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Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein–protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitropotency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59with a balanced profile, which are suitable for future development toward therapeutic use.

ABSTRACTThe translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivoMycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

AbstractResearch on common carp, Cyprinus carpio,is beneficial for zebrafish research because of resources available owing to its large body size, such as the availability of sufficient organ material for transcriptomics, proteomics, and metabolomics. Here we describe the shot gun sequencing of a clonal double-haploid common carp line. The assembly consists of 511891 scaffolds with an N50 of 17 kb, predicting a total genome size of 1.4–1.5 Gb. A detailed analysis of the ten largest scaffolds indicates that the carp genome has a considerably lower repeat coverage than zebrafish, whilst the average intron size is significantly smaller, making it comparable to the fugu genome. The quality of the scaffolding was confirmed by comparisons with RNA deep sequencing data sets and a manual analysis for synteny with the zebrafish, especially the Hox gene clusters. In the ten largest scaffolds analyzed, the synteny of genes is almost complete. Comparisons of predicted exons of common carp with those of the zebrafish revealed only few genes specific for either zebrafish or carp, most of these being of unknown function. This supports the hypothesis of an additional genome duplication event in the carp evolutionary history, which—due to a higher degree of compactness—did not result in a genome larger than that of zebrafish.

The most direct approach to study the physiological role of a protein of unknown function (Functional Genomics) is to change its expression pattern in an intact organism and analyze the phenotypic consequences of this manipulation. The introduction of a method to generate stably transgenic Xenopus laevis has paved the way to the use of tissue / cell- and developmental stage-specific promoters allowing to study the physiological function of proteins in a defined set of fully differentiated cells. Whereas stable (over)expression of proteins in Xenopus is now within reach, stable inhibition of protein expression can only be accomplished randomly, by gene trap approaches. We here report our efforts to induce stable RNA interference (RNAi) in X. laevis via transgene-driven expression of inverted repeats. Stable, and muscle- and neuron-specific knock-down of expression of exogenous green fluorescent protein (GFP) reporter was achieved via RNA polymerase II promoter-driven expression of long GFP RNA duplexes. Unfortunately, our attempts to induce RNAi directed against various endogenous targets, based on the use of RNA polymerase II and III promoters, and long and short inverted repeats have not resulted in a reliable protocol for stable, transgene-driven RNAi in Xenopus. In the second part, we present an example of the use of a cell-specific promoter for functional studies. Cell-specific transgene overexpression of a GFP-tagged member of the p24 family thought to be involved in intracellular protein transport was achieved and this manipulation of the intermediate pituitary melanotrope cell had a phenotypic consequence at its physiological target, the skin melanophore. Thus, the traditional experimental advantages of X. laevis combined with the recently developed technique of stable, non-mosaic Xenopus transgenesis make this lower vertebrate an attractive model organism for Functional Genomics.

We have discovered two new exons in the mouse fibroblast growth factor 2 (FGF-2 or bFGF) gene that can be alternatively spliced to the second coding exon of the gene. The newly identified exons 1b and 1c are located at, respectively, approximately 19 and 32 kb downstream of the canonical exon 1a. Using RT-PCR analysis, mRNAs containing exon 1c and canonical exons 2 and 3 were identified in embryonic limb, placenta, face, carcass and ocular tissues. A 3.7-kb transcript present in placenta and embryonic limb hybridizes with an exon 1c-derived probe in Northern blot analysis. Alternative splicing of exon 1c to exon 2 creates a transcript for which the predicted alternative FGF-2 (altFGF-2) polypeptide contains a novel N-terminal domain. Our data indicate that in mouse embryos multiple novel mRNA variants are transcribed from the FGF-2 locus using alternative splicing. These data suggest that proteins arising from these alternative transcripts may play a role in mouse embryogenesis.

