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Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.
Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET–based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.
Designed polypharmacology presents as an attractive strategy to increase therapeutic efficacy in multi-factorial diseases by a directed modulation of multiple involved targets with a single molecule. Such an approach appears particularly suitable in non-alcoholic steatohepatitis (NASH) which involves hepatic steatosis, inflammation and fibrosis as pathological hallmarks. Among various potential pharmacodynamic mechanisms, activation of the farnesoid X receptor (FXRa) and inhibition of leukotriene A4 hydrolase (LTA4Hi) hold promise to counteract NASH according to preclinical and clinical observations. We have developed dual FXR/LTA4H modulators as pharmacological tools, enabling evaluation of this polypharmacology concept to treat NASH and related pathologies. The optimized FXRa/LTA4Hi exhibits well-balanced dual activity on the intended targets with sub-micromolar potency and is highly selective over related nuclear receptors and enzymes rendering it suitable as tool to probe synergies of dual FXR/LTA4H targeting.
Designed multitarget ligands are a popular approach to generating efficient and safe drugs, and fragment-based strategies have been postulated as a versatile avenue to discover multitarget ligand leads. To systematically probe the potential of fragment-based multiple ligand discovery, we have employed a large fragment library for comprehensive screening on five targets chosen from proteins for which multitarget ligands have been successfully developed previously (soluble epoxide hydrolase, leukotriene A4 hydrolase, 5-lipoxygenase, retinoid X receptor, farnesoid X receptor). Differential scanning fluorimetry served as primary screening method before fragments hitting at least two targets were validated in orthogonal assays. Thereby, we obtained valuable fragment leads with dual-target engagement for six out of ten target combinations. Our results demonstrate the applicability of fragment-based approaches to identify starting points for polypharmacological compound development with certain limitations.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Dual- or multi-target ligands have gained increased attention in the past years due to several advantages, including more simple pharmacokinetic and phamarcodynamic properties compared to a combined application of several drugs. Furthermore multi-target ligands often possess improved efficacy. We present a new approach for the discovery of dual-target ligands using aligned pharmacophore models combined with a shape-based scoring. Starting with two sets of known active compounds for each target, a number of different pharmacophore models is generated and subjected to pairwise graph-based alignment using the Kabsch-Algorithm. Since a compound may be able to bind to different targets in different conformations, the algorithm aligns pairs of pharmacophore models sharing the same features which are not necessarily at the exactly same spatial distance. Using the aligned models, a pharmacophore search on a multi-conformation-database is performed to find compounds matching both models. The potentially “dual” ligands are scored by a shape-based comparison with the known active molecules using ShaEP.
Using this approach, we performed a prospective fragment-based virtual screening for dual 5-LO/sEH inhibitors. Both enzymes play an important role in the arachidonic acid cascade and are involved in inflammatory processes, pain, cardiovascular diseases and allergic reactions. Beside several new selective inhibitors we were able to find a compound inhibiting both enzymes in low micromolar concentrations. The results indicate that the idea of aligned pharmacophore models can be successfully employed for the discovery of dual-target ligands.
Lipide sind essentielle Strukturelemente von Zellen. Sie sind unter anderem Hauptbestandteil von Membranen, die einerseits verschiedene Kompartimente innerhalb der Zelle gegeneinander abgrenzen und andererseits die Zelle nach außen abschotten. Membranen regulieren den Transport von Ionen, kleinen polaren Molekülen sowie peptidartigen Botenstoffen, da sie für viele Bestandteile des Organismus nicht oder nur wenig durchlässig (permeabel) sind.
We developed the Pharmacophore Alignment Search Tool (PhAST), a text-based technique for rapid hit and lead structure searching in large compound databases. For each molecule, a two-dimensional graph of potential pharmacophoric points (PPPs) is created, which has an identical topology as the original molecule with implicit hydrogen atoms. Each vertex is coloured by a symbol representing the corresponding PPP. The vertices of the graph are canonically labelled. The symbols associated with the vertices are combined to a so-called PhAST-Sequence beginning with the vertex with the lowest canonical label. Due to the canonical labelling the created PhAST-Sequence is characteristic for each molecule. For similarity assessment, PhAST-Sequences are compared using the sequence identity in their global pairwise alignment. The alignment score lies between 0 (no similarity) and 1 (identical PhAST-Sequences). In order to use global pairwise sequence alignment, a score matrix for pharmacophoric symbols was developed and gap penalties were optimized. PhAST performed comparably and sometimes superior to other similarity search tools (CATS2D, MOE pharmacophore quadruples) in retrospective virtual screenings using the COBRA collection of drugs and lead structures. Most importantly, the PhAST alignment technique allows for the computation of significance estimates that help prioritize a virtual hit list.