The Fmoc/t-Bu solid-phase synthesis of three difficult peptide sequences (a 9-mer, 15-mer, and 24-mer) was performed using N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on ...polystyrene, Tentagel, and ChemMatrix resins. In order to obtain an insight into the specific role of the elevated temperature and/or the electromagnetic field for peptide syntheses carried out using microwave irradiation, peptide couplings and Fmoc-deprotection steps were studied under microwave and conventionally heated conditions at the same temperature. While room temperature couplings/deprotections generally produced the difficult peptides in rather poor quality, excellent peptide purities were obtained using microwave heating at a temperature of 86 °C for both the coupling and deprotection steps in only 10 and 2.5 min reaction time, respectively. While for most amino acids no significant racemization was observed, the high coupling temperatures led to considerable levels of racemization for the sensitive amino acids His and Cys. It was demonstrated for all three peptide sequences that when performing the coupling/deprotection steps at the same reaction temperature using conventional heating, nearly identical results in terms of both peptide purity and racemization levels were obtained. It therefore appears that the main effect of microwave irradiation applied to solid-phase peptide synthesis is a purely thermal effect not related to the electromagnetic field.
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IJS, KILJ, NUK, PNG, UL, UM
In the emerging era of antimicrobial resistance, the susceptibility to co-infections of patients suffering from either acquired or inherited hemolytic disorders can lead to dramatic increase in ...mortality rates. Closely related, heme liberated during hemolysis is one of the major sources of iron, which is vital for both host and invading microorganisms. While recent intensive research in the field has demonstrated that heme exerts diverse local effects including impairment of immune cells functions, it is almost completely unknown how it may compromise key molecules of our innate immune system, such as antimicrobial host defense peptides (HDPs). Since HDPs hold great promise as natural therapeutic agents against antibiotic-resistant microbes, understanding the effects that may modulate their action in microbial infection is crucial. Here we explore how hemin can interact directly with selected HDPs and influence their structure and membrane activity. It is revealed that induced helical folding, large assembly formation, and altered membrane activity is promoted by hemin. However, these effects showed variations depending mainly on peptide selectivity toward charged lipids, and the affinity of the peptide and hemin to lipid bilayers. Hemin-peptide complexes are sought to form semi-folded co-assemblies, which are present even with model membranes resembling mammalian or bacterial lipid compositions. In vitro cell-based toxicity assays supported that toxic effects of HDPs could be attenuated due to their assembly formation. These results are in line with our previous findings on peptide-lipid-small molecule systems suggesting that small molecules present in the complex in vivo milieu can regulate HDP function. Inversely, diverse effects of endogenous compounds could also be manipulated by HDPs.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The elaboration of potent delivery systems for peptides/proteins is still a challenge but is increasingly needed in advanced therapy. In the present research, we have developed a nanoencapsulation ...system for peptides/proteins, which is suitable for the delivery of hydrophilic bioactive compounds. The preparation method combines the advantageous properties of reverse nanoemulsion and nanoprecipitation, resulting in the fonnation of nanoparticles in the size range of 350-500 nm. The polymeric coating composed of polycaprolactone allows chemical functionalization and protection while the inner microenvironment (containing 1 -decanoyl-rac-glycerol and N,N-dimethyldodecylamine N-oxide surfactants) provides aqueous surrounding for the active with structural fidelity. Hen egg white lysozyme and β-lactoglobulin were successfully encapsulated, achieving protein contents of 10-60 µg/mg and encapsulation efficiencies ranging from 5-50% depending on the protein type and loading concentration. In vitro release measurement showed a biphasic sustained release profile of both proteins in the time range of one month. In vitro cytotoxicity investigation of protein-loaded nanoparticles exhibited good cell viability (above 95% at the highest treatment concentration of 0.3 mg/ml). The encapsulated membrane-active peptides have shown improved bioactivity.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate ...(PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the ...formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors.
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•A broad library of cysteine targeting warheads was explored and characterized.•Diverse chemical reactions are suitable for specific covalent protein modification.•Warheads impact the reactivity and specificity of covalent fragments.•A road map for the optimization of cysteine targeting inhibitors is proposed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Nanoparticles consisting of biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) are promising carriers for drug molecules to improve the treatment of tuberculosis. Surface modifiers, such as ...Pluronic F127, are essential for biocompatibility and for the protection against particle aggregation. This study demonstrates a successful approach to conjugate Pluronic F127 coated PLGA nanoparticles with Tuftsin, which has been reported as a macrophage-targeting peptide. Transformation of Pluronic F127 hydroxyl groupswhich have limited reactivityinto aldehyde groups provide a convenient way to bind aminooxy-peptide derivatives in a one-step reaction. We have also investigated that this change has no effect on the physicochemical properties of the nanoparticles. Our data showed that coating nanoparticles with Pluronic-Tuftsin conjugate markedly increased the internalization rate and the intracellular activity of the encapsulated drug candidate against Mycobacterium tuberculosis. By employing this approach, a large variety of peptide targeted PLGA nanoparticles can be designed for drug delivery.
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IJS, KILJ, NUK, PNG, UL, UM
The complex immunopathology ofMycobacterium tuberculosis(Mtb) is one of the main challenges in developing a novel vaccine against this pathogen, particularly regarding eliciting protection against ...both active and latent stages. Multistage vaccines, which contain antigens expressed in both phases, represent a promising strategy for addressing this issue, as testified by the tuberculosis vaccine clinical pipeline. Given this approach, we designed and characterized a multistage peptide-based vaccine platform containing CD4+ and CD8+ T cell epitopes previously validated for inducing a relevant T cell response against Mtb. After preliminary screening, CFP10 (32–39), GlfT2 (4–12), HBHA (185–194), and PPE15 (1–15) were selected as promising candidates, and we proved that the PM1 pool of these peptides triggered a T cell response in Mtb-sensitized human peripheral blood mononuclear cells (PBMCs). Taking advantage of the use of thiol-maleimide chemoselective ligation, we synthesized a multiepitope conjugate (Ac-CGHP). Our results showed a structure–activity relationship between the conjugation and a higher tendency to fold and assume an ordered secondary structure. Moreover, the palmitoylated conjugate (Pal-CGHP) comprising the same peptide antigens was associated with an enhanced cellular uptake in human and murine antigen-presenting cells and a better immunogenicity profile. Immunization study, conducted in BALB/c mice, showed that Pal-CGHP induced a significantly higher T cell proliferation and production of IFNγ and TNFα over PM1 formulated in the Sigma Adjuvant System.
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IJS, KILJ, NUK, PNG, UL, UM
Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory ...Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 μM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.
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Carbon quantum dots (CQDs) are a novel family of fluorescent materials that could be employed as non-toxic alternatives to molecular fluorescent dyes in biological research and also ...in medicine. Four different preparation approaches, including microwave assisted heating and solvent refluxing, were explored. In addition to the widely used microwave assisted methods, a simple convenient new procedure is presented here for the particle synthesis. A detailed X-ray photoelectron spectroscopic (XPS) analysis was employed to characterize the composition, and more importantly, the chemical structure of the CQD samples and the interrelation of the characteristic surface chemical groups with the fluorescence properties and with surface polarity was unambiguously established. In vitro cellular internalization experiments documented their applicability as fluorescence labels while non-toxic properties were also approved. It was demonstrated that the adequate water-dispersibility of the particles plays a crucial role in their biological application. The synthetized CQD samples turned to be promising for cellular imaging applications both in laser illuminated flow cytometric measurements and in fluorescence microscopy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds ...with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral β
-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.
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