Quorum sensing-associated bacterial phenazines are potential ligands of human α1 -acid glycoprotein.

α1 -Acid glycoprotein (AGP) is a prominent acute phase component of blood plasma and extravascular fluids. As a member of the immunocalins, AGP exerts protective effects against Gram-negative bacterial infections but the underlying molecular mechanisms still need to be elucidated. Notably, the chemical structures of phenothiazine, phenoxazine and acridine type ligands of AGP are similar to those of phenazine compounds excreted by the opportunistic human pathogen Pseudomonas aeruginosa and related bacterial species. These molecules, like pyocyanin, act as quorum sensing-associated virulence factors and are important contributors to bacterial biofilm formation and host colonisation. Molecular docking simulations revealed that these agents fit into the multi-lobed cavity of AGP. The binding site is decorated by several aromatic residues which seem to be essential for molecular recognition of the ligands allowing multifold π-π and CH-π interactions. The estimated affinity constants (~105 M-1 ) predict that these secondary metabolites could be trapped inside the ß-barrel of AGP which in turn could reduce their cytotoxic effects and disrupt the microbial QS network, facilitating the eradication of bacterial infections.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

An overlooked UV spectroscopic tool for sensing coil-to-helix and helix-to-coil conformational transitions of proteins and peptides.

A simple spectrophotometric approach is proposed for sensing coil-to-helix and helix-to-coil conformational transitions of intrinsically disordered and folded peptide/protein sequences. Helix formation induced by a variety of physico-chemical factors results in a substantial intensity reduction (hypochromism) of the intense far-UV absorption band associated with the π-π* transition of amide chromophores. Conversely, the same band exhibits intensity increase (hyperchromism) as the consequence of unfolding events. This method, faded into obscurity several decades ago, may obtain widespread applications in the field of protein science.

Far-UV circular dichroism signatures indicate fluorophore labeling induced conformational changes of penetratin.

Fluorescent labeling is a broadly utilized approach to assess in vitro and in vivo behavior of biologically active, especially cell-penetrating and antimicrobial peptides. In this communication, far-UV circular dichroism (CD) spectra of penetratin (PEN) fluorophore conjugates reported previously have been re-evaluated. Compared to the intrinsically disordered native peptide, rhodamine B and carboxyfluorescein derivatives of free and membrane-bound PEN exhibit extrinsic CD features. Potential sources of these signals displayed above 220 nm are discussed suggesting the contributions of both intra- and intermolecular chiral exciton coupling mechanisms. Careful evaluation of the CD spectra of fluorophore-labeled peptides is a valuable tool for early detection of labeling-provoked structural alterations which in turn may modify the membrane binding and cellular uptake compared to the unconjugated form.

Chiroptical detection of self-aggregating fluorescent rhodamine conjugates: Mistakes and prospects.

Fluorescent conjugation can be considered as the chromophoric derivatization of the target, and as such, it may provide additional structure-related information available by using circular dichroism (CD) spectroscopy. In this essay, peculiar CD spectroscopic data reported earlier for thyroid hormone-rhodamine conjugates have been re-evaluated. Contrary to the original proposal on the intramolecular folding of the labelled hormone, the bisignate motif observed in the CD spectrum is a clear evidence of dye-dye intermolecular chiral exciton coupling, indicating supramolecular self-association of the conjugates. This anomalous solution behaviour undermines the credibility of experimental results reported with such conjugates still being used in the laboratory practice. The extension of routine far-ultraviolet (UV) CD spectroscopic scans of chiral fluorophore conjugates into the near-UV and visible spectral region is strongly recommended.

Sulfobutylation of Beta-Cyclodextrin Enhances the Complex Formation with Mitragynine: An NMR and Chiroptical Study.

Mitragynine (MTR), the main indole alkaloid of the well-known plant kratom (Mitragyna speciosa), is one of the most studied natural products nowadays, due to its remarkable biological effects. It is a partial agonist on the opioid receptors, and as such relieves pain without the well-known side-effects of the opioids applied in the clinical practice. MTR and its derivatives therefore became novel candidates for drug development. The poor aqueous solubility and low bioavailability of drugs are often improved by cyclodextrins (CyDs) as excipients through host-guest type complex formation. Among the wide variety of CyDs, sulfobutylether-beta-cyclodextrin (SBEßCyD) is frequently used and official in the European and U.S. Pharmacopoeia. Herein, the host-guest complexation of MTR with ßCyD and SBEßCyD was studied using chiroptical and NMR spectroscopy. It was found by NMR measurements that MTR forms a rather weak (logß11 = 0.8) 1:1 host-guest complex with ßCyD, while the co-existence of the 2MTR∙SBEßCyD and MTR∙SBEßCyD species was deducted from 1H NMR titrations in the millimolar MTR concentration range. Sulfobutylation of ßCyD significantly enhanced the affinity towards MTR. The structure of the formed inclusion complex was extensively studied by circular dichroism spectroscopy and 2D ROESY NMR. The insertion of the indole moiety was confirmed by both techniques.