PDGF is an important polypeptide growth factor that plays an essential role during early vertebrate development and is associated with tissue repair and wound healing in the adult vertebrate. Moreover, PDGF is thought to play a role in a variety of pathological phenomena, such as cancer, fibrosis and atherosclerosis. PDGF is expressed as a dimer of A and/or B chains, the precursors of which are encoded by two single copy genes. Although the PDGF genes are expressed coordinately in a number of cell types, they are independently expressed in a majority of cell types. The expression of either PDGF gene can be affected by very diverse extracellular stimuli and the type of response is dependent on the cell type that is exposed to the stimulus. Expression of the PDGF chains can be modulated at every imaginable level: by regulating accessibility of the transcription start site, by varying the transcription initiation rate, by using alternative transcription start sites, by alternative splicing, by using alternative polyadenylation signals, by varying mRNA decay rates, by regulating efficiency of translation, by protein modification, and by regulating secretion. Even upon secretion, the activity of PDGF can be modulated by non-specific or specific PDGF-binding proteins. This review provides an overview of the cell types in which the PDGF genes are expressed, of the factors that are known to affect the expression of PDGF, and of the various levels at which the expression of PDGF genes can be regulated.

The transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat γD-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced)in vitrodifferentiation of rat lens fiber cells.In vitro,in the presence of bFGF only, the endogenous γD mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and γD mRNA starts to accumulate at Day 8. Demethylation of the γD promoter region, as assessed by measuring the methylation state of theThaI site at −16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: −141/−131, −88/−71, −55/−45, and −15/−4. Site-directed mutagenesis of the G residues at −55 and −46 resulted in a three- to fivefold decrease in promoter activity of transfected γD/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G→T mutation at −43 had no effect. The −55/−45 footprint thus derives from a proximal activator. The −88/−71 footprint identifies a silencer of the γD promoter in late fiber cell differentiation, as a tetramer of the −85/−67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, ofin vitrodifferentiation. To time the appearance of regulatory factors, the activity of a −73/+45 γD/CAT (containing the activator region) and of a −1100/+45 γD/CAT construct was measured during fiber cell differentiation. The −73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the −1100/+45 construct constrained γD promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the γD-crystallin gene promoter.

By in vivo DMS footprint and reporter gene analyses we identified two transcription factor binding sites in the human c-sis/PDGF B gene promoter. The low basal activity of the PDGF B promoter in HeLa and undifferentiated K562 cells, which express low PDGF B mRNA levels, and in PC3 cells, which express a high PDGF B mRNA level, results from binding of a weak transcriptional activator between positions -64 and -61 relative to the transcription start site. Cytotrophoblast-like JEG-3 cells, which do not express the 3.5 kb PDGF B mRNA, contain a transcriptional activator directed at the -64/-61 sequence, but DNA methylation may render the endogenous promoter inaccessible to this activator. A CCACCCAC element at position -611/-54 was identified as the in vivo binding site for a strong transcriptional activator in phorbol ester-treated megakaryocytic K562 cells, which express a high PDGF B mRNA level. Primary human fibroblasts, which do not transcribe the PDGF B gene, contain a transcriptional activator that recognizes an element between positions -60 and -45 but does not bind to the endogenous unmethylated promoter. Our results show that the complex expression pattern of the human PDGF B gene involves the cell type-specific expression of weak and strongtranscriptional activatore and regulation of promoter accessibility to these factors.

The human platelet-derived growth factor (PDGF) B chain precursor is usually translated from a 3.5 kb c-sis/PDGF B gene transcript. The first exon of the c-sis gene contains the code for the signal peptide of the PDGF B chain precursor, preceded by a 1 kb long untranslated sequence with potent translation Inhibitory activity. In this paper we show that a novel 2.6 kb c-sis mRNA present in the human choriocarcinoma cell line JEG-3 initiates at an alternative exon 1, which we refer to as exon 1a. The 90 bp long exon 1a is located in the center of the first intron of the gene. It coincides with a very pronounced DNase-hhypersensitive site and is preceded by a functional promoter. Of the three ATG codons present in exon 1 a, the third one perfectly matches the criteria of a consensus start codon. It Initiates an open reading frame that is continuous with the code for the PDGF B chain precursor but lacks the code for a signal peptide. We conclude that this novel 2.6 kb c-sis mRNA species lacks the strong translation inhibitory potential of the regular exon 1 and contains the code for a PDGF B-IIke protein that may be targeted to the cell nucleus.