Identification of a novel ALDH1A3-selective inhibitor by a chemical probe with unrelated bioactivity: An approach to affinity-based drug target discovery.

The identification of biologically active target compounds and their binding proteins is important in mechanism-of-action studies for drug development. Additionally, the newly discovered binding proteins provide unforeseen ideas for novel drug discovery and for subsequent structural transformation to improve target specificity. Based on the lead and final candidate compounds related to the type 5 phosphodiesterase (PDE5) inhibitor E4021, we designed chemical probes and identified their target proteins by the affinity chromatography approach. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3), currently reported as a cancer stem cell target, was clearly isolated as a binding protein of the lead 'immature' inhibitor probe against PDE5. In the early derivatization to the closely related structure, Compound 5 (ER-001135935) was found to significantly inhibit ALDH1A3 activity. The discovery process of a novel ALDH1A3-selective inhibitor with affinity-based binder identification is described, and the impact of this identification method on novel drug discovery is discussed.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking.

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model.

Phosphodiesterase (PDE) 4 inhibition is a well-known anti-inflammatory mechanism, but the development of PDE4 inhibitors has been hampered by side effects such as nausea and emesis. Local delivery of a PDE4 inhibitor to the site of inflammation may overcome these issues. The purpose of this study was to assess the therapeutic potential of E6005 (methyl 4-[({3-[6,7-dimethoxy-2-(methylamino)quinazolin-4-yl]phenyl}amino)carbonyl]benzoate), a novel PDE4 inhibitor developed as a topical agent for atopic dermatitis (AD). E6005 potently and selectively inhibited human PDE4 activity with an IC50 of 2.8 nM and suppressed the production of various cytokines from human lymphocytes and monocytes with IC50 values ranging from 0.49 to 3.1 nM. In mice models, the topical application of E6005 produced an immediate antipruritic effect as well as an anti-inflammatory effect with reduced expression of cytokines/adhesion molecules. On the basis of these observed effects, topical E6005 ameliorated the appearance of atopic dermatitis-like skin lesions in two types of AD models, hapten- and mite-elicited models, exhibiting inhibitory effects comparable to that of tacrolimus. The use of ¹4C-labeled E6005 showed rapid clearance from the blood and low distribution to the brain, contributing to the low emetic potential of this compound. These results suggest that E6005 may be a promising novel therapeutic agent with antipruritic activity for the treatment of AD.

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization.

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.

Divergent subunit interactions among fungal mRNA 5'-capping machineries.

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1+) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.