Structure-based exploration and pharmacological evaluation of N-substituted piperidin-4-yl-methanamine CXCR4 chemokine receptor antagonists.

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects.

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.

Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae.

Expression of the BAP3 gene of Saccharomyces cerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium. This induction is mediated via an upstream activating sequence (called UAS(aa)) in the BAP3 promoter, and dependent on Stp1p, a nuclear protein with zinc finger domains, suggesting that Stp1p is a transcription factor involved in BAP3 expression. In this paper, we show that Stp2p, a protein with considerable similarity to Stp1p, is also involved in the induction of BAP3 expression. To gain more insight into the roles of STP1 and STP2, we have overexpressed both Stp1p and Stp2p in yeast cells. Gel shift assays with the UAS(aa)as a probe show that the UAS(aa)can form two major complexes. One complex is dependent on Stp2p overexpression and the other is formed independently of STP1 or STP2, suggesting that the UAS(aa)is also bound by another factor. Here we show that the other factor is Abf1p, which binds specifically to the UAS(aa)of BAP3.

Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae.

The BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high-affinity permease for branched-chain amino acids. In this paper, we show that, like BAP2, the expression of the BAP3 gene in S. cerevisiae is induced by the addition of branched-chain amino acids to the medium. Unexpectedly, most other naturally occurring L-amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3. The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre-tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1 - mutant. The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a Deltaleu3 mutant. By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from - 418 to - 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p-dependent induction of BAP3 expression by (most) amino acids. We have therefore named this sequence UASaa (amino acid-dependent upstream activator sequence). Neither Stp1p nor Leu3p appear to bind to the UASaa, at least in vitro, as judged from gel retardation assays. Sequences similar to the UASaa can be found in the promoters of BAP2, PTR2 and TAT1; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.

Signalling pathways leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast.

Addition of glucose to yeast cells growing on less preferred carbon sources triggers profound changes in the expression levels of several genes. This paper focuses on the signal transduction pathways leading to transcriptional activation of the glycolysis in Saccharomyces cerevisiae during the transition from respiratory to fermentative growth conditions. To this end, we studied the transcriptional regulation of glycolytic genes (PFK1, PYK1 and PDC), one gluconeogenic gene (FBP1) and the two genes encoding the 6-phosphofructo-2-kinase isoenzymes (PFK26 and PFK27) during this transition. The results of experiments using glycolysis mutants, different fermentable carbon sources and 2-deoxyglucose indicate that proper transcriptional regulation of these genes is dependent on the ability to form glucose 6-phosphate by any one of the three hexose kinases. In addition, we conclude that signalling via the Ras-adenylate cyclase pathway is not necessary for the proper transcriptional response of glycolytic and gluconeogenic genes to glucose, because the transcription of these genes is not significantly affected in mutants having either high or low activities of this pathway. In contrast, the transcriptional regulation of the PFK26 and PFK27 genes is significantly altered in several of the Ras-adenylate cyclase pathway mutants studied, indicating that protein kinase A plays an important role in the transcriptional regulation of these genes.

Gastric chief cell-specific transcription of the pepsinogen A gene.

The molecular mechanisms underlying the regulation of pepsinogen A (PGA) gene expression in mammalian cells are poorly understood. In this paper we describe the structural and functional analysis of the pepsinogen A gene promoter in the pig. By genomic Southern analyses we demonstrate that, in contrast with human PGA genes which are amplified and organized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediating the gastric mucosa cell-specific transcription of the PGA gene in pig, we isolated a PGA gene from a porcine genomic library. The nucleotide sequence of the first exon and 1.7 kb of the upstream DNA region were determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter activity of the PGA gene a functional assay was developed: we succeeded in obtaining primary monolayer cultures of porcine gastric mucosal chief cells, suitable for transfection. Fragments of 5'-flanking and noncoding first exon sequences of the porcine and human PGA genes were linked to the chloramphenicol acetyltransferase (CAT) gene. The transcriptional activity of these hybrid genes was assessed in transient expression assays upon transfection (lipofection) of gastric and nongastric cells. Whereas PGA 5'-flanking sequences showed no promoter activity in nongastric cell types, the DNA region from -205 to +21 was found to be sufficient to direct expression of the porcine PGA constructs in a cell-specific manner. Further deletion analysis of the proximal promoter fragment identified several regions (-205 to -167, -127 to -67 and +2 to +21) acting synergistically in the transcriptional regulation of the PGA gene. In contrast, all human PGA-CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating species-specific control of PGA gene expression. In addition, the transcriptional activity of the porcine PGA promoter in chief cells from pig was completely abolished by in vitro CpG methylation. Footprint analyses of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable differences between both extracts, which might reflect the interaction with (a) cell-specific factor(s).

Family and population studies on the human pepsinogen A multigene family.

Human pepsinogen A (PGA) displays highly polymorphic isozymogen patterns after polyacrylamide gel electrophoresis and activity staining. The patterns differ with respect to the presence and the relative intensity of the individual fractions. Family studies strongly suggest that these isozymogen patterns are encoded by allelic haplotypes, encompassing different numbers and types of PGA genes. In this paper, we confirm the essential features of this multigene model. We establish the relationship between the haplotypes and the corresponding isozymogen patterns by determination of the PGA polymorphism at both the DNA and the protein level in 117 Dutch individuals, 60 of whom were unrelated. The combination of HindIII and EcoRI restriction fragment length polymorphisms (RFLPs) has enabled us to define different haplotypes, which are shown to segregate within families. Most genes are characterized by their specific EcoRI fragments. The HindIII RFLP is in strong linkage disequilibrium with PGA genes showing strong expression of the relevant isozymogen. Although a general picture of the relationship between genotypes and phenotypes is emerging, there are exceptions, suggesting that rare haplotypes evolve by unique crossover events.

Nucleotide sequence comparison of five human pepsinogen A (PGA) genes: evolution of the PGA multigene family.

To unravel the genetic basis for the pepsinogen A (PGA) protein polymorphism, we have isolated and characterized a number of PGA genes, distinguishable by polymorphic EcoRI fragments of 12.0, 15.0, and 16.6 kb. Using a HindIII or AvaII polymorphism, we can discriminate between different 15.0 (15.0 and 15.0*) and 12.0 (12.0s and 12.0l) genes, respectively. The coding sequences of a 15.0 and a 16.6 gene were determined, together with considerable stretches of the 5'- and 3'-flanking regions and introns. The genes were demonstrated to encode Pg5 and Pg4, respectively. Because substitutions in codons 43 and 207 appeared to be critical in the determination of the encoded proteins, we sequenced only these regions in the two 12.0 genes and the 15.0* gene. On the basis of these partial sequences, we assume that these genes encode Pg3. In the evolutionary model of the PGA gene cluster presented here, the 12.0 genes arose by an unequal, but homologous crossover. The results of sequence analysis of the second intron of the 12.0s, 12.0l, 15.0, and 16.6 genes suggest that the two 12.0 genes have arisen from two different crossover events.

Cloning and sequencing of rhesus monkey pepsinogen A cDNA.

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Genomic structure and evolution of the human pepsinogen A multigene family.

A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0-16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.