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.

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.

[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.

[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.

A marine analgesic peptide, Contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties.

Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

Expanding chemical diversity of conotoxins: peptoid-peptide chimeras of the sodium channel blocker µ-KIIIA and its selenopeptide analogues.

The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys(7) residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys(7) with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfide-depleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Nav1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar(7)]KIIIA, the most potent in blocking the channels while favouring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.

Selenopeptide analogs of EETI-II retain potent trypsin inhibitory activities.

Three-disulfide-bridged Ecballium elaterium trypsin inhibitor II (EETI-II) is a 28-residue peptide that belongs to the squash family of canonical trypsin inhibitors. Herein, we report synthesis and biological activity of three EETI-II analogs. In each of analog, a pair of cysteine residues forming a native disulfide bridge was individually replaced by a pair of selenocysteine residues. All selenopeptide analogs were chemically synthesized using the Fmoc protocol and subsequently folded in the presence of oxidized and reduced glutathione. The analogs containing a diselenide bridge displayed association constants with trypsin that ranged from 2.6 x 10(9) to 5.1 x 10(9) [M(-1) ]. Our results suggest that the selenopeptide analogs retained low nanomolar inhibitory potencies, and only the diselenide bridge adjacent to the inhibitory binding loop weakened the interactions with trypsin by approximately fivefold. We discuss these findings in the context of a broader use of selenopeptide analogs as proxies to study cysteine-rich peptides.

Disulfide-Depleted Selenoconopeptides: a Minimalist Strategy to Oxidative Folding of Cysteine-Rich Peptides.

Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged mu-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide's ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of mu-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide's selectivity in blocking a neuronal (Na(v)1.2) over a muscle (Na(v)1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.

Cooccupancy of the outer vestibule of voltage-gated sodium channels by micro-conotoxin KIIIA and saxitoxin or tetrodotoxin.

The guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX) are classic ligands of voltage-gated sodium channels (VGSCs). Like TTX and STX, micro-conotoxin peptides are pore blockers but with greater VGSC subtype selectivity. micro-Conotoxin KIIIA blocks the neuronal subtype Na(V)1.2 with nanomolar affinity and we recently discovered that KIIIA and its mutant with one fewer positive charge, KIIIA[K7A], could act synergistically with TTX in a ternary peptide x TTX x Na(V) complex. In the complex, the peptide appeared to trap TTX in its normal binding site such that TTX could not readily dissociate from the channel until the peptide had done so; in turn, the presence of TTX accelerated the rate at which peptide dissociated from the channel. In the present study we examined the inhibition of Na(V)1.2, exogenously expressed in Xenopus oocytes, by STX (a divalent cation) and its sulfated congener GTX2/3 (with a net +1 charge). Each could form a ternary complex with KIIIA and Na(V)1.2, as previously found with TTX (a monovalent cation), but only when STX or GTX2/3 was added before KIIIA. The KIIIA x alkaloid x Na(V) complex was considerably less stable with STX than with either GTX2/3 or TTX. In contrast, ternary KIIIA[K7A] x alkaloid x Na(V) complexes could be formed with either order of ligand addition and were about equally stable with STX, GTX2/3, or TTX. The most parsimonious interpretation of the overall results is that the alkaloid and peptide are closely apposed in the ternary complex. The demonstration that two interacting ligands ("syntoxins") occupy adjacent sites raises the possibility of evolving a much more sophisticated neuropharmacology of VGSCs.

Integrated oxidative folding of cysteine/selenocysteine containing peptides: improving chemical synthesis of conotoxins.

Building bridges: The use of diselenide and selectively ((15)N/(13)C)-labeled disulfide bridges is combined to give improvements in oxidative folding and disulfide mapping. Conotoxin analogues, each with a pair of selenocysteines (Sec) and labeled cysteines (see scheme, red), exhibited significantly improved folding and the labeled cysteines allow correctly folded species to be rapidly identified by NMR spectroscopy.

Structurally minimized mu-conotoxin analogues as sodium channel blockers: implications for designing conopeptide-based therapeutics.

Disulfide bridges that stabilize the native conformation of conotoxins pose a challenge in the synthesis of smaller conotoxin analogues. Herein we describe the synthesis of a minimized analogue of the analgesic mu-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal Na(V)1.2 and skeletal muscle Na(V)1.4. Three disulfide-deficient analogues of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15, or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide's bioactivity. To further minimize this analogue, the N-terminal residue was removed and two nonessential serine residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting "polytide" analogue retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogues containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.

NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA.

Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.

Role of hydroxyprolines in the in vitro oxidative folding and biological activity of conotoxins.

Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.