A major challenge to the treatment of advanced prostate cancer (PCa) is the development of resistance to androgen-deprivation therapy (ADT) and chemotherapy. It is imperative to discover effective therapies to overcome drug resistance and improve clinical outcomes. We have developed a novel class of silicon-containing compounds and evaluated the anticancer activities and mechanism of action using cellular and animal models of drug-resistant PCa. Five organosilicon compounds were evaluated for their anticancer activities in the NCI-60 panel and established drug-resistant PCa cell lines. GH1504 exhibited potent in vitro cytotoxicity in a broad spectrum of human cancer cells, including PCa cells refractory to ADT and chemotherapy. Molecular studies identified several potential targets of GH1504, most notably androgen receptor (AR), AR variant 7 (AR-v7) and survivin. Mechanistically, GH1504 may promote the protein turnover of AR, AR-v7 and survivin, thereby inducing apoptosis in ADT-resistant and chemoresistant PCa cells. Animal studies demonstrated that GH1504 effectively inhibited the in vivo growth of ADT-resistant CWR22Rv1 and chemoresistant C4-2B-TaxR xenografts in subcutaneous and intraosseous models. These preclinical results indicated that GH1504 is a promising lead that can be further developed as a novel therapy for drug-resistant PCa.

We have investigated the effect of deuteration of non-exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well-known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G-binding domain of protein G (GB3). Reversed-phase high-performance liquid chromatography (RP-HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP-HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross-correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub-nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at Cα -Cβ sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.

We designed de novo and synthesized two series of five 26-residue amphipathic α-helical cationic antimicrobial peptides (AMPs) with five or six positively charged residues (D-Lys, L-Dab (2,4-diaminobutyric acid) or L-Dap (2,3-diaminopropionic acid)) on the polar face where all other residues are in the D-conformation. Hemolytic activity against human red blood cells was determined using the most stringent conditions for the hemolysis assay, 18h at 37°C, 1% human erythrocytes and peptide concentrations up to 1000 μg/mL (~380 μM). Antimicrobial activity was determined against 7 Acinetobacter baumannii strains, resistant to polymyxin B and colistin (antibiotics of last resort) to show the effect of positively charged residues in two different locations on the polar face (positions 3, 7, 11, 18, 22 and 26 versus positions 3, 7, 14, 15, 22 and 26). All 10 peptides had two D-Lys residues in the center of the non-polar face as “specificity determinants” at positions 13 and 16 which provide specificity for prokaryotic cells over eukaryotic cells. Specificity determinants also maintain excellent antimicrobial activity in the presence of human sera. This study shows that the location and type of positively charged residue (Dab and Dap) on the polar face are critical to obtain the best therapeutic indices.

We have designed de novo and synthesized ten 26-residue D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), seven with "specificity determinants", which provide specificity for prokaryotic cells over eukaryotic cells. The ten AMPs contain five or six positively charged residues (d-Arg, d-Lys, d-Orn, l-Dab, or l-Dap) on the polar face to understand their role in hemolytic activity against human red blood cells and antimicrobial activity against seven Acinetobacter baumannii strains, resistant to polymyxin B and colistin, and 20 A. baumannii worldwide isolates from 2016 and 2017 with antibiotic resistance to 18 different antibiotics. AMPs with specificity determinants and with l-Dab and l-Dap residues on the polar face have essentially no hemolytic activity at 1000 μg/mL (380 μM), showing for the first time the importance of these unusual amino acid residues in solving long-standing hemolysis issues of AMPs. Specificity determinants maintained excellent antimicrobial activity in the presence of human sera.

