Kirsten Andersen

Computer-aided discovery of aromatic l-α-amino acids as agonists of the orphan G protein-coupled receptor GPR139.

GPR139 is an orphan G protein-coupled receptor expressed mainly in the central nervous system. We developed a pharmacophore model based on known GPR139 surrogate agonists which led us to propose aromatic-containing dipeptides as potential ligands. Upon testing, the dipeptides demonstrated agonism in the Gq pathway. Next, in testing all 20 proteinogenic l-α-amino acids, L-tryptophan and l-phenylalanine were found to have EC50 values of 220 and 320 μM, respectively, making them the first putative endogenous agonists for GPR139.

Inheritance of Resistance To Heterodera Avenae in Barley

Tests of F2 barley populations showed that the gene for resistance to race 1 of Heterodera avenae and the gene for resistance to both race 1 and race 2 are located at different loci.

Novel Agonist Bioisosteres and Common Structure-Activity Relationships for The Orphan G Protein-Coupled Receptor GPR139

GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity and Parkinson’s disease. The two aromatic amino acids L-Trp and L-Phe have been proposed as putative endogenous agonists, and three structurally related benzohydrazide, glycine benzamide, and benzotriazine surrogate agonist series have been published. Herein, we assayed 158 new analogues selected from a pharmacophore model, and identified 12 new GPR139 agonists, containing previously untested bioisosteres. Furthermore, we present the first combined structure-activity relationships, and a refined pharmacophore model to serve as a rationale for future ligand identification and optimization.

The orphan G protein-coupled receptor GPR139 is activated by the peptides: Adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW

Abstract GPR139 is an orphan G protein‐coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l‐tryptophan and l‐phenylalanine (EC50 values of 220 &mgr;M and 320 &mgr;M, respectively), as well as di‐peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle “similar targets bind similar ligands”, we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and &agr;‐ and &bgr;‐melanocyte stimulating hormone (&agr;‐MSH and &bgr;‐MSH) on CHO‐k1 cells stably expressing the human GPR139 in a Fluo‐4 Ca2+‐assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low micromolar range. Moreover, we found that peptides consisting of nine or ten N‐terminal residues of &agr;‐MSH activate GPR139 in the submicromolar range. &agr;‐MSH1‐9 was found to correspond to the product of a predicted cleavage site in the pre‐pro‐protein pro‐opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, &agr;‐MSH, &bgr;‐MSH, the conserved core motif HFRW as well as a potential endogenous peptide &agr;‐MSH1‐9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides. HighlightsUsing GPCRdb we found that the binding cavity of GPR139 is 49% similar to MC4R.ACTH, &agr;‐MSH and &bgr;‐MSH activate GPR139 in the low &mgr;M‐range.We predicted a novel possible cleavage site in POMC leading to the peptide &agr;‐MSH1‐9.&agr;‐MSH1‐9 activates GPR139 in the high nM range.

The GPR139 reference agonists 1a and 7c, and tryptophan and phenylalanine share a common binding site

GPR139 is an orphan G protein-coupled receptor expressed in the brain, in particular in the habenula, hypothalamus and striatum. It has therefore been suggested that GPR139 is a possible target for metabolic disorders and Parkinson’s disease. Several surrogate agonist series have been published for GPR139. Two series published by Shi et al. and Dvorak et al. included agonists 1a and 7c respectively, with potencies in the ten-nanomolar range. Furthermore, Isberg et al. and Liu et al. have previously shown that tryptophan (Trp) and phenylalanine (Phe) can activate GPR139 in the hundred-micromolar range. In this study, we produced a mutagenesis-guided model of the GPR139 binding site to form a foundation for future structure-based ligand optimization. Receptor mutants studied in a Ca2+ assay demonstrated that residues F1093×33, H1875×43, W2416×48 and N2717×38, but not E1083×32, are highly important for the activation of GPR139 as predicted by the receptor model. The initial ligand-receptor complex was optimized through free energy perturbation simulations, generating a refined GPR139 model in agreement with experimental data. In summary, the GPR139 reference surrogate agonists 1a and 7c, and the endogenous amino acids l-Trp and l-Phe share a common binding site, as demonstrated by mutagenesis, ligand docking and free energy calculations.

The Inhibitory Effect of Natural Products on Protein Fibrillation May Be Caused by Degradation Products – A Study Using Aloin and Insulin

Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also complicates the manufacturing and use of protein drugs. As a case study, the inhibitory activity of the natural compound aloin against insulin fibrillation was investigated. Based on Thioflavin T assays, high-performance liquid chromatography and transmission electron microscopy it was found that a degradation product of aloin, formed over weeks of storage, was able to significantly inhibit insulin fibrillation. The activity of the stored aloin was significantly reduced in the presence of small amounts of sodium azide or ascorbic acid, suggesting the active compound to be an oxidation product. A high-performance liquid chromatography method and a liquid chromatography-mass spectrometry method were developed to investigate the degradation products in the aged aloin solution. We found that the major compounds in the solution were aloin A and aloin B. In addition, 10-hydroxy aloin and elgonica dimers were detected in smaller amounts. The identified compounds were isolated and tested for activity by means of Thioflavin T assays, but no activity was observed. Thus, the actual fibrillation inhibitor is an as yet unidentified and potentially metastable degradation product of aloin. These results suggest that degradation products, and in particular oxidation products, are to be considered thoroughly when natural products are investigated for activity against protein fibrillation.

Longevity of Cereal Seeds

Abstract Cereal crops were harvested at different stages of maturity and threshed immediately or after drying in the field. The threshed seeds were dried and stored at different moisture contents. Low moisture content increased seed longevity, but threshing at a premature stage caused some damage. Procedures for handling seeds in the Danish genebank are reported.

Heterodera Avenae: Virulence and Resistance

Cereals and grasses are damaged by several species of cyst forming nematodes. One species, Heterodera avenae Woll. (the oat nematode = cereal root eelworm), is common in countries in Northwest Europe, and is found in Canada, North Africa, India and Australia. Control is possible by use of resistant cultivars, but different pathotypes are found, and the resistance of a cultivar must correspond to the pathotype of the nematode.