Rein Dijkema

ERβ: Identification and characterization of a novel human estrogen receptor

A novel estrogen receptor (hereinafter referred to as ERβ) was cloned using degenerate PCR primers. A comparison of the amino acid sequence of ERβ with the ‘classical’ ER (ERα) shows a high degree of conservation of the DNA‐binding domain (96%), and of the ligand‐binding domain (58%). In contrast, the A/B domain, the hinge region and the F‐domain are not conserved. Northern blot analysis revealed that ERβ is expressed in human thymus, spleen, ovary and testis. Transient transfections of an ERβ expression construct together with an ERE‐based reporter construct in CHO cells clearly demonstrated transactivation of ERβ by 17β‐estradiol. In addition, the ERα antagonist ICI‐164384 is a potent antagonist for ERβ as well. Interestingly, the level of transactivation by 17β‐estradiol is higher for ERα than for ERβ, which may reflect suboptimal conditions for ERβ at the level of the ligand, responsive element or cellular context.

Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella

A heterodisperse family of antigens, previously detected on sporozoites and merozoites of Eimeria tenella, has been localised to the microneme organelles within the sporozoite. Sequencing of genomic and cDNA clones shows that the gene for this antigen family contains 4 exons separated by 3 short (519, 226 and 156 nucleotides) intervening sequences and that the predicted polypeptide from the longest open reading frame has 4 structural domains. One of these contains 5 copies of the thrombospondin-like motif, previously identified in the partial sequence of the gene, which is conserved in a variety of molecules which have been demonstrated to have adhesive properties. A second domain of the polypeptide has strong similarity to a conserved region that occurs in another group of molecules which have adhesive properties, including the alpha subunits of several integrins, complement factor Bb and a number of extracellular matrix glycoproteins. Overall the antigen resembles the thrombospondin-related anonymous protein identified in the erythrocytic stage of Plasmodium falciparum. The structure of the gene supports a role for this microneme antigen in cell-cell or cell-matrix interactions.

X-ray structure of antistasin at 1.9 A resolution and its modelled complex with blood coagulation factor Xa.

The three‐dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Å resolution by X‐ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55‐residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two β‐strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N‐terminal domain being wedge‐shaped and the C‐terminal domain flat. Docking studies suggest that differences in domain shape enable the N‐terminal, but not C‐terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N‐terminal domain of antistasin, comprising residues 15‐17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C‐terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.

Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

Mutational analysis of antistasin, an inhibitor of blood coagulation factor xa derived from the mexican leech Haementeria officinalis

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.

Identification of Ligands for the Orphan Nuclear Receptor GCNF

HTS has been executed with a luciferase reporter assay in CHO cells stably expressing an ERa/GCNF chimer. Confirmed hits could be optimized some 10 to 100 fold. Partial agonists identified during the hit optimization process behaved as antagonists in a competition based variant of the reporter assay. The latter assay format was also used to identify some full antagonists by HTS. GCNF binding of agonists and antagonists was confirmed by GCNF/cofactor interactions studies in solution.