Wild-type Receptor Research Articles

Structural basis for ligand modulation and partial agonism of the 5-HT3R.

Type-3 serotonin receptors (5-HT3Rs) are pentameric ligand gated ion channels that mediate fast synaptic signaling in response to binding of the neurotransmitter serotonin. 5-HT3Rs play a major role in regulating gut motility, secretion, and visceral perception. Hyperactivity of 5-HT3Rs underlies pathologies such as chemotherapy-induced nausea and vomiting, irritable bowel syndrome, depression, anxiety, bipolar disorder, and excessive visceral pain, making it an important drug target. Previous research has resulted in the structures of the 5-HT3AR in complex with the agonist serotonin and setrons, 5-HT3R antagonists. Recently, 5-HT3AR partial agonists have been proposed to treat 5-HT3R pathologies with less severe side effects than full antagonists. However, the molecular mechanisms of partial agonism by orthosteric ligands of the 5-HT3R are still poorly understood. Here we present structures of the 5-HT3AR in complex with two novel orthosteric partial agonists generated from cryo-electron microscopy imaging. Structural stability of the ligand binding poses was assessed by molecular dynamic simulations, and ligand function was assessed by two-electrode voltage clamp in wild type and mutant receptors. Using our ligand-bound models of the 5-HT3AR, we propose structure-based mechanisms for the functional differences between orthosteric partial agonists, full agonist, and antagonists.

NKG2D Ligand Expression Induced by Oxidative Stress Mitigates Cutaneous Ischemia-Reperfusion Injury.

Pressure ulcers represent a crucial clinical problem, especially in hospitalized patients. Ischemia-reperfusion (I-R) is an important cause of these lesions. Natural killer (NK), invariant NK T (iNKT), and dendritic epidermal T-cells, which express the natural killer group 2, member D (NKG2D) receptor, have been reported to have physiological roles in skin tissue repair and wound healing. However, a role for NKG2D-NKG2D ligand interactions in I-R-induced skin injury has not been determined. Using a murine pressure ulcer model, we demonstrated that I-R-induced ulcers in NKG2D-deficient mice were larger than those in wild-type or T-cell receptor δ knockout mice. Histopathological evaluation revealed that accumulation of macrophages and neutrophils at the peripheral deep dermis and subcutaneous tissue of the ulcers was enhanced in NKG2D-deficient mice. Rae-1 mRNA, which encodes an NKG2D ligand, was induced, and RAE-1 protein was detected immunohistochemically in fibroblasts and inflammatory cells in the dermis after reperfusion. RAE-1 expression was also increased in primary mouse fibroblasts treated with sodium arsenite. These results suggested that NKG2D ligand expression was induced by oxidative stress after I-R injury and support a putative role for this ligand in wound repair. Furthermore, the influx of NKG2D-positive cells at I-R sites may mitigate pressure ulcers via NKG2D-NKG2D ligand interactions.

Posttreatment with dexmedetomidine aggravates LPS-induced myocardial dysfunction partly via activating cardiac endothelial α2A-AR in mice

BackgroundDexmedetomidine (DEX) administered before or at 30 min after sepsis induction was reported to alleviate septic cardiomyopathy in experimental models. However, sepsis is a life-threatening organ dysfunction due to infection-induced dysregulated host response, whether DEX treatment in the presence of organ dysfunction affects septic cardiomyopathy is unknown. This study investigated the effect of DEX posttreatment on septic cardiomyopathy. MethodsMale wild-type and α2A-adrenergic receptor (AR) knockout mice were exposed to lipopolysaccharide (LPS) or cecal ligation puncture (CLP), and cultured cardiac endothelial cells were used. Mouse survival, myocardial function, inflammatory response and related signaling pathways were determined. ResultsDEX treatment at 6, 9 h after LPS challenge significantly reduced survival rate of LPS-challenged mice, especially at 9 h. DEX administered at 9 h after LPS injection or CLP significantly reduced survival in LPS or CLP-induced sepsis in wild-type mice, but not in α2A-AR knockout mice. LPS treatment for 20 h decreased the left ventricle + dp/dt, increased myocardial interleukin (IL)-1β and IL-6 concentrations as well as cardiac endothelial tumor necrosis factor (TNF)-α, vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1 expression, which were enhanced by DEX treated at 9 h after LPS injection in wild-type mice, but not in α2A-AR knockout mice. Furthermore, DEX posttreatment increased p38 phosphorylation, c-Fos nuclear translocation and VCAM-1 expression in LPS-treated cardiac endothelial cells, which were eliminated by α2A-AR knockout or PKC inhibitor. ConclusionsDEX posttreatment aggravates LPS-induced cardiac inflammation and myocardial dysfunction, at least in part, via activating cardiac endothelial α2A-AR-mediated PKC signal pathway.

TRPV4-induced Neurofilament Injury Contributes to Memory Impairment after High Intensity and Low Frequency Noise Exposures

ObjectiveExposure to high intensity, low frequency noise (HI-LFN) causes vibroacoustic disease (VAD), with memory deficit as a primary non-auditory symptomatic effect of VAD. However, the underlying mechanism of the memory deficit is unknown. This study aimed to characterize potential mechanisms involving morphological changes of neurons and nerve fibers in the hippocampus, after exposure to HI-LFN. MethodsAdult wild-type and transient receptor potential vanilloid subtype 4 knockout (TRPV4-/-) mice were used for construction of the HI-LFN injury model. The new object recognition task and the Morris water maze test were used to measure the memory of these animals. Hemoxylin and eosin and immunofluorescence staining were used to examine morphological changes of the hippocampus after exposure to HI-LFN. ResultsThe expression of TRPV4 was significantly upregulated in the hippocampus after HI-LFN exposure. Furthermore, memory deficits correlated with lower densities of neurons and neurofilament-positive nerve fibers in the cornu ammonis 1 (CA1) and dentate gyrus (DG) hippocampal areas in wild-type mice. However, TRPV4-/- mice showed better performance in memory tests and more integrated neurofilament-positive nerve fibers in the CA1 and DG areas after HI-LFN exposure. ConclusionTRPV4 up-regulation induced neurofilament positive nerve fiber injury in the hippocampus, which was a possible mechanism for memory impairment and cognitive decline resulting from HI-LFN exposure. Together, these results identified a promising therapeutic target for treating cognitive dysfunction in VAD patients.

Systematic analysis and comparison of peptide specificity and selectivity between their cognate receptors and noncognate decoys.

Protein-peptide interactions (PpIs) play an important role in cell signaling networks and have been exploited as new and attractive therapeutic targets. The affinity and specificity are two unity-of-opposite aspects of PpIs (and other biomolecular interactions); the former indicates the absolute binding strength between the peptide ligand and its cognate protein receptor in a PpI, while the latter represents the relative recognition selectivity of the peptide ligand for its cognate protein receptor in a PpI over those noncognate decoys that could be potentially encountered by the peptide in cell. Although the PpI binding affinity has been widely investigated over the past decades, the peptide recognition specificity (and selectivity) still remains largely unexplored to date. In this study, we classified PpI specificity into three types: (i) class-I specificity: peptide selectivity for its cognate wild-type protein receptor over the noncognate mutant decoys of this receptor, (ii) class-II specificity: peptide selectivity for its cognate protein receptor over other noncognate decoys that are homologous with this receptor, and (iii) class-III specificity: peptide selectivity for its cognate protein receptor over other noncognate decoys that are the cognate receptors of other peptides. We performed affinity and selectivity analysis for the three types of PpI specificity and revealed that the PpIs generally exhibit a moderate or modest specificity; peptide selectivity increases in the order: class-I < class-II < class-III. All the three types of PpI specificity were observed to have no statistically significant correlation with peptide length and hydrophobicity, but the class-I and class-II specificities can be influenced considerably by peptide secondary structures; the high specificity is preferentially associated with ordered structure types as compared to undefined structure types. In addition, the mutation distribution (for class-I specificity), sequence conservation (for class-II specificity), and structural similarity (for class-III specificity) seem also to address effects on peptide selectivity.

Extensive diet-induced atherosclerosis in scavenger receptor class B type 1-deficient mice is associated with substantial leukocytosis and elevated vascular cell adhesion molecule-1 expression in coronary artery endothelium.

High levels of low density lipoprotein (LDL) cholesterol and low levels of high density lipoprotein (HDL) cholesterol are risk factors for cardiovascular disease. Mice that lack genes involved in the clearance of LDL from the bloodstream, such as the LDL receptor and apolipoprotein E, are widely used models of experimental atherosclerosis. Conversely, mice that lack the HDL receptor, scavenger receptor class B type I, and therefore have disrupted HDL functionality, also develop diet-inducible atherosclerosis but are a seldom-used disease model. In this study, we compared atherosclerosis and associated phenotypes in scavenger receptor class B type I knockout mice with those of wild type, LDL receptor knockout, and apolipoprotein E knockout mice after 20weeks of being fed an atherogenic diet containing sodium cholate. We found that while scavenger receptor class B type I knockout mice had substantially lower plasma cholesterol than LDL receptor and apolipoprotein E knockout mice, they developed atherosclerotic plaques with similar sizes and compositions in their aortic sinuses, and more extensive atherosclerosis in their descending aortas and coronary arteries. This was associated with elevated tumor necrosis factor alpha levels in scavenger receptor class B type I knockout mice compared to wild type and LDL receptor knockout mice, and lymphocytosis, monocytosis, and elevated vascular cell adhesion molecule expression in coronary artery endothelial cells compared to the other mice examined. We conclude that extensive atherosclerosis in arteries that are not generally susceptible to atherosclerosis in scavenger receptor class B type I knockout mice is driven by factors in addition to hypercholesterolemia, including inflammation, dysregulation of the immune system and increased sensitivity of endothelial cells in arteries that are normally resistant to atherosclerosis. Scavenger receptor class B type I knockout mice fed a cholate containing atherogenic diet may prove to be a useful model to study mechanisms of atherosclerosis and evaluate treatments that rely on intact LDL clearance pathways.

Targeting Ligand Independent Tropism of siRNA-LNP by Small Molecules for Directed Therapy of Liver or Myeloid Immune Cells.

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild-type (WT) and low-density lipoprotein receptor (Ldlr)-/- mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high-density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA-LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA-LNP by the BLT1i in Ldlr-/- mice by in vivo imaging and discover an induction of specific uptake-related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA-LNP induced RNA-binding and transport-related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA-LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA-LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

PS-BPB08-2: DEVELOPMENT OF NON-PEPTIDE TYPE OF ANGIOTENSIN II TYPE 1 RECEPTOR LIGAND AS A BIASED AGONIST

Objective: Peptide type of angiotensin II (Ang II) type 1 (AT1) receptor ligand as a biased agonist antagonized Ang II-stimulated G protein signaling, but stimulated several kinase pathways. Here, we developed non-peptide type of AT1 receptor compound as a biased ligand. Design and method: We synthesized three non-peptide type of AT1 receptor ligands (Compound A, B and C) as candidates of biased ligands. These compounds were provided by Daiichi Sankyo Co. LTD. In vitro studies, inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) 1/2 activation were measured using AT1 receptor overexpressing cell system. We mutated the AT1 receptor (L112A, Q257A, Y292A and N295A receptors) and examined binding modes of receptor-ligand by competition binding study, and subsequently analyzed whether these ligands would induce IP production and ERK1/2 activation. Results: Kd of Compound A, B and C to AT1 wild-type receptor by competition binding study were 0.8, 21 and 48 nM, respectively. Kd of Compound A, B and C to L112A receptor were 37, 23 and 31 nM, respectively. Compound B and C decreased and increased IP production, respectively, whereas Compound A did not change IP production. Compound B and C, but not Compound A, activated ERK1/2. L112A had a key role of IP production. Conclusions: Compound A, B and C were a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, Compound B and C, but not Compound A, were agonists to ERK1/2 activation. Thus, we developed non-peptide type of AT1 receptor compound as a biased ligand.

Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors.

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.

Vitamin D3 eradicates Helicobacter pylori by inducing VDR-CAMP signaling.

