A System-Wide Investigation of the Dynamics of Wnt Signaling Reveals Novel Phases of Transcriptional Regulation

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression

Positive Evolutionary Selection of an HD Motif on Alzheimer Precursor Protein Orthologues Suggests a Functional Role

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the “transcription binding site turnover.” CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities. © 2013 Stuckenholz et al

Complexes of Trypanosoma cruzi Sterol 14 -Demethylase (CYP51) with Two Pyridine-based Drug Candidates for Chagas Disease: Structural basis for pathogen selectivity

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(triflu-oromethyl)phenyl)-piperazin-1-yl) -2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperidyl] pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α- demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azolebound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51- targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens

Structural basis for rational design of inhibitors targeting Trypanosoma cruzi sterol 14α-demethylase: two regions of the enzyme molecule potentiate its inhibition

Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14 alpha-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of beta-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50 = 1.2 nM, vs (S)-2/(S)-3, EC50 = 1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development of more effective antiparasitic drugs

Generation of multiple pharmacophore hypotheses using multiobjective optimisation techniques

Pharmacophore methods provide a way of establishing a structure--activity relationship for a series of known active ligands. Often, there are several plausible hypotheses that could explain the same set of ligands and, in such cases, it is important that the chemist is presented with alternatives that can be tested with different synthetic compounds. Existing pharmacophore methods involve either generating an ensemble of conformers and considering each conformer of each ligand in turn or exploring conformational space on-the-fly. The ensemble methods tend to produce a large number of hypotheses and require considerable effort to analyse the results, whereas methods that vary conformation on-the-fly typically generate a single solution that represents one possible hypothesis, even though several might exist. We describe a new method for generating multiple pharmacophore hypotheses with full conformational flexibility being explored on-the-fly. The method is based on multiobjective evolutionary algorithm techniques and is designed to search for an ensemble of diverse yet plausible overlays which can then be presented to the chemist for further investigation