Thomas Giller

“Scaffold‐Hopping” by Topological Pharmacophore Search: A Contribution to Virtual Screening

A chemically advanced template search (CATS) based on topological pharmacophore models has been developed as a technique for virtual screening. This technique has successfully identified novel potent Ca(2+) antagonists (such as 2) that have a similar activity to 1 (a known T-channel blocking agent) in a library of several hundred thousand compounds on the basis of a correlation vector representation.

A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor

We have identified the human counterpart of the corticotropin-releasing factor receptor subtype 2beta. Its functional response to human urocortin was demonstrated after stable expression in HEK-293 cells. The receptor was also shown to bind sauvagine, corticotropin-releasing factor and urocortin. In contrast to rodents, the human CRF(2beta) receptor is only weakly expressed in heart and skeletal tissues, where the CRF(2alpha) isoform is predominant. Moreover, we have identified additional mRNAs of the CRF(2beta) type which are probably a consequence of aberrant splicing events.

Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species

Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin-Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.

A new family of orphan G protein‐coupled receptors predominantly expressed in the brain1

The cloning of a cDNA encoding a G protein‐coupled receptor homologous to the endothelin type B receptor, but unable to bind endothelin, was recently reported and termed ETBR‐LP. We report here the isolation of a human cDNA encoding a receptor that is highly related to ETBR‐LP and which was therefore termed ETBR‐LP‐2. Comparison of the two amino acid sequences revealed 68% overall homology and 48% identity. As is the case for ETBR‐LP, the new receptor is strongly expressed in the human central nervous system (e.g. in cerebellar Bergmann glia, cerebral cortex, internal capsule fibers). Membranes of HEK‐293 cells stably expressing ETBR‐LP‐2 did not bind endothelin‐1, endothelin‐2, endothelin‐3, bombesin, cholecystokinin‐8 or gastrin‐releasing peptide.

Human napsin A: expression, immunochemical detection, and tissue localization

A novel aspartic proteinase, called napsin, has recently been found in human and mouse. Due to high similarity with cathepsin D a structural model of human napsin A could be built. Based on this model a potential epitope SFYLNRDPEEPDGGE has been identified, which was used to immunize rabbits. The resulting antibody was employed in monitoring the expression of recombinant human napsin A in HEK293 cell line. Western blot analysis confirmed the specificity of the antibody and showed that human napsin A is expressed as a single chain protein with the molecular weight of approximately 38 kDa. Immunohistochemical studies revealed high expression levels of napsin A in human kidney and lung but low expression in spleen.

Discovery of Ro 48-5695: A potent mixed endothelin receptor antagonist optimized from bosentan

Abstract Implementation of a pyridylcarbamoyl group and an isopropylpyridylsulfonamide substituent as key components in the scaffold of Bosentan resulted in the identification of the potent orally active endothelin receptor antagonist Ro 48-5695. It shows affinities for ETA and ETB receptors in the low nanomolar range and high functional antagonistic potency in vitro.

ABSENCE OF ETB-MEDIATED CONTRACTION IN PIEBALD-LETHAL MICE

Activation of the endothelin (ET) ET(B) receptor can mediate opposite effects, endothelium-dependent vasodilation but also direct vasoconstriction. So far one gene encoding an ET(B) receptor has been identified and associated with endothelium-dependent relaxation. It has been suspected that the presence of another ET(B) gene could explain ET(B)-mediated contraction. The goal of the present study was to evaluate in Piebald-lethal (s[1]) mice, a naturally occurring mutant with deletion of the known ET(B) receptor gene, whether ET(B) receptor-mediated constriction is lost. Piebald-lethal (s[1]) mice, in contrast to control mice, completely lacked ET(B) specific ligand binding. The pressor effect of the ET(B) receptor selective agonist sarafotoxin S6c was completely absent. In vitro, contraction of stomach strips induced by sarafotoxin S6c was also abolished in Piebald-lethal (s[1]) mice. These results demonstrate the responsibility of the known ET(B) receptor gene in ET(B)-mediated constriction.

Detection of immunoreactive napsin A in human urine

Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.

Cloning, expression and functional characterization of rat napsin.

A full-length cDNA clone coding for rat napsin was identified by homology search of the ZooSeq rat EST database (Incyte). Northern blot analysis revealed high expression of napsin mRNA transcripts in kidney, lung and spleen. Western blot analysis showed that rat napsin is expressed in kidney as a 50-kDa, highly glycosylated, monomeric protein. Lysates prepared from human embryonic kidney cells (HEK293) transfected with rat napsin showed increased enzymatic activity which was inhibited by pepstatin.

Cloning and characterization of marmoset renin: comparison with human renin.

The poor interspecies conservation of the renin-angiotensin system prevents the use of nonprimate in vivo models to test renin inhibitors. Thus the small New-World monkey marmoset is used in many instances as a model. However, large differences between the potencies of renin inhibitors as measured in human and marmoset plasma were observed. To understand this phenomenon, we cloned marmoset renin and angiotensinogen. They were highly homologous to their human counterparts, except for a six-residue deletion in the marmoset renin propeptide. Human and marmoset recombinant renins were found in vitro to display comparable activities, suggesting that the observed differences in plasma apparent affinity of inhibitors could be due to different plasma protein binding of the inhibitors.