Vitamin D3 [VitD3, 1,25 (OH)2D3] is known to have immunomodulatory and anti-microbial properties; however, its activity against Helicobacter pylori is unclear. In this study, we established H. pylori infection models in wild-type and VitD3 receptor (VDR) knockdown mice and analyzed the effects of VitD3 and their underlying mechanisms. VDR+/+ and VDR+/- mice were intragastrically infected with the H. pylori SS1 strain. After confirmation of H. pylori infection, mice were treated with different doses of VitD3. The infection levels in stomach tissues were quantified using the colony-forming assay, and the expression levels of the VDR and cathelicidin antimicrobial peptide (CAMP) in the gastric mucosa were analyzed by immunohistochemistry and western blotting. The gastric mucosa of VDR+/- mice was more susceptible to H. pylori colonization and had lower levels of VDR and CAMP expression than that of VDR+/+ mice. H. pylori infection upregulated VDR and CAMP expression in the stomach of both wild-type and mutant mice, and VitD3 treatment resulted in further increase of VDR and CAMP levels, while significantly and dose-dependently decreasing the H. pylori colonization rate in both mouse groups, without affecting blood calcium or phosphorus levels. Our data indicate that oral administration of VitD3 reduces the H. pylori colonization rate and upregulates VDR and CAMP expression in the gastric mucosa, suggesting a role for VitD3/VDR/CAMP signaling in the eradication of H. pylori in the stomach. These findings provide important insights into the mechanism underlying the anti-H. pylori activity of VitD3 and should be useful in the development of measures to eradicate H. pylori.

Insights Into the Differential Desensitization of α4β2 Nicotinic Acetylcholine Receptor Isoforms Obtained With Positive Allosteric Modulation of Mutant Receptors.

The development of highly efficacious positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChR) has proven useful in defining the ligand dependence of the conformational dynamics of α7 receptors. No such effective modulators are known to exist for the α4β2 nAChR of the brain, limiting our ability to understand the importance of desensitization for the activity profile of specific ligands. In this study, we used mutant β2 subunits that allowed the use of the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) to probe the desensitizing effects of nicotinic ligands on the two forms of α4β2 receptors; high sensitivity (HS) (two α4 and three β2 subunits) and low sensitivity (LS) (three α4 and two β2 subunits). A total of 28 different ligands of 8 different categories, based on activity and selectivity, were tested for their ability to induce TQS-sensitive desensitization of HS and LS α4β2 receptors. Results confirm that HS α4β2 receptor responses are strongly limited by desensitization, by at least an order of magnitude more so than the responses of LS receptors. The activation of α4β2 receptors by the smoking-cessation drugs cytisine and varenicline is strongly limited by desensitization, as is the activation of LS receptors by the HS-selective agonists 6-[5-[(2S)-2-Azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol dihydrochloride and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate. The evaluation of drugs previously identified as α7-selective agonists revealed varying patterns of α4β2 cross-desensitization that were predictive of the effects of these drugs on the activation of wild-type α4β2 receptors by acetylcholine, supporting the utility of TQS-sensitive receptors for the development of focused therapeutics. SIGNIFICANCE STATEMENT: To varying degrees, ligands regulate the balance of active and desensitized states of the two forms of the primary nAChR subtypes in brain. Using mutant beta subunits, an allosteric modulator can reverse ligand-induced desensitization, revealing the differential desensitization of the receptors by specific ligands. This study shows that drugs believed to be selective for therapeutic targets may cross-desensitize other targets and that, within a class of drugs, improved specificity can be achieved by using agents that reduce such cross-desensitization.

Macrocyclization of Quinazoline-Based EGFR Inhibitors Leads to Exclusive Mutant Selectivity for EGFR L858R and Del19.

Activating mutations in the epidermal growth factor receptor (EGFR) are frequent oncogenic drivers of non-small-cell lung cancer (NSCLC). The most frequent alterations in EGFR are short in-frame deletions in exon 19 (Del19) and the missense mutation L858R, which both lead to increased activity and sensitization of NSCLC to EGFR inhibition. The first approved EGFR inhibitors used for first-line treatment of NSCLC, gefitinib and erlotinib, are quinazoline-based. However, both inhibitors have several known off-targets, and they also potently inhibit wild-type (WT) EGFR, resulting in side effects. Here, we applied a macrocyclic strategy on a quinazoline-based scaffold as a proof-of-concept study with the goal of increasing kinome-wide selectivity of this privileged inhibitor scaffold. Kinome-wide screens and SAR studies yielded 3f, a potent inhibitor for the most common EGFR mutation (EGFR Del19: 119 nM) with selectivity against the WT receptor (EGFR: >10 μM) and the kinome.

Engineered Stem Cell Antibody Paired Evasion-2 (ESCAPE-2): Paired HSC Epitope Engineering and Direct Editing of Sickle Allele for Antibody-Mediated Autologous Hematopoietic Stem Cell Therapy Conditioning for the Treatment of Sickle Cell Disease

Introduction of the naturally-occurring Hb G-Makassar variation in the human betaglobin gene (HBB) through base editing to eliminate the polymerizing sickle protein, HbS, the major molecular driver for sickle cell disease (SCD), represents a promising new paradigm for the potential treatment of individuals with this disease. While several ex vivo delivery of gene editing technologies for SCD are advancing in the clinic, several challenges remain for this potentially transformative cell therapy, namely the requirement of myeloablative conditioning necessary for autologous hematopoietic stem cell transplant (HSCT) to be fully effective. Aiming to address this, we have developed a strategy whereby a monoclonal antibody (mAb) that binds to CD117, a critical receptor on HSPCs essential for viability, is coupled with multiplexed engineered HSCs (eHSCs). Our eHSCs are designed to both evade mAb binding and harbor the Makassar therapeutic edit. Our engineered stem cell antibody paired evasion (ESCAPE) strategy is designed to provide a non-genotoxic alternative to current conditioning regimens and a potential curative therapy for the treatment of SCD. In our ESCAPE-1 approach, we demonstrated that we could successfully engineer epitopes of the stem cell marker and receptor, CD117, by introducing missense mutations in the ligand binding domain that preserved normal CD117 function and prevented the binding of a high affinity antibody against wildtype (WT) CD117. We leveraged this selective advantage and screened guides that could install missense mutations that covered the entirety of the CD117 ectodomain and were compatible in a multiplexed edited HSC with a next-generation adenine base editor that could install the therapeutic Hb G-Makassar edit. We generated a library of CD117 variants via highly-efficient base editing of the CD117 gene, in mobilized peripheral blood CD34+ HSPCs and then counter-screened these variants against a library of mAbs that bound to wild-type CD117. We identified an antagonist mAb that interacts in the ligand binding domain of CD117, blocking normal SCF ligand binding to the WT receptor, but was unable to bind eHSCs. In vitro biological de-risking of this engineered epitope showed no significant changes in receptor function in response to SCF, or during in vitro differentiation and colony forming unit (CFU) assays when compared to WT CD117. Furthermore, we could demonstrate that this engineered epitope allowed for evasion of cytotoxicity in vitro when edited HSCs were exposed to increasing concentrations of the mAb. We next optimized the multiplex editing conditions in HSCs and achieved outcomes where all cells harboring a CD117 edit, also included at least one Makassar edit. We previously demonstrated that sickle cells harboring even a single Makassar edit exhibited reduced sickling when exposed to hypoxia in vitro. Subsequent Fc receptor silencing modifications were also optimized to reduce any mast cell degranulation that could be the result of antibody-based binding to Fc-receptor on mast cells. Altogether, our ESCAPE-2 strategy is compatible with the installation of the therapeutic Makassar edit that, when combined with a mAb as a conditioning agent, represents a promising potential alternative to busulfan-based myeloablative regimens in an autologous HSCT setting for the treatment of SCD.

Tumour necrosis factor plays a deleterious role in the pathogenesis of chikungunya virus infection.