Molecular Dynamics Approaches Dissect Molecular Mechanisms Underlying Methylene Blue-Glycosaminoglycan Interactions.

Glycosaminoglycans (GAGs) are a class of periodic anionic linear polysaccharides involved in a number of biologically relevant processes in the extracellular matrix via interactions with various types of molecules including proteins, peptides and small organic molecules. The metachromatic dye methylene blue (MB) is a GAG binding agent. This molecule possesses a tricyclic, monocationic phenothiazine ring system, while the terminal methyl groups attached to the nitrogen atoms bear the most positive charges of the cation and, therefore, represent potential binding sites for negatively charged GAGs. In this study, we rigorously explored molecular mechanisms underlying these interactions for several GAG types: heparin, heparan and chondroitin sulfates. We found that GAG-MB interactions are predominantly electrostatically driven, with the particularly important role of sulfate groups. MB oligomeric stack formation was favored in the presence of GAGs. Furthermore, the impact of MB binding on the conformation of GAGs was also evaluated. The novel results allow for better quantitative analytics of GAG composition in the studied biochemical systems using MB dye as a GAG-specific marker. Our data add to the knowledge on small molecule-GAG interactions and could be potentially useful for novel developments in drug design and putative disease therapies in which GAGs are involved.

Human host-defense peptide LL-37 targets stealth siderophores.

A growing number of evidence shows that human-associated microbiota is an important contributor in health and disease. However, much of the complexity of host-microbiota interaction remains to be elucidated both at cellular and molecular levels. Siderophores are chemically diverse, ferric-specific chelators synthesized and secreted by microbes to secure their iron acquisition. The host defense peptide LL-37 is ubiquitously produced at epithelial surfaces modulating microbial communities and suppressing pathogenic strains. The present work demonstrates that LL-37 binds tightly siderocalin-resistant stealth siderophores which are important contributors to the virulence of several pathogens. As indicated by circular dichroism spectroscopic experiments, addition of aerobactin and rhizoferrin increases the membrane active α-helical conformation of the partially folded peptide. The cationic nature of LL-37 (+6 net charge at pH 7.4) and the multiple carboxylate groups present in siderophores refer to the dominant contribution of electrostatic interactions in the stabilization of peptide-chelator adducts. It is proposed that aside siderocalin proteins, LL-37 may be a complementary, less specific component of the siderophore scavenging repertoire of the innate immune system.

Drug Conjugation Induced Modulation of Structural and Membrane Interaction Features of Cationic Cell-Permeable Peptides.

Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.

Manipulating Active Structure and Function of Cationic Antimicrobial Peptide CM15 with the Polysulfonated Drug Suramin: A Step Closer to in Vivo Complexity.

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder-to-order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide-to-drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15⋅suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small-molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP-based antimicrobial agents.

The molecular mechanism of structural changes in the antimicrobial peptide CM15 upon complex formation with drug molecule suramin: a computational analysis.

Dynamic increase of resistant bacterial infectious diseases continuously requires development of novel compounds against them. The molecular level understanding of the mechanism and interactions of natural host-defense peptides or antimicrobial peptides (AMPs) is an important step towards rational design and development of compounds inspired by their function. A particular set of these peptides have disordered structure, the ordering of which may modify their antimicrobial properties. Recent experiments demonstrate that such conformational transitions of AMPs could be mediated by the presence of small organic compounds, such as approved drug molecules. However, the molecular mechanisms underlying these structural changes are unclear. In this study, we apply molecular docking and molecular dynamics-based approaches to rigorously analyze the interactions between the drug suramin and the AMP CM15, a synthetic unstructured hybrid peptide. We characterize the energetic properties of putative CM15-suramin complexes revealing particular impacts of CM15 residues as well as the parts of suramin on these interactions. We find that α-helical content of the peptide is increased in the presence of suramin, which is in agreement with the experimental data. Kinetics analysis from canonical molecular dynamics and metadynamics simulations suggest that the effect of suramin does not promote the formation of α-helix but rather results from its ability to stabilize the α-helical population in the conformational pool of the peptide. Potentially, understanding the physico-chemical basis underlying the interactions between drug molecules and disordered AMPs will prove useful in strategies for antimicrobial compound development. Further on, the given computational protocol for the analysis of such flexible systems provide a basis for future theoretical investigation of similar biomolecular complexes.

The Un(f)told Story of General Anesthesia.

Inhalational anesthetics are routinely employed in clinical practice to accomplish general anesthesia. Concerns have recently emerged regarding the deleterious impact of these volatile agents on cognitive performance, immune functions, and tumor recurrence and metastasis. These agents have been shown to modify the gene-expression pattern as well as cell signaling in tumor cells, but the underlying molecular mechanisms remain a matter of conjecture. Regulatory/signaling proteins either of cytosolic or membrane origin abundantly contain intrinsically disordered sequences, the conformational pliability of which is pivotal in their biological functions. It is well known that chloroform (an anesthetic itself), trifluoroethanol, hexafluoroisopropanol, and related haloalcohols markedly affect the structure of disordered proteins and protein regions by inducing folding, misfolding, or even aggregation. Taking into consideration the physicochemical similarities and protein interaction modes of these volatile solvents and inhaled anesthetics, it is postulated that administration of these drugs can also modify the secondary structure of disordered protein segments. Accordingly, pharmacological effects of anesthetics may, at least in part, be mediated by conformational perturbations of intrinsic disorder-based regulatory protein networks of cells.

Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane-Associated Bio-macromolecules Studied with Polarized Light.

Extracellular vesicles (EVs) are currently in scientific focus, as they have great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, and thus, additional insight into their molecular-level properties, membrane-protein interactions, and membrane rigidity is still needed. We here demonstrate the use of red-blood-cell-derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular-level structural information on these systems. Flow-linear dichroism (flow-LD) measurements show that EVs can be oriented by shear force and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage, this interaction ceases; this is coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight into vesicle rigidity, which decreases in time parallel to changes in protein conformation. Overall, we propose that both linear dichroism and circular dichroism spectroscopy can provide simple, rapid, yet efficient ways to track changes in the membrane-protein interactions of EV components at the molecular level, which may also give insight into processes occurring during vesiculation.

Hemin and bile pigments are the secondary structure regulators of intrinsically disordered antimicrobial peptides.

The interaction of protoporphyrin compounds of human origin with the major bee venom component melittin (26 a.a., Z +6) and its hybrid derivative (CM15, 15 a.a., Z +6) were studied by a combination of various spectroscopic methods. Throughout a two-state, concentration-dependent process, hemin and its metabolites (biliverdin, bilirubin, bilirubin ditaurate) increase the parallel ß-sheet content of the natively unfolded melittin, suggesting the oligomerization of the peptide chains. In contrast, α-helix promoting effect was observed with the also disordered but more cationic CM15. According to fluorescence quenching experiments, the sole Trp residue of melittin is the key player during the binding, in the vicinity of which the first pigment molecule is accommodated presumably making indole-porphyrin π-π stacking interaction. As circular dichroism titration data suggest, cooperative association of additional ligands subsequently occurs, resulting in multimeric complexes with an apparent dissociation constant ranged from 20 to 65 µM. Spectroscopic measurements conducted with the bilirubin catabolite urobilin and stercobilin refer to the requirement of intact dipyrrinone moieties for inducing secondary structure transformations. The binding topography of porphyrin rings on a model parallel ß-sheet motif was evaluated by absorption spectroscopy and computational modeling showing a slipped-cofacial binding mode responsible for the red shift and hypochromism of the Soret band. Our results may aid to recognize porphyrin-responsive binding motifs of biologically relevant, intrinsically disordered peptides and proteins, where transient conformations play a vital role in their functions.

Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup.

Cationic peptides proved fundamental importance as pharmaceutical agents and/or drug carrier moieties functioning in cellular processes. The comparison of the in vitro activity of these peptides is an experimental challenge and a combination of different methods, such as cytotoxicity, internalisation rate, haemolytic and antibacterial effect, is necessary. At the same time, several issues need to be addressed as the assay conditions have a great influence on the measured biological effects and the experimental setup needs to be optimised. Therefore, critical comparison of results from different assays using representative examples of cell penetrating and antimicrobial peptides was performed and optimal test conditions were suggested. Our main goal was to identify carrier peptides for drug delivery systems of antimicrobial drug candidates. Based on the results of internalisation, haemolytic, cytotoxic and antibacterial activity assays, a classification of cationic peptides is advocated. We found eight promising carrier peptides with good penetration ability of which Penetratin, Tat, Buforin and Dhvar4 peptides showed low adverse haemolytic effect. Penetratin, Transportan, Dhvar4 and the hybrid CM15 peptide had the most potent antibacterial activity on Streptococcus pneumoniae (MIC lower than 1.2 µM) and Transportan was effective against Mycobacterium tuberculosis as well. The most selective peptide was the Penetratin, where the effective antimicrobial concentration on pneumococcus was more than 250 times lower than the HC50 value. Therefore, these peptides and their analogues will be further investigated as drug delivery systems for antimicrobial agents.

Structural features of human DJ-1 in distinct Cys106 oxidative states and their relevance to its loss of function in disease.

DJ-1 (PARK7) is a multifunctional protein linked to the onset and progression of a number of diseases, most of which are associated with high oxidative stress. The Cys106 of DJ-1 is unusually reactive and thus sensitive to oxidation, and due to high oxidative stress it was observed to be in various oxidized states in disease condition. The oxidation state of Cys106 of DJ-1 is believed to determine the specific functions of the protein in normal and disease conditions. Here we report molecular dynamics simulation and biophysical experimental studies on DJ-1 in reduced (Cys106, S-), oxidized (Cys106, SO2-), and over-oxidized (Cys106, SO3-) states. To simulate the different oxidation states of Cys106 in DJ-1, AMBER related force field parameters were developed and reported for 3-sulfinoalanine and cysteine sulfonic acid. Our studies found that the overall structure of DJ-1 in different oxidation states was similar globally, while it differed locally significantly, which have implications on its stability, function and its link to disease on-set. Importantly, the results suggest that over-oxidation may trigger loss of functions due to local structural modification in the Cys106 containing pocket of DJ-1 and structurally destabilize the dimeric state of DJ-1, which is believed to be its bioactive conformation. Such loss of functions would result in reduced ability of DJ-1 to protect from oxidative stress insults and may lead to increased progression of disease.

Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

OBJECTIVE AND DESIGN: Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. MATERIALS: The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. METHODS: Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RESULTS: RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. CONCLUSIONS: RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

Inclusion excluded: Chiroptical sensing of the external surface of sulfated cyclodextrins.

It is shown that the heparin antagonist bis-aminoquinoline derivative surfen interacts with sulfated cyclodextrins in a unique fashion. Analysis of the UV spectroscopic data revealed exceptionally strong association (K(a) ∼ 10(7) M(-1)) of several surfen molecules to the external surface of the cyclodextrin hosts. H-bonded to the sulfate groups in 1:1 stoichiometry, the drug molecules form a chiral layer around the macrocycles. Due to the steric proximity, dipole-dipole coupling occurs between the adjacent aminoquinoline rings that accounts for the large UV hypochromism and the induced exciton couplet in the circular dichroism spectra.

Glycosaminoglycans are potential pharmacological targets for classic DNA minor groove binder drugs berenil and pentamidine.

It is shown that the antiprotozoal drugs berenil and pentamidine, conventional minor groove binders of DNA, form non-covalent complexes with polyanionic glycosaminoglycans. Induced circular dichroism (CD) spectra as well as UV hypochromism confirmed drug binding to the asymmetric template of heparin and chondroitin 6-sulfate. The biphasic nature of the CD signals refers to intermolecular chiral exciton coupling between the dicationic guest molecules forming a right- or a left-handed helical array along the GAG chains. Quantitative evaluation of the spectroscopic data measured in pH 7.0 buffer solution (80 mM NaCl) indicated a higher (Ka ∼ 10(6) M(-1) for berenil) and a lower (Ka ∼ 10(5) M(-1) for pentamidine) affinity heparin binding of these agents, similar to that reported for DNA. Drug-chondroitin sulfate complexes (Ka ∼ 10(4)-10(5) M(-1)) could be detected only at low ionic strength. These results imply that besides nucleic acids, GAGs may be another pharmacological targets for diarylamidine drugs.

Glycosaminoglycan and DNA Binding Induced Intra- and Intermolecular Exciton Coupling of the bis-4-Aminoquinoline Surfen.

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High-affinity (Ka ~ 10(7) M(-1)) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right-handed heparin template, binding of surfen to the left-helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf-thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists.