• 1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols: a new class of potent and selective aldosterone synthase inhibitors.

      Grombein, Cornelia M; Hu, Qingzhong; Heim, Ralf; Rau, Sabrina; Zimmer, Christina; Hartmann, Rolf W; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Campus C23, D-66123 Saarbrücken, Germany. (2015-01-07)
      1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols and related compounds were synthesized and evaluated for inhibition of aldosterone synthase (CYP11B2), a potential target for cardiovascular diseases associated with elevated plasma aldosterone levels like congestive heart failure and myocardial fibrosis. Introduction of substituents at the phenylsulfinyl moiety and changes of the substitution pattern at the naphthalene core were examined. Potent compounds were further examined for selectivity versus other important steroidogenic CYP enzymes, i.e. the highly homologous 11β-hydroxylase (CYP11B1), CYP17 and CYP19. The most potent compound (IC50 = 14 nM) discovered was the meta-trifluoromethoxy derivative 11, which also exhibited excellent selectivity toward CYP11B1 (SF = 415), and showed no inhibition of CYP17 and CYP19.
    • 17β-Hydroxysteroid Dehydrogenase Type 2 Inhibition: Discovery of Selective and Metabolically Stable Compounds Inhibiting Both the Human Enzyme and Its Murine Ortholog.

      Gargano, Emanuele M; Allegretta, Giuseppe; Perspicace, Enrico; Carotti, Angelo; Van Koppen, Chris; Frotscher, Martin; Marchais-Oberwinkler, Sandrine; Hartmann, Rolf W; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2015)
      Design and synthesis of a new class of inhibitors for the treatment of osteoporosis and its comparative h17β-HSD2 and m17β-HSD2 SAR study are described. 17a is the first compound to show strong inhibition of both h17β-HSD2 and m17β-HSD2, intracellular activity, metabolic stability, selectivity toward h17β-HSD1, m17β-HSD1 and estrogen receptors α and β as well as appropriate physicochemical properties for oral bioavailability. These properties make it eligible for pre-clinical animal studies, prior to human studies.
    • 2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 2. Structure-Based Optimization and Investigation of Effects Specific to the Allosteric Mode of Action.

      Bestgen, Benoît; Kufareva, Irina; Seetoh, Weiguang; Abell, Chris; Hartmann, Rolf W; Abagyan, Ruben; Le Borgne, Marc; Filhol, Odile; Cochet, Claude; Lomberget, Thierry; et al. (American Chemical Society, 2019-02-28)
      Protein CK2 has gained much interest as an anticancer drug target in the past decade. We had previously described the identification of a new allosteric site on the catalytic α-subunit, along with first small molecule ligands based on the 4-(4-phenylthiazol-2-ylamino)benzoic acid scaffold. In the present work, structure optimizations guided by a binding model led to the identification of the lead compound 2-hydroxy-4-((4-(naphthalen-2-yl)thiazol-2-yl)amino)benzoic acid (27), showing a submicromolar potency against purified CK2α (IC
    • 3-Pyridyl substituted aliphatic cycles as CYP11B2 inhibitors: aromaticity abolishment of the core significantly increased selectivity over CYP1A2.

      Yin, Lina; Hu, Qingzhong; Hartmann, Rolf W; Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany. (2012)
      Aldosterone synthase (CYP11B2) is a promising therapeutic target for the treatment of cardiovascular diseases related to abnormally high aldosterone levels. On the basis of our previously identified lead compounds I-III, a series of 3-pyridinyl substituted aliphatic cycles were designed, synthesized and tested as CYP11B2 inhibitors. Aromaticity abolishment of the core was successfully applied to overcome the undesired CYP1A2 inhibition. This study resulted in a series of potent and selective CYP11B2 inhibitors, with compound 12 (IC(50) = 21 nM, SF = 50) as the most promising one, which shows no inhibition toward CYP1A2 at 2 µM. The design conception demonstrated in this study can be helpful in the optimization of CYP inhibitor drugs regarding CYP1A2 selectivity.
    • Absorption of Anthocyanin Rutinosides after Consumption of a Blackcurrant ( Ribes nigrum L.) Extract.