Arthralgia is a hallmark of chikungunya virus (CHIKV) infection and can be very debilitating and associated with a robust local inflammatory response. Many pathophysiological aspects associated with the disease remain to be elucidated. Here, we describe a novel model of CHIKV infection in immunocompetent mice and evaluate the role of tumour necrosis factor in the pathogenesis of the disease. C57BL/6 wild type (WT) or TNF receptor 1 deficient (TNFR1-/- ) mice were inoculated with 1 × 106 PFU of CHIKV in the paw. Alternatively, etanercept was used to inhibit TNF in infected WT mice. Hypernociception, inflammatory and virological analysis were performed. Inoculation of CHIKV into WT mice induced persistent hypernociception. There was significant viral replication in target organs and local production of inflammatory mediators in early time-points after infection. CHIKV infection was associated with specific humoral IgM and IgG responses. In TNFR1-/- mice, there was a decrease in the hypernociception threshold, which was associated with a milder local inflammatory response in the paw but delayed viral clearance. Local or systemic treatment with etanercept reduced CHIKV-induced hypernociception. This is the first study to describe hypernociception, a clinical correlation of arthralgia, in immunocompetent mice infected with CHIKV. It also demonstrates the dual role of TNF in contributing to viral clearance but driving tissue damage and hypernociception. Inhibition of TNF may have therapeutic benefits but its role in viral clearance suggests that viral levels must be monitored in CHIKV-infected patients and that TNF inhibitors should ideally be used in combination with anti-viral drugs.

Discovery of Potent and Selective Inhibitors of Wild-Type and Gatekeeper Mutant Fibroblast Growth Factor Receptor (FGFR) 2/3.

Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.

Post-Transcriptional regulation of wild-type and variant androgen receptors during prostate cancer progression

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Anti-seizure mechanisms of midazolam and valproate at the β2(L51M) variant of the GABAA receptor

Genetic sequencing is identifying an expanding number of variants of GABAA receptors associated with human epilepsies. We identified a new de novo variant of the β2 subunit (β2L51M) of the inhibitory GABAA receptor associated with seizures. Our analysis determined the pathogenicity of the variant and the effects of anti-seizure medications. Our data demonstrates that the variant reduced cell surface trafficking and peak GABA-gated currents. Synaptic currents mediated by variant-containing receptors decayed faster than wild-type and single receptor currents showed that the variant shortened the duration of receptor activity by decreasing receptor open times. We tested the effects of the anti-seizure medications, midazolam, carbamazepine and valproate and found that all three enhance variant receptor surface expression. Additionally, midazolam restored receptor function by increasing single receptor active periods and synaptic current decay times towards wild-type levels. By contrast, valproate increased synaptic peak currents, event frequency and promoted synaptic bursting. Our study identifies a new disease-causing variant to the GABAA receptor, profiles its pathogenic effects and demonstrates how anti-seizure drugs correct its functional deficits.

Effects of maternal HF diet and absence of TRPC1 gene on mouse placental growth and fetal intrauterine growth retardation (IUGR)

Placental tissue intracellular calcium (Ca2+) regulates placental development and growth. Maternal high-fat diet (HFD) results in placental lipid accumulation, increased inflammation, reduced nutrient transport expression, and intrauterine growth restriction (IUGR). Currently, whether maternal HFD differentially affects placental and fetal growth and development under reduced Ca2+ influx is not yet known. We hypothesized that maternal HFD feeding decreases placental growth and development resulting in IUGR and that reduction of Ca2+ influx in the placenta worsens maternal HFD-induced placental dysfunction and IUGR. Three-week-old female B6129SF2/J wild type (WT) and transient receptor potential canonical 1 (TRPC1) protein deficient (KO) mice were fed normal fat (NF, 16 kcal % fat) and high fat (HF, 45 kcal % fat) diets for 12 weeks prior to mating with NF diet fed male mice. Fetuses and placentae were examined at mid- (D12) and late- (D18) gestation. At D12, maternal HFD had no effects on placental or fetal weight changes in WT and TRPC1 KO mice while absence of TRPC1 resulted in decreased placental and fetal weights. At D18, maternal HFD increased placental weights in both TRPC1 KO and WT mice, in part, by moderately increasing placental tissue triacylglyceride (TAG, P=.0632). At D12, mRNA expression of key placental growth factors including IGF1, PLGF, and VEGF were increased in WT compared to TRPC1 KO mice while IGF2 and VEGF mRNA expression were increased at D18. Results presented in our study demonstrated that maternal HFD increased placental weight, in part, due to increased lipid concentration resulting in IUGR and via an additive adverse effect of genotype and maternal HFD. Future studies are needed to determine the signaling mechanism underlying Ca2+ influx reduction-induced placental dysfunction and IUGR.

MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.