studies estimated that our genome contains over 1600 ITIM-bearing transmembrane proteins. However, testing and development of these candidates requires increased understanding of their expression patterns and likelihood to function as inhibitory receptor. Therefore, we designed a novel bioinformatics pipeline integrating machine learning-guided structural predictions and sequence-based likelihood models to identify putative inhibitory receptors. Using transcriptomics data of immune cells, we determined the expression of these novel inhibitory receptors, and classified them into previously proposed functional categories. Known and putative inhibitory receptors were expressed across different immune cell subsets with cell type-specific expression patterns. Furthermore, putative immune inhibitory receptors were differentially expressed in subsets of tumour infiltrating T cells. In conclusion, we present an inhibitory receptor pipeline that identifies 51 known and 390 novel human inhibitory receptors. This pipeline will support future drug target selection across diseases where therapeutic targeting of immune inhibitory receptors is warranted.

A novel bioinformatics pipeline for the identification of immune inhibitory receptors as potential therapeutic targets

Despite major successes with inhibitory receptor blockade in cancer, the identification of novel inhibitory receptors as putative drug targets is needed due to lack of durable responses, therapy resistance, and side effects. Most inhibitory receptors signal via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and previous studies estimated that our genome contains over 1600 ITIM-bearing transmembrane proteins. However, testing and development of these candidates requires increased understanding of their expression patterns and likelihood to function as inhibitory receptor. Therefore, we designed a novel bioinformatics pipeline integrating machine learning-guided structural predictions and sequence-based likelihood models to identify putative inhibitory receptors. Using transcriptomics data of immune cells, we determined the expression of these novel inhibitory receptors, and classified them into previously proposed functional categories. Known and putative inhibitory receptors were expressed across different immune cell subsets with cell type-specific expression patterns. Furthermore, putative immune inhibitory receptors were differentially expressed in subsets of tumour infiltrating T cells. In conclusion, we present an inhibitory receptor pipeline that identifies 51 known and 390 novel human inhibitory receptors. This pipeline will support future drug target selection across diseases where therapeutic targeting of immune inhibitory receptors is warranted.

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.

No Evidence for Endocannabinoid-Induced G Protein Subtype Selectivity at Human and Rodent Cannabinoid CB1 Receptors.

Introduction: The endocannabinoid system (ECS) is a widespread neurotransmitter system. A key characteristic of the ECS is that there are multiple endogenous ligands (endocannabinoids). Of these, the most extensively studied are arachidonoyl ethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG), both act as agonists at the cannabinoid CB1 receptor. In humans, three CB1 variants have been identified: hCB1, considered the most abundant G protein-coupled receptor in the brain, alongside the less abundant and studied variants, hCB1a and hCB1b. CB1 exhibits a preference for coupling with inhibitory Gi/o proteins, although its interactions with specific members of the Gi/o family remain poorly characterized. This study aimed to compare the AEA and 2-AG-induced activation of various G protein subtypes at CB1. Furthermore, we compared the response of human CB1 (hCB1, hCB1a, hCB1b) and explored species differences by examining rodent receptors (mCB1, rCB1). Materials and Methods: Activation of individual G protein subtypes in HEK293 cells transiently expressing CB1 was measured with G protein dissociation assay utilizing TRUPATH biosensors. The performance of the TRUPATH biosensors was evaluated using Z-factor analysis. Pathway potencies and efficacies were analyzed using the operational analysis of bias to determine G protein subtype selectivity for AEA and 2-AG. Results: Initial screening of TRUPATH biosensors performance revealed variable sensitivities within our system. Based on the biosensor performance, the G protein subtypes pursued for further characterization were Gi1, Gi3, GoA, GoB, GZ, G12, and G13. Across all pathways, AEA demonstrated partial agonism, whereas 2-AG exhibited full or high-efficacy agonism. Notably, we provide direct evidence that the hCB1 receptor couples to G12 and G13 proteins. Our findings do not indicate any evidence of G protein subtype selectivity. Similar observations were made across the human receptor variants (hCB1, hCB1a, hCB1b), as well as at mCB1 and rCB1. Discussion: There was no evidence suggesting G protein subtype selectivity for AEA and 2-AG at CB1, and this finding remained consistent across human receptor variants and different species.

8599 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

9285 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

8627 Viral Hormones: The Role Of Viral Insulin/IGF-1 Like Peptides In Host-Pathogen Interactions

Abstract Disclosure: A. Chuard: None. Viruses, encode proteins that are similar to host proteins to manipulate the host. Until our recent discovery of viral hormones, examples of viral mimicry were limited to immunomodulatory proteins and growth factors. We recently showed that six viruses in Iridoviridae family encode genes mimicking human insulin and IGF-1. We previously chemically synthesizing these viral insulin/IGF-1 like peptides (VILPs) and showed that VILPs can bind to human insulin and IGF-1 receptor and further stimulate post-receptor signaling. VILPs also stimulate glucose uptake and proliferation in mammalian cells and lower the blood glucose in mice. Originally, VILP-carrying viruses were isolated from fish; however, the role of VILPs in viral pathogenesis is not studied in depth. Here, we examined the effects of Grouper Iridovirus (GIV, one of the VILP carrying viruses) infection and GIV-VILP on host insulin/IGF signaling system using fish cells.To this end, we chemically synthesized GIV-VILP in its single chain (sc, IGF-1 like) and double chain (dc, insulin-like) forms. Stimulation of grouper kidney (GK) and AB9 zebrafish cells with these peptides, insulin and IGF-1, showed that IGF-1 was the most potent ligand in these cells indicating an abundance of IGF-1 receptor (IGF1R). Further, both forms of the VILPs were as potent as insulin in its activity on receptor phosphorylation and post-receptor signaling. Examining the viral kinetics, we also showed that GIV32 gene (encoding GIV VILP) is an early viral gene in both cell types. Virion analysis through mass spectrometry indicated that VILPs are not viral structural proteins. On the other hand, analysis of the supernatant from infected cells indicated that VILPs are secreted peptides during the viral cycle. Our results with supernatant transfer experiments from infected cells to uninfected cells support the same conclusion that VILPs are secreted. The secreted VILPs initiate autophosphorylation of IGF1R and/or insulin receptor (IR), consequently triggering downstream cellular signaling and metabolism. Through the use of specific inhibitors for each receptor, we illustrated that IR signaling is crucial for GIV viral replication. Surprisingly, inhibition of IGF1R increased viral replication and we are currently examining potential inhibitory effects of GIV VILP. Furthermore, VILPs appear to exert a biased effect on the AKT pathway compared to the Erk pathway. Furthermore, the inhibition of the AKT pathway significantly reduces GIV viral replication. In summary, our findings reveal that GIV-VILP is actively produced during infection and contributes to the viral replication. To our knowledge, VILPs are the first hormone-mimics characterized in viral replication. Elucidating the role of VILPs in host-pathogen interactions, we propose a novel viral pathogenesis mechanism where viruses mimic host hormones to manipulate the host's endocrine system. Presentation: 6/3/2024

The Role of Human Adiponectin Receptor 1 in 2-Ethylhexyl Diphenyl Phosphate Induced Lipid Metabolic Disruption.

Epidemiological evidence links exposure to 2-ethylhexyl diphenyl phosphate (EHDPP) with lipid metabolic disruption, typically attributed to nuclear receptors, while the role of membrane receptors remains underexplored. This study explored the role of adiponectin receptor 1 (AdipoR1) in EHDPP-induced lipid metabolic disturbances. We examined EHDPP's binding affinity and transcriptional impact on AdipoR1. AdipoR1 knockdown (AdipoR1kd) human liver cells and coculture experiments with AdipoR1 activator (AdipoRon) were used to investigate the effect and the mechanism. EHDPP disrupted triglyceride and phospholipid synthesis and altered corresponding gene expression, mirroring effects in AdipoR1kd cells but diminishing in EHDPP-treated AdipoR1kd cells. RNA sequencing revealed that EHDPP primarily disrupted oxidative phosphorylation and insulin signaling dependent on AdipoR1. Mechanistically, EHDPP interacted with AdipoR1 and reduced AdipoR1 protein levels at 10-7 mol/L or higher, weakening the activation of the calmodulin dependent protein kinase β (CaMKKβ)/AMPK/acetyl CoA carboxylase pathway. Furthermore, EHDPP pretreatment blocked the increase in Ca2+ flux and the corresponding kinase CaMKKβ, as well as liver kinase B1 (LKB1) activation induced by AdipoRon, which is necessary for AMPK activation. Collectively, these findings demonstrate that EHDPP-induced lipid imbalance is partially dependent on AdipoR1, expanding the understanding of environmental metabolic disruptors beyond nuclear receptors.

Race/ethnicity and human epidermal growth hormone receptor 2–targeted therapy for breast cancer.

91 Background: Breast cancer is the most common cancer in the US. Up to 25% of breast cancers are classified as human epidermal growth factor receptor 2 (HER2)-positive and associated with an aggressive tumor burden. HER2-targeting monoclonal antibodies improves survival in patients with HER2-positive breast cancer. Prior work has demonstrated disparities in access to oncologic therapies among older women. Our study seeks to examine racial/ethnic disparities in receiving HER2-targeted therapies for patients with HER2-positive breast cancer and trends in use over time. Methods: In the Surveillance, Epidemiology and End Results (SEER) Medicare-linked database we identified women diagnosed with breast cancer from 2010 to 2019. We included those >66 years old who survived 12 months after diagnosis, were enrolled in Medicare Parts A/B for 12 months pre and post-diagnosis, and had local or regional stage HER2-positive cancer. Our primary outcome was receipt of HER2-targeted therapies in the 12 months after diagnosis. Our independent variable was race/ethnicity as measured by the validated Research Triangle Institute race/ethnicity codes in Medicare. Due to small sample sizes, we focus our analysis on White, Black, or Hispanic women. We used multivariable Modified Poisson regression to evaluate changes in the probability of HER2-targeted therapy receipt by race/ethnicity over time, adjusted for sociodemographics, cancer factors, and medical comorbidities. Results: Our cohort comprised 13,887 patients including 8.7% Black, 7.5% Hispanic, and 83.8% White (mean age 74.9). Overall, 7,351 (52.9%) received HER2-targeted therapies in the 12 months post-diagnosis, with use increasing over time in all racial/ethnic groups (Table). Compared with White patients, Black and Hispanic patients had a 19% (adjusted risk ratio [aRR] 0.81, 95% CI:0.69-0.96) and 27% (aRR 0.73, 95% CI 0.61-0.89) lower likelihood, respectively, of receiving HER2-targeted therapies from 2010-2011 (Table). By 2018-2019, no significant differences in receiving HER2-targeted therapies were observed across racial/ethnic groups. Conclusions: In a national cohort of Medicare enrollees with HER2-positive breast cancer, we observed significant racial/ethnic disparities which narrowed over time. Overall utilization rates were low. Future work is needed to understand how improved access to breast cancer treatments impact downstream outcomes. Use of human epidermal growth hormone receptor 2–targeted therapy from 2010-2011 and 2018-2019 by race/ethnicity. Race and Ethnicity Unadjusted Rates Adjusted Risk Ratios (95% CI)* 2010-2011 2018-2019 2010-2011 2018-2019 Black 36% 62% 0.81 (0.69-0.96) 0.97 (0.87-1.08) Hispanic 31% 68% 0.73 (0.61-0.89) 1.01 (0.92-1.12) White 42% 64% Reference Reference *Adjusted for age, marital status, region, breast cancer stage and year of diagnosis, and medical comorbidities.

Cryo-EM structures of the human P2X1 receptor reveal subtype-specific architecture and antagonism by supramolecular ligand-binding

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.

Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4.

Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally regulate paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE complexes. Claudin/CpE complexes are the building blocks of oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here, we present the structures of CpE in complex with its native claudin receptor in humans, claudin-4, using cryogenic electron microscopy. The structures reveal the architecture of the claudin/CpE complex, the residues used in binding, the orientation of CpE relative to the membrane, and CpE-induced changes to claudin-4. Further, structures and modeling allude to the biophysical procession from claudin/CpE complexes to cytotoxic β-barrel pores during pathogenesis. In full, this work proposes a model of claudin/CpE assembly and provides strategies to obstruct its formation to treat CpE diseases.

From Millets to Medicine: ADMET Insights into Diabetes Management with P. sumatrense Compounds

Diabetes mellitus, a prevalent metabolic disorder characterised by impaired blood glucose regulation, has escalated into a global health crisis, exacerbated by high-glycaemic diets and unhealthy eating habits. While therapeutic agents such as acarbose mitigate diabetes by inhibiting alpha-amylase and alpha-glucosidase, their use is marred by adverse side effects, underscoring the need for safer, plant-based alternatives. This study explores the antidiabetic potential of Little Millet, an underutilised cereal grain with a low glycaemic index, rich in bioactive compounds. Unlike common staples such as wheat and rice, Little Millet contains unique compounds, including octodrine, 18,19-secoyohimban-19-oic acid, and phenylephrine, which were investigated for their novel application in diabetes management. Gas chromatography-mass spectrometry (GCMS) analysis identified 120 bioactive compounds, selected for their abundance and potential bioactivity. These compounds span fatty acids, phenols, flavonoids, organic acids, sugar alcohols, and amino acids, all of which were rigorously docked to alpha-amylase and human maltase-glucoamylase receptors. Notably, key ligands such as chlorogenic acid and 18,19-secoyohimban-19-oic acid exhibited superior binding affinities compared to acarbose, with promising drug-likeness and ADMET profiles, suggesting higher absorption and fewer side effects. A 100-ns dynamics simulation further validated the stability of these compounds, positioning them as viable candidates for diabetes treatment. These findings advance drug design and highlight the potential of incorporating Little Millet into diets for diabetes prevention. Further validation through In-vitro, In-vivo, and clinical studies is essential to fully realise their therapeutic potential. This research also opens avenues for exploring Little Millet in managing related conditions and enhancing its health benefits through breeding programmes.

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

BackgroundHuman interleukin-22 (IL-22) is known as a “dual function” cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis.MethodsWe used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis.ResultsWe demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A.ConclusionsWe developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

High-resolution crystal structure of human coronavirus HKU1 receptor binding domain bound to TMPRSS2 receptor

High-resolution crystal structure of human coronavirus HKU1 receptor binding domain bound to TMPRSS2 receptor