      Röhrig, Teresa; Kirsch, Verena; Schipp, Dorothea; Galan, Jens; Richling, Elke; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2019-06-19)
      The dominant anthocyanins in blackcurrant are delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside. Data on their absorption and distribution in the human body are limited. Therefore, we performed a human pilot study on five healthy male volunteers consuming a blackcurrant (Ribes nigrum L.) extract. The rutinosides and their degradation products gallic acid and protocatechuic acid were determined in plasma and urine. The rutinosides’ concentrations peaked in both plasma and urine samples within 2 h of extract ingestion. The recoveries of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside from urine samples were 0.040 ± 0.011% and 0.048 ± 0.016%, respectively, over a 48 h period. Protocatechuic acid concentration increased significantly after ingestion of the blackcurrant extract. Our results show that after ingestion of a blackcurrant extract containing delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, significant quantities of biologically active compounds circulated in the plasma and were excreted via urine. Furthermore, these results contribute to the understanding of anthocyanin metabolism in humans.
    • Anti-biofilm Agents against Pseudomonas aeruginosa: A Structure-Activity Relationship Study of C-Glycosidic LecB Inhibitors

      Sommer, Roman; Rox, Katharina; Wagner, Stefanie; Hauck, Dirk; Henrikus, Sarah S; Newsad, Shelby; Arnold, Tatjana; Ryckmans, Thomas; Brönstrup, Mark; Imberty, Anne; et al. (American Chemical Society, 2019-10-24)
      Biofilm formation is a key mechanism of antimicrobial resistance. We have recently reported two classes of orally bioavailable C-glycosidic inhibitors of the Pseudomonas aeruginosa lectin LecB with antibiofilm activity. They proved efficient in target binding, were metabolically stable, nontoxic, selective, and potent in inhibiting formation of bacterial biofilm. Here, we designed and synthesized six new carboxamides and 24 new sulfonamides for a detailed structure-activity relationship for two clinically representative LecB variants. Sulfonamides generally showed higher inhibition compared to carboxamides, which was rationalized based on crystal structure analyses. Substitutions at the thiophenesulfonamide increased binding through extensive contacts with a lipophilic protein patch. These metabolically stable compounds showed a further increase in potency toward the target and in biofilm inhibition assays. In general, we established the structure-activity relationship for these promising antibiofilm agents and showed that modification of the sulfonamide residue bears future optimization potential.
    • Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.

      Castoldi, Arianna; Empting, Martin; De Rossi, Chiara; Mayr, Karsten; Dersch, Petra; Hartmann, Rolf; Müller, Rolf; Gordon, Sarah; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2018-12-05)
      The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.
    • Benzamidobenzoic acids as potent PqsD inhibitors for the treatment of Pseudomonas aeruginosa infections.

      Hinsberger, Stefan; de Jong, Johannes C; Groh, Matthias; Haupenthal, Jörg; Hartmann, Rolf W (2014-04-09)
      Targeting PqsD is a promising novel approach to disrupt bacterial cell-to-cell-communication in Pseudomonas aeruginosa. In search of selective PqsD inhibitors, two series of benzamidobenzoic acids - one published as RNAP inhibitors and the other as PqsD inhibitors - were investigated for inhibitory activity toward the respective other enzyme. Additionally, novel derivatives were synthesized and biologically evaluated. By this means, the structural features needed for benzamidobenzoic acids to be potent and, most notably, selective PqsD inhibitors were identified. The most interesting compound of this study was the 3-Cl substituted compound 5 which strongly inhibits PqsD (IC₅₀ 6.2 μM) while exhibiting no inhibition of RNAP.
    • Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials.

      Brengel, Christian; Thomann, Andreas; Schifrin, Alexander; Allegretta, Giuseppe; Kamal, Ahmed A M; Haupenthal, Jörg; Schnorr, Isabell; Cho, Sang Hyun; Franzblau, Scott G; Empting, Martin; et al. (2017-10-09)
      The development of novel antimycobacterial agents against Mycobacterium tuberculosis (Mtb) is urgently required due to the appearance of multidrug resistance (MDR) combined with complicated long-term treatment. CYP121 was shown to be a promising novel target for inhibition of mycobacterial growth. In this study, we describe the rational discovery of new CYP121 inhibitors by a systematic screening based on biophysical and microbiological methods. The best hits originating from only one structural class gave initial information about molecular motifs required for binding and activity. The initial screening procedure was followed by mode-of-action studies and further biological characterizations. The results demonstrate superior antimycobacterial efficacy and a decreased toxicity profile of our frontrunner compound relative to the reference compound econazole. Due to its low molecular weight, promising biological profile, and physicochemical properties, this compound is an excellent starting point for further rational optimization.
    • Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains.

      Müller, Stefan; Rachid, Shwan; Hoffmann, Thomas; Surup, Frank; Volz, Carsten; Zaburannyi, Nestor; Müller, Rolf; Helmholtz Institute for Pharmaceutical Research Saarland,Saarbru¨ cken, Saarland 66123, Germany. (2014-07-17)
      The crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay.
    • Catechol-based substrates of chalcone synthase as a scaffold for novel inhibitors of PqsD.

      Allegretta, Giuseppe; Weidel, Elisabeth; Empting, Martin; Hartmann, Rolf W; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), PO Box 15 11 50, D-66041 Saarbrücken, Germany. (2015-01-27)
      A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach led to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by Surface Plasmon Resonance (SPR) experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo.
    • Comparing the Self-Assembly of Sexiphenyl-Dicarbonitrile on Graphite and Graphene on Cu(111).

      Schmidt, Nico; Li, Jun; Gottardi, Stefano; Moreno-Lopez, Juan Carlos; Enache, Mihaela; Monjas, Leticia; van der Vlag, Ramon; Havenith, Remco W A; Hirsch, Anna K H; Stöhr, Meike; et al. (Wiley-Blackwell, 2019-04-01)
      A comparative study on the self-assembly of sexiphenyl-dicarbonitrile on highly oriented pyrolytic graphite and single-layer graphene on Cu(111) is presented. Despite an overall low molecule-substrate interaction, the close-packed structures exhibit a peculiar shift repeating every four to five molecules. This shift has hitherto not been reported for similar systems and is hence a unique feature induced by the graphitic substrates.
    • Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

      Weidel, Elisabeth; Negri, Matthias; Empting, Martin; Hinsberger, Stefan; Hartmann, Rolf W (2014)
      In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required.
    • Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps.

      Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W; Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany. (2011-09)
      17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.
    • Crystal structure of 4-methyl-sulfanyl-2-(2H-tetra-zol-2-yl)pyrimidine.

      Thomann, Andreas; Huch, Volker; Hartmann, Rolf W; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-12-01)
      The title compound, C6H6N6S, crystallized with two independent mol-ecules (A and B) in the asymmetric unit. The conformation of the two mol-ecules differs slightly. While the tetra-zole ring is inclined to the pyrim-idene ring by 5.48 (7) and 4.24 (7)° in mol-ecules A and B, respectively, the N-C-S-C torsion angles of the thio-methyl groups differ by ca 180°. In the crystal, the A and B mol-ecules are linked via a C-H⋯N hydrogen bond. They stack along the b-axis direction forming columns within which there are weak π-π inter-actions present [shortest inter-centroid distance = 3.6933 (13) Å].
    • Delivery system for budesonide based on lipid-DNA.

      Liu, Yun; Bos, I Sophie T; Oenema, Tjitske A; Meurs, Herman; Maarsingh, Harm; Hirsch, Anna K H; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-09-01)
      Budesonide is a hydrophobic glucocorticoid with high anti-inflammatory activity for the treatment of asthma, inflammatory bowel disease and rheumatoid arthritis. A micellar drug-delivery system based on lipid-DNA may provide a strategy to maximize its drug efficacy and reduce adverse effects. In this work, we report the use of lipid-DNAA (UU11mer), featuring two hydrophobic alkyl chains and forming micelles at a comparatively low critical micelle concentration, to render budesonide water-soluble with a high loading capacity (LC). The inhibition of interleukin-8 (IL-8) release shows that the new delivery system retains the inhibitory activity in cell-based assays. In conclusion, this research provides a novel approach to formulate and administer budesonide in a non-invasive manner, which dramatically improves its water-solubility while retaining its bioavailability.
    • Design and synthesis of a library of lead-like 2,4-bisheterocyclic substituted thiophenes as selective Dyrk/Clk inhibitors.

      Schmitt, Christian; Kail, Dagmar; Mariano, Marica; Empting, Martin; Weber, Nadja; Paul, Tamara; Hartmann, Rolf W.; Engel, Matthias; Helmholtz Institute für Pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2014)
      The Dyrk family of protein kinases is implicated in the pathogenesis of several diseases, including cancer and neurodegeneration. Pharmacological inhibitors were mainly described for Dyrk1A so far, but in fewer cases for Dyrk1B, Dyrk2 or other isoforms. Herein, we report the development and optimization of 2,4-bisheterocyclic substituted thiophenes as a novel class of Dyrk inhibitors. The optimized hit compounds displayed favorable pharmacokinetic properties and high ligand efficiencies, and inhibited Dyrk1B in intact cells. In a larger selectivity screen, only Clk1 and Clk4 were identified as additional targets of compound 48, but no other kinases frequently reported as off-targets. Interestingly, Dyrk1A is implicated in the regulation of alternative splicing, a function shared with Clk1/Clk4; thus, some of the dual inhibitors might be useful as efficient splicing modulators. A further compound (29) inhibited Dyrk1A and 1B with an IC50 of 130 nM, showing a moderate selectivity over Dyrk2. Since penetration of the central nervous system (CNS) seems possible based on the physicochemical properties, this compound might serve as a lead for the development of potential therapeutic agents against glioblastoma. Furthermore, an inhibitor selective for Dyrk2 (24) was also identified, which might be are suitable as a pharmacological tool to dissect Dyrk2 isoform-mediated functions.
    • Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal derivatives possessing potent diversified pharmacological properties.

      Bansal, Ranju; Guleria, Sheetal; Thota, Sridhar; Bodhankar, Subhash L; Patwardhan, Moreshwar R; Zimmer, Christina; Hartmann, Rolf W; Harvey, Alan L; University Institute of Pharmaceutical Sciences, Sector-14, Panjab University, Chandigarh 160014, India. ranju29in@yahoo.co.in (2012-05)
      As a part of our investigations into the structural-activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC50=0.18 μM and IC50=0.168 μM, respectively, approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007 μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity K(i)=25 nM, approximately 115-fold higher in comparison to standard drug galanthamine (K(i)=2.9 μM).
    • Development and validation of a UHPLC-MS/MS procedure for quantification of the Pseudomonas Quinolone Signal in bacterial culture after acetylation for characterization of new quorum sensing inhibitors.

      Maurer, Christine K; Steinbach, Anke; Hartmann, Rolf W; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany. Electronic address: christine.maurer@helmholtz-hzi.de. (2013-12)
      The appearance of antibiotic resistance requires novel therapeutic strategies. One approach is to selectively attenuate bacterial pathogenicity by interfering with bacterial cell-to-cell communication known as quorum sensing. The PQS quorum sensing system of Pseudomonas aeruginosa employs as signal molecule the Pseudomonas Quinolone Signal (PQS; 2-heptyl-3-hydroxy-4-(1H)-quinolone), a key contributor to virulence and biofilm formation. Thus, interference with PQS production is considered as promising approach for the development of novel anti-infectives. Therefore, in this study, we developed and validated an ultra-high performance liquid chromatographic-tandem mass spectrometric approach for reliable quantification of PQS in P. aeruginosa cultures for activity determination of new quorum sensing inhibitors. The poor chromatographic properties of PQS reported by others could be overcome by fast microwave-assisted acetylation. The validation procedure including matrix effects, recovery, process efficiency, selectivity, carry-over, accuracy and precision, stability of the processed sample, and limit of quantification demonstrated that the method fulfilled all requirements of common validation guidelines. Its applicability was successfully proven in routine testing. In addition, two-point calibration was shown to be applicable for fast and reliable PQS quantification saving time and resources. In summary, the described method provides a powerful tool for the discovery of new quorum sensing inhibitors as potential anti-infectives and illustrated the usefulness of chemical derivatization, acetylation, in liquid chromatography-mass spectrometry analysis.
    • Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers.

      Köberle, Verena; Pleli, Thomas; Schmithals, Christian; Augusto Alonso, Eduardo; Haupenthal, Jörg; Bönig, Halvard; Peveling-Oberhag, Jan; Biondi, Ricardo M; Zeuzem, Stefan; Kronenberger, Bernd; et al. (2013)
      MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.