Non-target screening of surface water samples to identify exposome-related pollutants: a case study from Luxembourg

peer reviewedBackground Non-target screening of surface water samples collected over an extended period can reveal interesting temporal patterns in exposome-related pollutants. Additionally, geographical data on pollution sources close to the sampling sites, chemical classification data and the consideration of flow paths can provide valuable information on the origins and potential threat of tentatively identified chemical compounds. In this study, 271 surface water samples from 20 sampling sites across Luxembourg were analysed using high-resolution mass spectrometry, complementing routine target monitoring efforts in 2019–2022. Data analysis was performed using the open source R-package patRoon, which offers a customizable non-target workflow. By employing open source workflows featuring scoring terms, like spectral match and applying identification levels, tentative identifications can be prioritized, e.g. based on spectral similarity. Furthermore, by utilizing supplementary database information such as PubChemLite annotation categories and classification software such as classyFire, an overall assessment of the potential threats posed by the tentatively identified chemicals was conducted, enabling the prioritization of chemicals for future confirmation through targeted approaches. Results The study tentatively identified 378 compounds associated with the exposome including benzenoids, organoheterocyclic compounds, and organic phosphoric acids and derivatives (11 classyFire superclasses, 50 subclasses). The classification analysis not only revealed temporal variations in agrochemicals, with the majority of identifications occurring in May to July, but also highlighted the prevalence of pharmaceuticals such as venlafaxine in surface waters. Furthermore, potential sources of pollutants, like metallurgic industry or household products were explored by considering common uses and geographical information, as commercial uses of almost 100% of the identified chemicals are known. 41 chemicals were suggested for potential inclusion to governmental monitoring lists for further investigation. Conclusions The findings of this study complement existing knowledge on the pollution status of surface water in Luxembourg and highlight the usefulness of non-target screening for identifying temporal and spatial trends in pollutant levels. This approach, performed in a complementary manner to routine monitoring, can help to tentatively identify chemicals of concern for potential inclusion in target monitoring methods following additional confirmation and quantification efforts.R-AGR-3703 - IAS - LuxTIME (01/06/2020 - 15/01/2025) - FICKERS Andrea

Non-Target Screening of Surface Water Samples to Identify Exposome-Related Pollutants: A Case Study From Luxembourg

peer reviewedTalk given by Dagny Aurich at the ICNTS in Erdin

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Conformationally constrained peptide ligands offer an attractive format for the development of therapeutics. They can bind with high affinity and selectivity to protein targets, they are small enough to diffuse into tissues, they can be synthesized chemically, and their degradation products are not toxic. In recent years, a number of novel techniques were innovated to develop peptide-based ligands. One such technique is the generation of bicyclic peptides by phage display. This technique is well established in our laboratory and has led to the isolation of high-affinity antagonist for a range of important therapeutic targets. In a first project I aimed at developing bicyclic peptide ligands of the epidermal growth factor receptor Her2. Aberrant expression of Her2 has been implicated in various malignancies including breast cancer. In the treatment of this cancer type several Her2 specific antibodies were proved to be efficient. Due to their small size, bicyclic peptides could potentially offer advantages over antibodies such as ease of synthesis and conjugation, higher molecule-permass ratios, and better tumor penetration. I panned a large bicyclic peptide phage library against the extra-cellular domain of Her2 and obtained a range of peptide sequences binding to Her2 in a specific manner. After affinity maturation, a bicyclic peptide that bound Her2 with a Kd of 304 nM could be obtained. This peptide ligand offers a valuable starting point for further improvement and the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. A second project was triggered by my observation that it is relatively difficult to generate high-affinity bicyclic peptide ligands to proteins with flat and featureless surfaces such as Her2. The major reason for the limited binding affinity of bicyclic peptides was supposed to lie in the lack of a defined secondary structure in solution and the resulting entropic penalty upon binding. To overcome this problem, I proposed to evolve ligands based on a-helices. Chemically stabilized a-helices, also named stapled peptides, were previously developed by rational design. I proposed to evolve a-helical peptides by phage display wherein the helix, is stabilized in a chemical reaction prior to phage panning. I tested this strategy by affinity maturing an alpha-helical peptide binding to beta-catenin. The project resulted with a 250-fold improved ligand of beta-catenin. In a third project, I developed a novel constrained peptide format that I dubbed âhelix-loopâ motif. A stabilized alpha-helix was expanded with a peptide loop in order to increase the number of amino acids that can formcontacts with a target. The loop was linked to either the N- or C-terminal end of the helix and via a cysteine residue to the chemical linker stabilizing the alpha-helix. Libraries were created adding randomized loops with different length to either side of the peptide. Ligands with improved affinity were isolated against the cancer target beta-catenin. In summary, I have exploited the existing bicyclic peptide format to evolve bicyclic peptide ligands of Her2. In addition, I have developed a new approach to affinity mature stabilized helical peptides by phage display as well as a new constrained peptide format. This new approach should be applicable for affinity maturation of any stabilized alpha-helix. The new peptide format promises the generation of high affinity ligands to any protein target, including Her2

Directed Evolution of Bicyclic Peptides for Therapeutic Application

Many naturally occurring cyclic peptides or derivatives thereof are used as therapeutics such as the human hormones vasopressin and oxytocin or the antibiotics vancomycin and daptomycin. The success of cyclic peptide therapeutics is based on their ability to bind with high affinity, their good target selectivity and their low toxicity. As nature provides cyclic peptides to only a small number of disease targets, strategies have been developed to generate cyclic peptide ligands with tailored specificity de novo. Our laboratory is specialized on the directed evolution of bicyclic peptide ligands by phage display. In this article, we review our recent work to in vitro evolve bicyclic peptide antagonists, the binding and pharmacokinetic properties of bicyclic peptides, as well as efforts to generate bicyclic peptides for therapeutic application

Phage selection of bicyclic peptides binding Her2

Aberrant expression of the epidermal growth factor receptor Her2 has been implicated in various malignancies including breast cancer. Monoclonal antibodies and an antibody drug conjugate targeting Her2 have found wide clinical application. Herein, we aimed at developing Her2-specifc ligands based on peptides that have a 100-fold smaller molecular weight than antibodies. Such peptides could potentially offer advantages in the development of ligand drug conjugates, such as ease of synthesis and conjugation, higher molecule-per-mass ratios, and better tumor penetration. Panning of large bicyclic peptide phage display libraries against Her2 yielded a range of Her2-specific ligands having different formats and binding motifs. Strong sequence similarities among several of the isolated peptides indicated that they interact with Her2 in a specific manner. The best bicyclic peptide obtained after affinity maturation bound Her2 with a K-D of 304 nM. The diverse peptide ligands may offer valuable starting points for the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. (C) 2014 Elsevier Ltd. All rights reserved

Bicyclization and tethering to albumin yields long-acting Peptide antagonists

Proteolytically stable peptide architectures are required for the development of long-acting peptide therapeutics. In this work, we found that a phage-selected bicyclic peptide antagonist exhibits an unusually high stability in vivo and subsequently deciphered the underlying mechanisms of peptide stabilization. We found that the bicyclic peptide was significantly more stable than its constituent rings synthesized as two individual macrocycles. The two rings protect each other from proteolysis when linked together, conceivably by constraining the conformation and/or by mutually shielding regions prone to proteolysis. A second stabilization mechanism was found when the bicyclic peptide was linked to an albumin-binding peptide to prevent its rapid renal clearance. The bicyclic peptide conjugate not only circulated 50-fold longer (t(1/2) = 24 h) but also became entirely resistant to proteolysis when tethered to the long-lived serum protein. The bicyclic peptide format overcomes a limitation faced by many peptide leads and appears to be suitable for the generation of long-acting peptide therapeutics

Occurrence and Distribution of Pharmaceuticals and their Transformation Products in Luxembourgish Surface Waters

This pre-print describes the analysis of pharmaceuticals and their transformation products in surface water samples collected in Luxembourg from 2019 to 2020. Details of the experimental and computational tools and workflows used are fully described in the manuscript. Links to the suspect lists, codes used, and data files are also provided.</p

Measuring net protease activities in biological samples using selective peptidic inhibitors

The measurement of activities from individual proteases in biological samples is difficult because of the numerous proteases, their overlapping activities, and the lack of specific substrates. We applied selective protease inhibitors based on bicyclic peptides (>2000-fold selective over related proteases) to block individual proteases, allowing the quantification of their net activities. In protease mixtures, activity contributions of the serine proteases plasma kallikrein and urokinase-type plasminogen activator (uPA) were accurately quantified. In a tumor extract, we could quantify uPA activity. Because bicyclic peptide inhibitors toward virtually any protease can be generated by phage display, the approach should be applicable to any protease

Occurrence and Distribution of Pharmaceuticals and Their Transformation Products in Luxembourgish Surface Waters

Pharmaceuticals and their transformation products (TPs) are continuously released into the aquatic environment via anthropogenic activity. To expand knowledge on the presence of pharmaceuticals and their known TPs in Luxembourgish rivers, 92 samples collected during routine monitoring events between 2019 and 2020 were investigated using nontarget analysis. Water samples were concentrated using solid-phase extraction and then analyzed using liquid chromatography coupled to a high-resolution mass spectrometer. Suspect screening was performed using several open source computational tools and resources including Shinyscreen (https://git-r3lab.uni.lu/eci/shinyscreen/), MetFrag (https://msbi.ipb-halle.de/MetFrag/), PubChemLite (https://zenodo.org/record/4432124), and MassBank (https://massbank.eu/MassBank/). A total of 94 pharmaceuticals, 88 confirmed at a level 1 confidence (86 of which could be quantified, two compounds too low to be quantified) and six identified at level 2a, were found to be present in Luxembourg rivers. Pharmaceutical TPs (12) were also found at a level 2a confidence. The pharmaceuticals were present at median concentrations up to 214 ng/L, with caffeine having a median concentration of 1424 ng/L. Antihypertensive drugs (15), psychoactive drugs (15), and antimicrobials (eight) were the most detected groups of pharmaceuticals. A spatiotemporal analysis of the data revealed areas with higher concentrations of the pharmaceuticals, as well as differences in pharmaceutical concentrations between 2019 and 2020. The results of this work will help guide activities for improving water management in the country and set baseline data for continuous monitoring and screening efforts, as well as for further open data and software developments

Chemical macrocyclization of peptides fused to antibody Fc fragments

To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates