Lipidation of Naturally Occurring α-Helical Antimicrobial Peptides as a Promising Strategy for Drug Design.

In this paper, we describe the chemical synthesis, preliminary evaluation of antimicrobial properties and mechanisms of action of a novel group of lipidated derivatives of three naturally occurring α-helical antimicrobial peptides, LL-I (VNWKKVLGKIIKVAK-NH2), LK6 (IKKILSKILLKKL-NH2), ATRA-1 (KRFKKFFKKLK-NH2). The obtained results showed that biological properties of the final compounds were defined both by the length of the fatty acid and by the structural and physico-chemical properties of the initial peptide. We consider C8-C12 length of the hydrocarbon chain as the optimal for antimicrobial activity improvement. However, the most active analogues exerted relatively high cytotoxicity toward keratinocytes, with the exception of the ATRA-1 derivatives, which had a higher selectivity for microbial cells. The ATRA-1 derivatives had relatively low cytotoxicity against healthy human keratinocytes but high cytotoxicity against human breast cancer cells. Taking into account that ATRA-1 analogues carry the highest positive net charge, it can be assumed that this feature contributes to cell selectivity. As expected, the studied lipopeptides showed a strong tendency to self-assembly into fibrils and/or elongated and spherical micelles, with the least cytotoxic ATRA-1 derivatives forming apparently smaller assemblies. The results of the study also confirmed that the bacterial cell membrane is the target for the studied compounds.

Bradykinin and Neurotensin Analogues as Potential Compounds in Colon Cancer Therapy.

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential.

Improving Fmoc Solid Phase Synthesis of Human Beta Defensin 3.

Human ß-defensin 3, HBD-3, is a 45-residue antimicrobial and immunomodulatory peptide that plays multiple roles in the host defense system. In addition to interacting with cell membranes, HBD-3 is also a ligand for melanocortin receptors, cytokine receptors and voltage-gated potassium channels. Structural and functional studies of HBD-3 have been hampered by inefficient synthetic and recombinant expression methods. Herein, we report an optimized Fmoc solid-phase synthesis of this peptide using an orthogonal disulfide bonds formation strategy. Our results suggest that utilization of an optimized resin, coupling reagents and pseudoproline dipeptide building blocks decrease chain aggregation and largely improve the amount of the target peptide in the final crude material, making the synthesis more efficient. We also present an alternative synthesis of HBD-3 in which a replacement of a native disulfide bridge with a diselenide bond improved the oxidative folding. Our work enables further biological and pharmacological characterization of HBD-3, hence advancing our understanding of its therapeutic potential.

Lipidation of Temporin-1CEb Derivatives as a Tool for Activity Improvement, Pros and Cons of the Approach.

The alarming raise of multi-drug resistance among human microbial pathogens makes the development of novel therapeutics a priority task. In contrast to conventional antibiotics, antimicrobial peptides (AMPs), besides evoking a broad spectrum of activity against microorganisms, could offer additional benefits, such as the ability to neutralize toxins, modulate inflammatory response, eradicate bacterial and fungal biofilms or prevent their development. The latter properties are of special interest, as most antibiotics available on the market have limited ability to diffuse through rigid structures of biofilms. Lipidation of AMPs is considered as an effective approach for enhancement of their antimicrobial potential and in vivo stability; however, it could also have undesired impact on selectivity, solubility or the aggregation state of the modified peptides. In the present work, we describe the results of structural modifications of compounds designed based on cationic antimicrobial peptides DK5 and CAR-PEG-DK5, derivatized at their N-terminal part with fatty acids with different lengths of carbon chain. The proposed modifications substantially improved antimicrobial properties of the final compounds and their effectiveness in inhibition of biofilm development as well as eradication of pre-formed 24 h old biofilms of Candida albicans and Staphylococcus aureus. The most active compounds (C5-DK5, C12-DK5 and C12-CAR-PEG-DK5) were also potent against multi-drug resistant Staphylococcus aureus USA300 strain and clinical isolates of Pseudomonas aeruginosa. Both experimental and in silico methods revealed strong correlation between the length of fatty acid attached to the peptides and their final membranolytic properties, tendency to self-assemble and cytotoxicity.

[Characteristic of AMP and the effects of chemical modifications on the modulation of their antimicrobial properties]. / Charakterystyka peptydów przeciwdrobnoustrojowych oraz wplyw modyfikacji chemicznych na modulowanie ich aktywnosci biologicznej.

Antibiotics have revolutionized the pharmacology market but their "golden area" passed away. The urgent need to develop new medicins with the mechanism of action different than those already used is constantly growing because of the drug-resistance of pathogenic microorganisms. The observed increasing resistance of microbes motivates scientists to design innovative strategies based on the natural peptides that exhibit antimicrobial activity. In this article we present general characterization of antimicrobial peptides and effects of chemical modifications on the modulation of their antimicrobial properties.

Evaluation of sample injection precision in respect to sensitivity in capillary electrophoresis using various injection modes.

A comparative study was conducted to assess the injection precision in capillary electrophoresis for cationic analytes (arecoline, codeine, papaverine). The precision was measured in respect to methods sensitivity in various injection modes in capillary electrophoresis: standard hydrodynamic injection (3.45 kPa for 6 s), large volume sample stacking (3.45 kPa for 40 s), and field-amplified sample injection (10 kV for 65 s). All measurements were conducted for aqueous solutions of standards to minimize the errors linked to the sample preparation step. The methods were submitted to precision assessment at three concentration levels: at the limit of quantification, three-fold and ten-fold of limit of quantification. The results were compared to those from high-performance liquid chromatography as a reference technique. The field-amplified sample injection method was shown to provide greatest sensitivity (quantification limits down to 4 ng/mL for all three tested compounds) but the lowest precision. High-performance liquid chromatography was established as the most reliable technique (coefficient of variation in all intraday experiments was below 1%). It was also shown that with a use of large volume sample injection technique, similar sensitivity as in high-performance liquid chromatography can be easily reached.

[The role of proprotein convertases in cancer diseases with particular focus on PACE4]. / Rola konwertaz probialkowych w chorobach nowotworowych ze szczególnym uwzglednieniem enzymu PACE4.

Cancer is one of the most common cause of death nowadays. Thorough knowledge of the mechanisms of tumorigenesis and invasiveness of tumor cells is crucial for the development of molecular targeted therapies, which are believed to be future treatment of this type of diseases. Proteolytic enzymes are one of the factors involved in the development of cancer cells, very often used as markers of tumor progression. In this paper we describe the role of enzymes termed proprotein convertases (PCs) in pathogenesis and progress of cancer diseases. Furthermore, we indicate potential directions for the development of therapeutic strategies designed based on PCs inhibitors.

Potent antidiuretic agonists, deamino-vasopressin and desmopressin, and their inverso analogs: NMR structure and interactions with micellar and liposomic models of cell membrane.

Deamination of vasopressin (AVP) enhances its antidiuretic activity. Moreover, introduction of D-Arg8 instead of its L enantiomer in deamino-vasopressin (dAVP) results in an extremely potent and selective antidiuretic agonist - desmopressin (dDAVP). In this study we describe the synthesis, pharmacological properties and structures of these two potent antidiuretic agonists, and their inverso analogs. The structures of the peptides are studied in micellar and liposomic models of cell membrane using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy supported by molecular dynamics simulations. Our conformational studies have shown that desmopressin in a membrane mimicking environment adopts one of the characteristic for vasopressin-like peptides ß-turn - in position 3,4. Furthermore, dDAVP shows the tendency to create a ß-turn in the Cys6-Gly9 C-tail, considered to be important for the antidiuretic activity, and also some tendency to adopt a 5,6 ß-turn. In desmopressin, in contrast to the native vasopressin, deamino-vasopressin and [D-Arg8]-vasopressin (DAVP), the Arg8 side chain, crucial for the pressor and antidiuretic activities, is very well exposed for interaction with the receptor, whereas Gly9, crucial for the pressor and uterotonic activities, is situated together with the C-terminal amide group very close to the tocin ring. The arrangements of the Gln4 and Asn5 side chains, being crucial for OT activity, also differ in desmopressin as compared to those of AVP, dAVP and DAVP. These differences in arrangement of the important for activities side chains are likely to explain extremely potent and selective antidiuretic activities of desmopressin. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 245-259, 2016.

[Proprotein convertases - family of serine proteases with a broad spectrum of physiological functions]. / Konwertazy probialkowe - rodzina proteaz serynowych o szerokim spektrum funkcji fizjologicznych.

A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.

Human ß-defensin 4 - defensin without the "twist".

ß-defensins are small, cysteine-rich, cationic peptides that contribute to various processes related to both arms of host defense, the innate and adaptive immunities. All ß-defensins are potent antimicrobials with activity targeting a broad range of pathogens. Some human ß-defensins (hBDs) are also capable of binding and activating specific chemokine receptors, leading to chemotaxis of receptor-presenting cells. Two receptors identified as targets of specific human ß-defensins are CCR2 and CCR6, both members of the seven-transmembrane family of chemokine receptors. Currently, around 50 open reading frames (ORFs) identified in the human genome encode proteins that have signatures characteristic of ß-defensins. Of those, only three, hBD1-3, have been thoroughly characterized to date, including a detailed structural description of their molecules. In addition, limited information on biological and bactericidal properties is available for hBD4, as well as the solution structure of hBD6. The crystal structure of hBD4, determined here at resolution of 1.60 Å, indicates significant structural differences between this molecule and those reported previously for other hBDs. Crystallographic studies indicate a possibility of unique dimerization of hBD4, confirmed by solution studies using analytical ultracentrifugation. In contrast to hBD1-3, hBD4 does not induce CCR6-mediated chemotaxis. This observation can be attributed to an unusual conformation of the hBD4 N-terminus. In agreement with previously published reports, hBD4 was shown to be a potent antibacterial agent, as demonstrated by results of assays with E. coli ATCC 25922 cells.

Arginine-, D-arginine-vasopressin, and their inverso analogues in micellar and liposomic models of cell membrane: CD, NMR, and molecular dynamics studies.

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [D-Arg(8)]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides ß-turns: in the 2-5 and 3-6 fragments. The inverso-analogues also adopt ß-turns in the 3-6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr(2), Phe(3), and Arg(8), important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr(2) and Phe(3) in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide-membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic-zwitterionic liposomes as well as the anionic-zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.

Improvement of derivatized amino acid detection sensitivity in micellar electrokinetic capillary chromatography by means of acid-induced pH-mediated stacking technique.

Derivatization is a frequently used sample preparation procedure applicable to the enhancement of analyte detection sensitivity. Amino acids mostly require derivatization prior to electrophoretic or chromatographic analysis, especially if spectrophotometric detection is used. This study presents an on-line preconcentration technique for derivatized amino acids. The sensitivity of the method was improved by the utilization of the proposed acid-induced pH-mediated stacking mechanism. The method is demonstrated by preconcentration of amino acids labeled with 2,4-dinitrofluorobenzene. Use of optimized conditions for a large sample volume injection (40 s, 13.8 kPa) followed by electrokinetic injection of 0.1 M HCl (20 s, 10 kV) gave a 20- to 30-fold enhancement of sensitivity. The significance of the sweeping mechanism and pseudo-isotachophoresis for the on-line sample focusing and the influence of parameters on the preconcentration process were discussed. The applicability of the elaborated method was demonstrated using human urine samples.

Electrochemical detection of bacterial endotoxin lipopolysaccharide (LPS) on gold electrode modified with DAL-PEG-DK5-PEG-OH - Antimicrobial peptide conjugate.

This work describes fabrication of gold electrodes modified with peptide conjugate DAL-PEG-DK5-PEG-OH that enables ultra-sensitive detection of lipopolysaccharide (LPS) isolated from the reference strain of Escherichia coli O26:B6. The initial step of the established procedure implies immobilization of the fully protected DAL-PEG-DK5-PEG-OH peptide on the surface of the gold electrode previously modified by cysteamine. Then side chain- and Fmoc-deprotection was performed in situ on the electrode surface, followed by its incubation in 1 % of BSA solution to block non-specific bindings sites before LPS detection. The efficiency of the modification was confirmed by X-ray Photoelectron Spectroscopy (XPS) measurements. Additionally, the cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS) were employed to monitor the effectiveness of each step of the modification. The obtained results confirmed that the presence of the surface-attached covalently bound peptide DAL-PEG-DK5-PEG-OH enables LPS detection by means of CV technique within the range from 5 × 10-13 to 5 × 10-4 g/mL in PBS solution. The established limit of detection (LOD) for EIS measurements was 4.93 × 10-21 g/mL with wide linear detection range from 5 × 10-21 to 5 × 10-14 g/mL in PBS solution. Furthermore, we confirmed the ability of the electrode to detect LPS in a complex biological samples, like mouse urine and human serum. The effectiveness of the electrodes in identifying LPS in both urine and serum matrices was confirmed for samples containing LPS at both 2.5 × 10-15 g/mL and 2.5 × 10-9 g/mL.

Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists.

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).

Spectroscopic characterization and in vitro studies of biological activity of bradykinin derivatives.

Eleven multiple analogs of bradykinin-a peptide that is a natural ligand of B1 and B2 receptors but does not bind or activate the B1 receptor unless Arg9 is removed from the sequence by the action of carboxypeptidase N-were synthesized. Their biological activity was examined on T-REx cell lines expressing B1 or B2 receptors using the intracellular IP1 assay. The mRNA expression of B1R and B2R in the lysate of tumor cell lines, e.g., U87-MG (human astrocytoma), SHP-77 (human small cell lung cancer), and H4 (human brain glioma), was determined. For five B1R antagonists, adsorption at the liquid/solid interface (Au nanoparticles (AuNPs) served as the solid surface) was discussed in terms of the vibrations of molecular fragments (structural factors) responsible for the biological properties of these analogs.

Understanding the Role of Self-Assembly and Interaction with Biological Membranes of Short Cationic Lipopeptides in the Effective Design of New Antibiotics.

This study investigates short cationic antimicrobial lipopeptides composed of 2-4 amino acid residues and C12-C18 fatty acids attached to the N-terminal part of the peptides. The findings were discussed in the context of the relationship among biological activity, self-assembly, stability, and membrane interactions. All the lipopeptides showed the ability to self-assemble in PBS solution. In most cases, the critical aggregation concentration (CAC) much surpassed the minimal inhibitory concentration (MIC) values, suggesting that monomers are the main active form of lipopeptides. The introduction of ß-alanine into the peptide sequence resulted in a compound with a high propensity to fibrillate, which increased the peptide stability and activity against S. epidermidis and C. albicans and reduced the cytotoxicity against human keratinocytes. The results of our study indicated that the target of action of lipopeptides is the bacterial membrane. Interestingly, the type of peptide counterion may affect the degree of penetration of the lipid bilayer. In addition, the binding of the lipopeptide to the membrane of Gram-negative bacteria may lead to the release of calcium ions necessary for stabilization of the lipopolysaccharide layer.

Novel analogues of bradykinin conformationally restricted in the C-terminal part of the molecule.

In the present work, achiral non-coded amino acids, N-(Bzl)-Gly, X(1) or X(2) , were substituted at position 7 of the model B(2) receptor antagonist [D-Arg(0) , Hyp(3) , Thi(5, 8) , D-Phe(7) ]-BK. The N-terminal amino group of the analogues was either free or acylated with 1-Aca or Aaa. Biological activity of the compounds was assessed in the in vitro rat uterus test and the in vivo rat blood pressure test. The X(1) (7) substitution resulted in a decrease in antagonistic potency of the new peptide in both assays. The X(2) (7) and N-(Bzl)-Gly(7) substituted analogues showed weak agonistic properties in the rat uterus test. Interestingly, the latter compound exhibited dual activity in the pressor test, i.e. intrinsic vasodepressor action and at the same time a weak antagonistic effect. Acylation of the N-terminus enhanced antagonistic properties of the resulting peptides in the rat blood pressure test in the case of compounds containing X(1) or X(2) modification. Our studies provide new information about structure-activity relationship of the BK antagonists which may be helpful for designing more potent B(2) receptor blockers.

Application of Alanine Scanning to Determination of Amino Acids Essential for Peptide Adsorption at the Solid/Solution Interface and Binding to the Receptor: Surface-Enhanced Raman/Infrared Spectroscopy versus Bioactivity Assays.

The article describes the application of the alanine-scanning technique used in combination with Raman, surface-enhanced Raman, attenuated total reflection Fourier transform infrared, and surface-enhanced infrared absorption (SEIRA) spectroscopies, which allowed defining the role of individual amino acid residues in the C-terminal 6-14 fragment of the bombesin chain (BN6-14) on the path of its adsorption on the surface of Ag (AgNPs) and Au nanoparticles (AuNPs). A reliable analysis of the SEIRA spectra of these peptides was possible, thanks to a curve fitting of these spectra. By combining alanine-scanning with biological activity studies using cell lines overexpressing bombesin receptors and the intracellular inositol monophosphate assay, it was possible to determine which peptide side chains play a significant role in binding a peptide to membrane-bound G protein-coupled receptors (GPCRs). Based on the analysis of spectral profiles and bioactivity results, conclusions for the specific peptide-metal and peptide-GPCR interactions were drawn and compared.

Design and Structure-Activity Relationship of a Potent Furin Inhibitor Derived from Influenza Hemagglutinin.

Furin plays an important role in various pathological states, especially in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH 2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to determine how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.

Novel analogues of arginine vasopressin containing alpha-2-indanylglycine enantiomers in position 2.

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with alpha-2-indanylglycine or its D-enantiomer (Igl or D-Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl(2) substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA(2) values ranging from 7.19 to 7.98) and practically did not interact with V(1a) and V(2) receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D-Igl(2) substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D-Igl peptides showed a strong antioxytocic potency (pA(2) values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues.