High-affinity Antagonist Research Articles

Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence.

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

M1 muscarinic receptors are necessary for retrieval of remote context fear memory

Several studies have investigated the transition of consolidation of recent memory to remote memory in aversively motivated tasks, such as contextual fear conditioning (CFC) and inhibitory avoidance (IA). However, the mechanisms that serve the retrieval of remote memories, has not yet been fully understood. Some evidences suggest that the central cholinergic system appears be involved in the modulation of these processes. Therefore, the present study aimed to investigate the effects of a pre-test administration of dicyclomine, a high-affinity M1 muscarinic receptor antagonist, on the retrieval of remote memories in fear conditioning and IA tasks. Male Wistar rats were trained, and after 1 or 28days, the rats received dicyclomine (16 or 32mg/kg, intraperitoneally, i.p.) and were tested in CFC, tone fear conditioning (TFC) and IA tasks. At both time intervals, 32mg/kg dicyclomine induced impairment of CFC. In TFC task only the performance of the rats 28days after training was impaired. The IA task was not affected in any of the studied intervals. These findings suggest a differential contribution of muscarinic receptors on recent and remote memories retrieval revealing a more generalized role in remote memory.

The 5-HT6 receptor antagonist idalopirdine potentiates the effects of donepezil on gamma oscillations in the frontal cortex of anesthetized and awake rats without affecting sleep-wake architecture

The 5-HT6 receptor is a promising target for cognitive disorders, in particular for Alzheimer's disease (AD). The high affinity and selective 5-HT6 receptor antagonist idalopirdine (Lu AE58054) is currently in development for mild-moderate AD as adjunct therapy to acetylcholinesterase inhibitors (AChEIs). We studied the effects of idalopirdine alone and in combination with the AChEI donepezil on cortical function using two in vivo electrophysiological methods. Neuronal network oscillations in the frontal cortex were measured during electrical stimulation of the brainstem nucleus pontis oralis (nPO) in the anesthetized rat and by an electroencephalogram (EEG) in the awake, freely moving rat. In conjunction with the EEG study, we investigated the effects of idalopirdine and donepezil on sleep-wake architecture using telemetric polysomnography. Idalopirdine (2 mg/kg i.v.) increased gamma power in the medial prefrontal cortex (mPFC) during nPO stimulation. Donepezil (0.3 and 1 mg/kg i.v.) also increased cortical gamma power and pretreatment with idalopirdine (2 mg/kg i.v.) potentiated and prolonged the effects of donepezil. Similarly, donepezil (1 and 3 mg/kg s.c.) dose-dependently increased frontal cortical gamma power in the freely moving rat and pretreatment with idalopirdine (10 mg/kg p.o.) augmented the effect of donepezil 1 mg/kg. Analysis of the sleep-wake architecture showed that donepezil (1 and 3 mg/kg s.c.) dose-dependently delayed sleep onset and decreased the time spent in both REM and non REM sleep stages. In contrast, idalopirdine (10 mg/kg p.o.) did not affect sleep-wake architecture nor the effects of donepezil. In summary, we show that idalopirdine potentiates the effects of donepezil on frontal cortical gamma oscillations, a pharmacodynamic biomarker associated with cognition, without modifying the effects of donepezil on sleep. The increased cortical excitability may contribute to the procognitive effects of idalopirdine in donepezil-treated AD patients.

Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging.

Serotonin-gated ionotropic 5-HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the "setrons") that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-11C)-N-granisetron ([11C]2) through N-alkylation with [11C]CH3I, respectively. Both compounds [18F]15 (chemical and radiochemical purity >95%, specific activity 41 GBq/μmol) and [11C]2 (chemical and radiochemical purity ≥99%, specific activity 170 GBq/μmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5-HT3 receptors in vivo.

Inhibition of tachykinin NK1 receptor using aprepitant induces apoptotic cell death and G1 arrest through Akt/p53 axis in pre-B acute lymphoblastic leukemia cells

Increasing number of genetic and cancer biology studies indicated a prominent role for tachykinin NK1 receptor (NK1R) in cancer cell growth and survival. Considering the fact that neoplastic lymphoid precursors in acute lymphoblastic leukemia (ALL) carry a three- to four-fold NK1R expression as compared to normal lymphocytes, using NK1R antagonist seems to be noteworthy in the treatment of ALL patients. In this study, we found that inhibition of NK1R with aprepitant, a selective high-affinity antagonist of the human NK1R, exerts cytotoxic and anti-proliferative effects against pre-B ALL-derived Nalm-6 cells either as single drug or in combination with doxorubicin. Our data showed that treatment of the cells with the inhibitor resulted in apoptotic cell death, at least partly, through abrogation of PI3K/Akt pathway, as revealed by the reduction of phospho/total Akt ratio. In agreement with the inhibitory effect on Akt, we also found that aprepitant increased the expression level of p21 and p27, which in turn leads to the induction of G1 cell cycle arrest. Overall, this study recommends mechanistic pathways by which inhibition of NK1R can augment apoptotic cell death through a plausible p53-dependent pathway rather than NF-κB-depended mechanism in pre-B ALL cells; however, further studies are needed to better characterize the application of NK1R inhibition in clinical cancer treatment.

Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia.

Various types of non-neuronal cells, including tumors, are able to produce acetylcholine (ACh), which acts as an autocrine/paracrine growth factor. T lymphocytes represent a key component of the non-neuronal cholinergic system. T cells-derived ACh is involved in a stimulation of their activation and proliferation, and acts as a regulator of immune response. The aim of the present work was to summarize the data about components of cholinergic machinery in T lymphocytes, with an emphasis on the comparison of healthy and leukemic T cells. Cell lines derived from acute lymphoblastic leukemias of T lineage (T-ALL) were found to produce a considerably higher amount of ACh than healthy T lymphocytes. Additionally, ACh produced by T-ALL is not efficiently hydrolyzed, because acetylcholinesterase (AChE) activity is drastically decreased in these cells. Up-regulation of muscarinic ACh receptors was also demonstrated at expression and functional level, whereas nicotinic ACh receptors seem to play a less important role and not form functional channels in cells derived from T-ALL. We hypothesized that ACh over-produced in T-ALL may act as an autocrine growth factor and play an important role in leukemic clonal expansion through shaping of intracellular Ca2+ signals. We suggest that cholinergic machinery may be attractive targets for new drugs against T-ALL. Specifically, testing of high affinity antagonists of muscarinic ACh receptors as well as antagomiRs, which interfere with miRNAs involved in the suppression of AChE expression, may be the first choice options.

Aspartate‐Based CXCR4 Chemokine Receptor Binding of Cross‐Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes

The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists.

Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists.

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.

Identification of 2-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)tricyclo[3.3.1.13,7]decane-2-carboxylic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.

Pharmacokinetics, pharmacodynamics and safety of CEP-26401, a high-affinity histamine-3 receptor antagonist, following single and multiple dosing in healthy subjects.

CEP-26401 is a novel orally active, brain-penetrant, high-affinity histamine H3 receptor (H3R) antagonist, with potential therapeutic utility in cognition enhancement. Two randomized, double-blind, placebo-controlled dose escalation studies with single (0.02 to 5 mg) or multiple administration (0.02 to 0.5 mg once daily) of CEP-26401 were conducted in healthy subjects. Plasma and urine samples were collected to investigate CEP-26401 pharmacokinetics. Pharmacodynamic endpoints included a subset of tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and nocturnal polysomnography. Population pharmacokinetic-pharmacodynamic modeling was conducted on one CANTAB and one polysomnography parameter of interest. CEP-26401 was slowly absorbed (median tmax range 3-6 hours) and the mean terminal elimination half-life ranged from 24-60 hours. Steady-state plasma concentrations were achieved within six days of dosing. CEP-26401 exhibits dose- and time-independent pharmacokinetics, and renal excretion is a major elimination pathway. CEP-26401 had a dose-dependent negative effect on sleep, with some positive effects on certain CANTAB cognitive parameters seen at lower concentrations. The derived three compartment population pharmacokinetic model, with first-order absorption and elimination, accurately described the available pharmacokinetic data. CEP-26401 was generally well tolerated up to 0.5 mg/day with most common treatment related adverse events being headache and insomnia. Further clinical studies are required to establish the potential of low-dose CEP-26401 in cognition enhancement.

Preclinical Evaluation of a P2X7 Receptor-Selective Radiotracer: PET Studies in a Rat Model with Local Overexpression of the Human P2X7 Receptor and in Nonhuman Primates.

The P2X7 receptor (P2X7R) orchestrates neuroinflammation, and this is the basis for an increased interest in the development of antagonists inhibiting P2X7R function in the brain. This study provides the preclinical evaluation of (11)C-JNJ-54173717, a PET tracer for P2X7R in both rats and nonhuman primates. (11)C-JNJ-54173717 is a high-affinity radiotracer for the human P2X7R (hP2X7R). Biodistribution and radiometabolite studies were performed. Viral vectors encoding either enhanced green fluorescent protein-hP2X7R or 3flag-hP2X7R were engineered and validated in cell culture. hP2X7R was regionally overexpressed in the rat striatum after stereotactic injection of viral vectors. Dynamic small-animal PET studies were performed in vector-injected rats and in healthy monkeys using (11)C-JNJ-54173717. The affinity of JNJ-54173717 was 1.6 ± 0.1 nM in a rat cortex P2X7R membrane binding assay. In a functional assay at the recombinant human and rat P2X7R orthologs, the half maximal inhibitory concentration (IC50) of JNJ-54173717 was 4.2 ± 0.01 nM and 7.6 ± 0.01 nM, respectively. The rat biodistribution study showed that (11)C-JNJ-54173717 crossed the blood-brain barrier and was cleared from plasma mainly via the hepatobiliary pathway. A polar radiometabolite was found in rat plasma. No radiometabolites were detected in rat brain. Dynamic small-animal PET showed binding of (11)C-JNJ-54173717 in the striatum expressing hP2X7R, with rapid washout from the noninjected control striatum and other brain regions. Likewise, (11)C-JNJ-54173717 PET signal was blocked by a chemically distinct P2X7R ligand, indicating specific binding to P2X7R in the monkey brain. JNJ-54173717 is a high-affinity P2X7R antagonist. An animal rat model stably expressing hP2X7R was developed and validated, identifying favorable characteristics for (11)C-JNJ-54173717 as a PET radioligand for in vivo visualization of hP2X7R. (11)C-JNJ-54173717 selectively visualized P2X7R in the monkey brain, and this radioligand will be further evaluated in a clinical setting to study P2X7R expression levels in neurodegenerative disorders.

Therapeutic Effect of Recombinant Mutated Interleukin 11 in the Mouse Model of Tuberculosis.

Earlier we demonstrated that blocking of interleukin 11 (IL-11) by systemic administration of anti-IL-11 antibodies attenuates severity of Mycobacterium tuberculosis infection in mice. The substitution W147A in the IL-11 molecule creates the form of cytokine capable to disrupt gp130/IL11R signaling complex formation, thus serving as a high-affinity specific antagonist of IL-11-mediated signaling. We hypothesized that this mutant form of IL-11 may serve as an effective tool for inhibition of native IL-11 activity in vivo. We established the recombinant W147A mutant form of IL-11 in an optimized Escherichia coli expression system and administered it as the aerosol in the lungs of M. tuberculosis-susceptible I/St mice infected with M. tuberculosis Our results show that this therapeutic approach markedly inhibits tuberculous inflammation in lungs, increases the survival time of infected animals, and decreases expression of key inflammatory factors at the RNA and protein levels. These findings are a step toward clinical evaluation of the anti-IL-11 therapy for tuberculosis.

BDNF acting in the hypothalamus induces acute pressor responses under permissive control of angiotensin II

Brain-derived neurotrophic factor (BDNF) expression increases in the paraventricular nucleus of the hypothalamus (PVN) during hypertensive stimuli including stress and hyperosmolarity, but its role in PVN cardiovascular regulatory mechanisms is unclear. Chronic BDNF overexpression in the PVN has been shown to elevate sympathetic tone and blood pressure in part by modulating central angiotensin (Ang) II mechanisms. However, the cardiovascular effects of short-term increases in PVN levels of BDNF and the mechanisms governing them are unknown. Therefore, we investigated whether acute BDNF microinjections into the PVN of conscious and anesthetized Sprague-Dawley rats induce blood pressure elevations and whether Ang II signaling is involved in these hypertensive responses. In conscious rats, unilateral BDNF (12.5ng) microinjections into the PVN increased mean arterial pressure (MAP) by 27±1mmHg (P<0.001 vs vehicle), which was significantly attenuated by intracerebroventricular infusion of the Ang II-type-1 receptor (AT1R) antagonist losartan and by ganglionic blockade with intravenous hexamethonium infusion. In anesthetized rats, unilateral PVN microinjection of BDNF increased MAP by 31±4mmHg (P<0.001 vs vehicle), which was prevented by PVN microinjection pretreatments with the high-affinity BDNF receptor TrkB antagonist ANA-12, losartan, the angiotensin converting enzyme inhibitor lisinopril, or by intravenous hexamethonium. Additional experiments in hypothalamic samples including the PVN revealed that BDNF-induced TrkB receptor phosphorylation was prevented by ANA-12 and losartan pretreatments. Collectively, these data indicate that BDNF acting within the PVN acutely raises blood pressure under permissive control of Ang II-AT1R mechanisms and therefore may play an important role in mediating acute pressor responses to hypertensive stimuli.

Pungent and Non-Pungent General Anesthetics Interact with TRPA1 via Distinct Binding Pocket in the Pore Domain Region

General anesthetics (GAs) suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile and intravenous GAs can excite peripheral sensory nerves to cause pain and irritation upon administration. Noxious GAs activate Transient Receptor Potential Ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms remain unknown. Here we show that in addition to activation by pungent GAs, a broad variety of noxious and non-noxious anesthetics inhibit agonist-evoked activation of mammalian but not drosophila TRPA1. Analysis of chimeric drosophila and mouse TRPA1 channels reveal that the 5th transmembrane domain (S5) is critical for anesthetic sensitivity. We identify two amino acids at the N-terminal S5 domain required for the inhibitory effects of GAs and introduction of these residues into dTRPA1 confers GA-induced block in these channels. Further, we show that activation by noxious GAs requires several amino acids in the S5, S6 and the first pore helix previously implicated in binding of the high-affinity antagonist, A-967079. Notably, isoflurane relieves A-967079-mediated inhibition consistent with a competitive interaction and in silico modeling predicts that pungent GAs occupy the A-967079 binding pocket. Thus inhibition and activation by GAs are governed by distinct amino acids in the pore domain region. These data reveal the mechanism by which TRPA1 discriminates between pungent and non-pungent GAs, and provide structural requirements for designing antagonists to counter the noxious properties of GAs.

N-terminally and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor.

Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here, we have characterized eight N-terminal truncations of human GIP(1-30)NH2 . COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with (125) I-labelled GIP(1-42), GIP(1-30)NH2 , GIP(2-30)NH2 or GIP(3-30)NH2 . GIP(1-30)NH2 displaced (125) I-GIP(1-42) as effectively as GIP(1-42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3-347 nM) with highest affinities for GIP(3-30)NH2 and GIP(5-30)NH2 (5-30)NH2 . Only GIP(1-30)NH2 (Emax 100% of GIP(1-42)) and GIP(2-30)NH2 (Emax 20%) were agonists. GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and Schild plot analyses identified GIP(3-30)NH2 and GIP(5-30)NH2 as competitive antagonists (Ki 15 nM). GIP(3-30) NH2 was a 26-fold more potent antagonist than GIP(3-42). Binding studies with agonist ((125) I-GIP(1-30)NH2 ), partial agonist ((125) I-GIP(2-30)NH2 ) and competitive antagonist ((125) I-GIP(3-30)NH2 ) revealed distinct receptor conformations for these three ligand classes. The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3-42) creates another naturally occurring, more potent, antagonist GIP(3-30)NH2 , which like GIP(5-30)NH2 , was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions.

The fast-off hypothesis revisited: A functional kinetic study of antipsychotic antagonism of the dopamine D2 receptor

Newer, “atypical” antipsychotics carry a lower risk of motor side-effects than older, “typical” compounds. It has been proposed that a ~100-fold faster dissociation from the dopamine D2 receptor (D2R) distinguishes atypical from typical antipsychotics. Furthermore, differing antipsychotic D2R affinities have been suggested to reflect differences in dissociation rate constants (koff), while association rate constants (kon) were assumed to be similar. However, it was recently demonstrated that lipophilic accumulation of ligand in the cell interior and/or membrane can cause underestimation of koff, and as high-affinity D2R antagonists are frequently lipophilic, this may have been a confounding factor in previous studies. In the present work, a functional electrophysiology assay was used to measure the recovery of dopamine-mediated D2R responsivity from antipsychotic antagonism, using elevated concentrations of dopamine to prevent the potential bias of re-binding of lipophilic ligands. The variability of antipsychotic kon was also reexamined, capitalizing on the temporal resolution of the assay. kon was estimated from the experimental recordings using a simple mathematical model assumed to describe the binding process. The time course of recovery from haloperidol (typical antipsychotic) was only 6.4- to 2.5-fold slower than that of the atypical antipsychotics, amisulpride, clozapine, and quetiapine, while antipsychotic kons were found to vary more widely than previously suggested. Finally, affinities calculated using our kon and koff estimates correlated well with functional potency and with affinities reported from radioligand binding studies. In light of these findings, it appears unlikely that typical and atypical antipsychotics are primarily distinguished by their D2R binding kinetics.

The Human Orexin/Hypocretin Receptor Crystal Structures.

The human orexin/hypocretin receptors (hOX1R and hOX2R) are G protein-coupled receptors (GPCRs) that mediate the diverse functions of the orexin/hypocretin neuropeptides. Orexins/hypocretins produced by neurons in the lateral hypothalamus stimulate their cognate GPCRs in multiple regions of the central nervous system to control sleep and arousal, circadian rhythms, metabolism, reward pathways, and other behaviors. Dysfunction of orexin/hypocretin signaling is associated with human disease, and the receptors are active targets in a number of therapeutic areas. To better understand the molecular mechanism of the orexin/hypocretin neuropeptides, high-resolution three-dimensional structures of hOX1R and hOX2R are critical. We have solved high-resolution crystal structures of both human orexin/hypocretin receptors bound to high-affinity antagonists. These atomic structures have elucidated how different small molecule antagonists bind with high potency and selectivity, and have also provided clues as to how the native ligands may associate with their receptors. The orexin/hypocretin receptor coordinates, now available to the broader academic and drug discovery community, will facilitate rational design of new therapeutics that modulate orexin/hypocretin signaling in humans.

Risperidone-induced skin rash.

Byline: Priya. Janardhana, Anil Kumar. Nagaraj, P. Basavanna Sir, Antipsychotic agents are known to cause adverse cutaneous reactions in approximately 5% of the individuals of which exanthematous eruptions, skin pigmentation changes, photosensitivity, urticarial, and pruritus are common. [sup][1] Risperidone, a benzisoxazole derivative is an atypical antipsychotic. It exhibits high-affinity antagonism at 5 HT [sub]2 and D [sub]2 receptors. It binds to alpha [sub]1 , alpha [sub]2 adrenergic receptors to a lesser extent. It has no affinity to cholinergic receptors. [sup][2] It is known to cause various adverse effects out of which cutaneous reaction is very rare. A thorough literature search revealed a few articles of risperidone-induced urticaria, angioneurotic edema, and photosensitivity reactions. We herein present a case of rare adverse effect, risperidone-induced eczematous skin rash. Mr. R, a 20-year-old male presented with complaints of aggressive behavior, self-injurious behavior, delusions of persecution, and third person auditory hallucinations of 2 weeks duration. He had a 3 years history of paranoid schizophrenia, diagnosed as per International Classification of Diseases 10, and was on irregular treatment. The current exacerbation happened after about 6 months of partial remission. Detailed history revealed no previous drug or food allergies. The patient was not on any concomitant drug therapy when he reported though he had taken olanzapine intermittently in the past. The patient was started on oral risperidone 6 mg/day in divided doses and was instructed follow-up after 1 week, as the caretakers were not ready to admit him. When he reported back, he showed clinical improvement but complained of generalized pruritus. Examination revealed excoriation of skin. Dermatologist opinion was taken, and he was treated with terbinafine and chlorpheniramine after total and differential white blood cell count and absolute eosinophil count, which were within normal limits. Simultaneously, the dose of oral risperidone was reduced to 4 mg/day suspecting a drug-induced rash. The patient came back in a week with no improvement in the dermatological complaints but significant reduction in psychotic symptoms. Examination revealed scaling and excoriation of skin. The patient continued to use terbinafine, and the dose of risperidone was reduced to 2 mg/day. In a span of 1 week, the patient reported again with exacerbation of the dermatological symptoms and now showed generalized scaling of the skin, papules, and ulcers with serous discharge over the dorsum of the hand. Risperidone was stopped completely and the dermatologist prescribed hydrocortisone acetate ointment. Follow-up after 1 week, showed considerable recovery of the scaly lesions and it completely disappeared after 2 weeks of discontinuation of risperidone. Even hydrocortisone was stopped after 2 weeks, and there were no fresh skin lesions during subsequent follow-up. …

ID: 182: Characterization of IL22RA2, a risk locus for multiple sclerosis

IL22RA2 (IL-22 binding protein, IL-22BP) is a soluble high-affinity antagonist of the cytokine IL-22, and has emerged recently with genome-wide significance as a risk locus for multiple sclerosis (MS). We performed a single nucleotide polymorphism (SNP) screen to identify putative causative variants, and identified a non-synonymous (NS) SNP significantly associated with MS in a multi-center screen including over 16,000 MS patients and controls. In order to evaluate if this SNP affected IL22RA2 protein processing and secretion, a site-directed mutagenesis was carried out, and both constitutive CMV-promoter-based and Tet-Express inducible vectors expressing C-terminally FLAG-tagged wild-type and mutant proteins were generated. Vectors were transfected in HEK293 cells and the intracellular and secreted protein fractions were analyzed by western blot. This showed that this NS SNP modulates secretion levels of the protein, with lower levels observed for the mutant than for the wild-type protein. As part of this work we performed a characterization of the N-glycosylation status of the recombinant protein and demonstrate that it contains uniquely high-mannose chains (43 kD) which are converted into complex sugar chains in the secreted form augmending its Mr to 50 kD. We have also investigated transcription, secretion and N-glycosylation of IL22RA2 produced by monocyte-derived dendritic cells and have characterized in detail the intracellular and secreted forms of the natural protein. We will present both a detailed overview of the genetic study and of the characterization of recombinant and natural IL22RA2.

Improvement of Outcome Measures of Dry Eye by a Novel Integrin Antagonist in the Murine Desiccating Stress Model.

We investigated the effects of GW559090, a novel, competitive, and high-affinity α4 integrin antagonist, in a murine model of dry eye. Through interaction with vascular cell adhesion molecule 1 (VCAM-1) and fibronectin α4β1 integrin is involved in leukocyte trafficking and activation. Female C57BL/6 mice, aged 6 to 8 weeks, were subjected to desiccating stress (DS). Bilateral topical twice daily treatment with GW559090 was compared to vehicle-treated controls. Treatment was initiated at the time of DS induction. Treatment effects were assessed on corneal staining with Oregon Green Dextran (OGD) and expression of inflammatory markers in ocular surface tissues by real time PCR. Dendritic cell activation was measured in draining cervical lymph nodes (CLN) by flow cytometry. Separate groups of mice received GW559090 subcutaneously to evaluate the effects of systemic administration on corneal staining and cells in CLN. Topical GW559090 significantly reduced corneal uptake of OGD compared to vehicle-treated disease controls in a dose-dependent manner (1, 3, 10, and 30 mg/mL) with 30 mg/mL showing the greatest reduction in OGD staining. When administered topically, corneal expression of IL-1α, matrix metalloproteinase (MMP)-9, chemokine ligand 9 (CXCL9), and TGF-β1 was reduced in GW559090-treated eyes. Topical treatment with GW559090 decreased dendritic cell activation in lymph nodes. The effects on corneal staining and cellular composition in CLN were not reproduced by systemic administration of GW559090, suggestive of a local role for integrin antagonism in the treatment of dry eye. The novel α4 integrin antagonist, GW559090, improved outcome measures of corneal staining and ocular surface inflammation in this murine model of dry eye. These results indicate the potential of this novel agent for the treatment of dry eye disease.