We are currently examining the potential of amphipathic cationic alpha-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed. Currently, we have designed de novo a mixture of six 26-residue all D-conformation amphipathic cationic alpha-helical peptides with a single, positively charged lysine residue in the center of the non-polar face and an increasing number of lysine residues (4-9 residues) replacing neutral residues in the polar face, resulting in an overall net positive charge of +5 to +10. Thus, the non-polar, preferred reversed-phase chromatography binding face remains constant, with only the polar face varying in hydrophilicity/hydrophobicity. Interestingly, even with the non-polar face remaining constant, reversed-phase columns of varying functional group properties (e.g., C-8, C-18, phenyl, polar endcapped, polar embedded) and porosity (porous versus superficially porous) were able to separate the six peptides in aq. TFA/acetonitrile gradients, albeit with different selectivities. The value of the standards in cation-exchange chromatography was expressed by monitoring the requirement of acetonitrile (0-40% in the mobile phase) to overcome hydrophobic interactions of the peptides with the cation-exchange matrix matrix when eluting with sodium perchlorate gradients at pH 6.5. Interestingly, the resolution of the higher charged peptides (+8,+9,+10) was particularly sensitive to acetonitrile levels. Our results clearly demonstrate the excellent potential of these novel peptide standards to enable optimal column choice and mobile phase conditions for reversed-phase chromatography and cation-exchange chromatography for peptide separations. (C) 2018 Elsevier B.V. All rights reserved.

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

We have developed a heterodimeric coiled-coil system based on two complementary peptides, namely (EVSALEK)5 and (KVSALKE)5, or E and K, for the attachment of E-tagged biomolecules onto K-decorated biomaterials. We here explore two approaches to control the strength and the stability of the E/K coiled-coil complex, and thus its potential for the controlled release of biomolecules. Those are Leucine-to-Alanine mutations in the K peptide (4 peptides with 0 to 3 mutations) and multivalent presentation of the E peptide (6 bio-objects from monomeric to dimeric and n-meric). Using E-tagged growth factors and nanoparticles as models, SPR-based assays performed under continuous flow indicated that the release rate was strongly affected by both approaches independently, and that the strength of the capture could be finely tuned over a wide range (apparent dissociation constant from 0.12 pM to 270 nM). Further release assays carried out in well-plates showed that the multivalent presentation only had a significant influence in this setup since the wells were not rinsed under continuous flow.

We have designed de novo and synthesized eight 26‐residue all D‐conformation amphipathic α‐helical cationic antimicrobial peptides (AMPs), four with “specificity determinants” which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin‐sensitive, and eight of which were methicillin‐resistant. The four AMPs without specificity determinants are extremely hemolytic. In contrast, the four AMPs with specificity determinants had dramatic improvements in therapeutic indices showing the importance of specificity determinants in removing eukaryotic cell toxicity. The specificity determinants combined with the location of positively charged residues on the polar face provide Gram‐negative pathogen selectivity between A. baumannii and S. aureus. Specificity determinants maintain excellent antimicrobial activity in the presence of human sera, whereas the AMPs without specificity determinants were inactive. This study clearly shows the potential of amphipathic α‐helical AMPs with specificity determinants as therapeutics to replace existing antibiotics.

Event Abstract Back to Event Coiled-coil interactions: a versatile bioaffinity system for the oriented immobilization and tunable release of biomolecules from biomaterials Frederic Murschel1*, Charles Fortier1, Robert Hodges2*, Mario Jolicoeur1 and Gregory De Crescenzo1* 1 Ecole Polytechnique de Montréal, Department of Chemical Engineering, Canada 2 University of Colorado, School of Medicine, Structural Biology and Biophysics Core Facilities, United States Introduction: The coiled-coil assembly – a natural motif found ubiquitously in proteins – has recently (re)gained attention as a powerful tool for the development of biomaterials, in particular peptide-based and peptide-polymer hybrid nanomaterials[1],[2]. We have successfully used de novo designed complementary E and K peptides for the grafting of E-tagged growth factors on various K-decorated biomaterials (Fig. 1A). The specific attachment of the molecules mediated by the E/K coiled-coil interactions has notably demonstrated more potency when compared to non-site specific grafting methods[3]. This tool has been since refined, and we report here our work on the fine-tuning of the stability and affinity of the assembly by (a) precise residue substitutions in the K peptide and by (b) changes in the numbers of available E moieties on the grafted molecule. Materials and Methods: Coil peptides and coil-tagged proteins were produced and purified as previously described[4]. Coil-decorated nanoparticles were produced by (i) auto-assembly of branched poly(ethylene imine) (PEI) and carboxymethylated dextran (CMD) bearing vinyl sulfone (VS) reactive groups then (ii) by reacting the cysteine-terminated E peptides with the VS groups. Surface plasmon resonance (SPR)-based assays were performed at 100 µL/min on a Biacore® T100 biosensor, using HBS-EP as running buffer. Results and Discussion: Three K peptide analogs were designed with 1 to 3 Leucine-to-Alanine substitutions as a means to destabilize the hydrophobic core of the E/K assembly (Fig. 1A-B). SPR-based assays of the interaction of the E peptide with the K analogs were performed (Fig. 2A), and the kinetic analysis of the sensorgrams indicated that a wide range of affinities could be obtained, with apparent dissociation constants ranging from 137 pM to 14.8 nM. Further SPR-based assays were performed with relevant biomolecules bearing a varying number of E moieties: E-tagged epidermal- and vascular endothelial growth factor, namely E-EGF and E-VEGF, as well as E-decorated PEI/CMD nanoparticles that could be used for nucleic acid encapsulation (Fig. 1C). The normalized responses obtained for a 50-nM injection of the tagged growth factors indicated that the number of E moieties affected both association and dissociation rates, as well as the amount that was recruited (data not shown). The enhancement of protein capture and stability was attributed to multivalent interactions occurring between the dimeric E-VEGF and the K-decorated surface, i.e., avidity causing an increase in the apparent affinity. Avidity was more plainly evidenced when nanoparticles bearing varying E densities were injected over the K peptide (Fig. 2B). Conclusion: Two levers – the sequence of the K peptide and the number of E moieties – were here identified in the use of coiled-coil interactions for the controlled grafting and release of biomolecules, be it for the direct protein attachment onto tissue engineering scaffold or the transport of drugs or nucleic acids within nanoparticles. This work was supported by the Canada Research Chair on Protein-Enhanced Biomaterials (G.D.C.), the Canada Research Chair in Applied Metabolic Engineering (M.J.), by the Natural Sciences and Engineering Research Council of Canada (G.D.C. and M.J.), by the Fonds de recherche du Québec - Nature et technologies (F.M. and C.F.) and by the MEDITIS training program (F.M. and C.F.).; We thank Josianne Lefebvre for technical support and fruitful discussionReferences:[1] Gerling-Driessen, U. I. M.; Mujkic-Ninnemann, N.; Ponader, D.; Schöne, D.; Hartmann, L., Exploiting Oligo(amido amine) Backbones for the Multivalent Presentation of Coiled-Coil Peptides. Biomacromolecules 2015, 16, (8), 2394-2402.[2] Aronsson, C.; Dånmark, S.; Zhou, F.; Öberg, P.; Enander, K.; Su, H.; Aili, D., Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties. Scientific Reports 2015, 5, 14063.[3] Lequoy, P.; Liberelle, B.; De Crescenzo, G.; Lerouge, S., Additive benefits of chondroitin sulfate and oriented tethered epidermal growth factor for vascular smooth muscle cell survival. Macromol. Biosci. 2014, 14, (5), 720-30.[4] Murschel, F.; Liberelle, B.; St-Laurent, G.; Jolicoeur, M.; Durocher, Y.; De Crescenzo, G., Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor. Acta Biomater. 2013, 9, (6), 6806-6813. Keywords: self-assembly, biosensing, growth factor, biofunctionalization Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Protein interactions with biomaterials Citation: Murschel F, Fortier C, Hodges R, Jolicoeur M and De Crescenzo G (2016). Coiled-coil interactions: a versatile bioaffinity system for the oriented immobilization and tunable release of biomolecules from biomaterials. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01520 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Frederic Murschel, Ecole Polytechnique de Montréal, Department of Chemical Engineering, Montréal, QC, Canada, Email1 Dr. Robert Hodges, University of Colorado, School of Medicine, Structural Biology and Biophysics Core Facilities, Aurora, CO, United States, robert.hodges@ucdenver.edu Dr. Gregory De Crescenzo, Ecole Polytechnique de Montréal, Department of Chemical Engineering, Montréal, QC, Canada, gregory.decrescenzo@polymtl.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Frederic Murschel Charles Fortier Robert Hodges Mario Jolicoeur Gregory De Crescenzo Google Frederic Murschel Charles Fortier Robert Hodges Mario Jolicoeur Gregory De Crescenzo Google Scholar Frederic Murschel Charles Fortier Robert Hodges Mario Jolicoeur Gregory De Crescenzo PubMed Frederic Murschel Charles Fortier Robert Hodges Mario Jolicoeur Gregory De Crescenzo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C-terminal domain. Orthologues with and without a C-terminal, α-helical coiled-coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)-dependent manner. The roles of phospholipids and the C-terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C-terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled-coil formation, the C-terminal domain bound liposomes and ProP concentrated at the cell poles in a CL-independent manner. The presence of the coiled-coil replaced those phenomena with CL-dependent binding and localization. The effects of amino acid replacements on lipid association of the C-terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C-terminal domain with the anionic lipid-enriched membrane at the cell poles. The coiled-coil domain present on only some orthologues renders that phenomenon CL-dependent.

We have utilized a de novo designed two-stranded α-helical coiled-coil template to display conserved α-helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface-exposed residues of the native α-helix in the native HA protein of interest displayed on the surface of the two-stranded α-helical coiled-coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α-helix in the native protein. We investigated the highly conserved sequence region 421-476 of HA by inserting 21 or 28 residue sequences from this region into our template. The cross-reactivity of the resulting rabbit polyclonal antibodies prepared to these immunogens was determined using a series of HA proteins from H1N1, H2N2, H3N2, H5N1, H7N7, and H7N9 virus strains which are representative of Group 1 and Group 2 virus subtypes of influenza A. Antibodies from region 449-476 were Group 1 specific. Antibodies to region 421-448 showed the greatest degree of cross-reactivity to Group 1 and Group 2 and suggested that this region has a great potential as a "universal" synthetic peptide vaccine for influenza A. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 144-159, 2016.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC. These materials are characterized by a solid core surrounded by a thin layer of porous material, and represent a technology for the future with continuing refinements. Such refinements are aided via the use of designed synthetic peptide standards during stationary-phase development. Concomitantly, such standards also enable the researcher to monitor RP-HPLC column performance and develop optimized separation protocols for peptides from a wide array of sources. © 2016 by John Wiley & Sons, Inc.

Antimicrobial peptides (AMPs) are widely distributed in nature, generally have broad-spectrum activity and represent a promising class of new antimicrobial agents. However, it is widely accepted that native AMPs lack specificity and may be too toxic (ability to lyse mammalian cells, normally expressed as hemolytic activity against human red blood cells) to be used for systemic treatment [1,2]. To overcome this problem, we developed the design concept of “specificity determinants” which refers to substituting positively charged residue(s) in the center of the non-polar face of amphipathic cyclic β-sheet [3,4] or amphipathic α-helical AMPs [5] to create selectivity between eukaryotic and prokaryotic membranes; that is, antimicrobial activity is improved or maintained and hemolytic activity or cell toxicity to mammalian cells is decreased or eliminated. We showed that a single valine to lysine substitution in the center of the non-polar face of an AMP dramatically reduced toxicity and increased the therapeutic index [5-7]. The question arose could we take such a broad spectrum AMP in the all-D conformation and use a rational design approach to enhance further the biological properties if the focus was to develop a better Gram-negative AMP rather than maintain broad-spectrum activity. Our final AMP had a 746fold improvement (i.e., decrease) in its hemolytic activity, improved antimicrobial activity and improved therapeutic indices by 1305-fold and 895-fold against Acinetobacter baumannii and Pseudomonas aeruginosa, respectively [7]. We applied this design concept to native AMPs, piscidin 1 (isolated from mast cells of hybrid striped bass Morone saxatilis male × Morone chrysops female) and dermaseptin S4 (isolated from the skin of tree-dwelling, South American frogs of the Phyllomedusa species), where substitution of one or two lysine residues at different positions in their non-polar faces enhanced or maintained Gram-negative activity, dramatically decreased hemolytic activity and significantly improved the therapeutic indices (55-fold and 730-fold for D-piscidin 1 I9K and D-dermaseptin S4 L7K, A14K against A. baumannii, respectively) [8]. In the current study, we used the above 2 native AMPs and tested their activity against 2 different pathogens: 11 and 20 diverse clinical isolates of A. baumannii, and Staphylococcus aureus (12 Methicillin-sensitive S. aureus strains and 8 Methicillin/Oxacillin-resistant S. aureus strains), respectively. We showed that substitution of “specificity determinant(s)” in broad spectrum AMPs, encode selectivity for Gram-negative pathogens and simultaneously remove both Gram-positive activity and hemolytic activity of these 2 diverse amphipathic α-helical AMPs which differ dramatically in amino acid composition, net positive charge and amphipathicity, showing generality of our approach.

Cancer is a major public health problem in the United States and throughout the world. It is currently the second leading cause of death in the United States and is expected to surpass heart diseases in the next few years to become the leading cause of death [1]. The estimated number of new cases of invasive cancer (all types) in the United States is 1,658,370 which is equivalent of more than 4,500 new cancer diagnoses each day. In addition, the estimated number of deaths from cancer in 2015 is 589,430 corresponding to about 1,600 deaths per day [1]. Though there has been a steady increase in survival for most cancers the death rate remains unacceptable and for certain cancers i.e. lung and pancreatic cancers the 5-year relative survival is currently 18% and 7%, respectively. Traditional chemotherapy drugs act against all actively dividing cells (normal and cancerous cells) whereas targeted cancer therapies are drugs that interfere with specific molecular targets involved in cancer cell growth, progression and spread of cancer. Most targeted therapies are either small molecules or monoclonal antibodies. However, therapeutic strategies that target single molecular pathways eventually succumb to problems of intrinsic or acquired resistance due to extensive signaling “cross talk”. Thus, combination targeted therapies are more attractive, as they synergistically inhibit multiple receptors. However, overlapping toxicities and pharmacological interactions limit patient compliance, feasibility and efficacy. Clearly, there is an urgent need to develop new first-line agents with enhanced efficacy and reduced toxicity. We support the concept that the ideal drug maybe a broad spectrum drug whose efficacy is based not on the inhibition of a single target but rather a multi-targeted drug that affects several proteins or events that contribute to the etiology, pathogenesis and progression of diseases [2]. In addition, multipathway targeting is one of the strategies to overcome chemo-resistance. To design novel anticancer drugs with unique structural properties we have taken an innovative and nontraditional approach where we combine pharmacophoric components to create new and highly potent small molecules with a simple three component “A-B-C” structure where each pharmacophore is known to have anticancer properties on its own or when incorporated as a component of an existing drug. Our multi-component “A-B-C” drugs can target simultaneously two or more different molecular targets or molecular mechanisms in a single entity which should reduce the likelihood of drug resistance.

Influenza A viruses spread rapidly, causing widespread seasonal epidemics of respiratory disease worldwide, which results in more than a billion cases and 500,000 deaths annually [1]. Current influenza vaccines primarily elicit antibodies against the receptor-binding region of the head domain of the hemagglutinin (HA) glycoprotein trimer (Figure 1). This region is hyper-variable and highly mutable, leading to new forms of the virus that can evade neutralizing antibodies. The stem region of HA contains highly conserved α-helical sequences as a result of their functional role in membrane fusion and virus entry. Studies have shown that a few rare neutralizing human monoclonal antibodies can recognize these highly conserved epitopes and neutralize both homotypic and heterotypic influenza strains [2,3]. There are some 18 HA subtypes in influenza A virus that infect humans, animals and birds (Figure 1) [4].

Conventional glioblastoma treatment still gives very modest results, thus development of the new treatment modalities is desperately needed for these disease.The present study was aimed at analyzing the effect of bradykinin (BK) antagonists on proliferation of the human glioblastoma cells U373.Methods.MTT-based cell proliferation assay.Results.BKM-570 revealed a significant antiproliferative activity in the U373 cells with LC 50 3,8 mM.Conclusions.The antiproliferative properties of BK antagonists were shown in vitro using the glioma cells.Further investigations of the molecular mechanisms of their action and pre-clinical studies on animal models are needed for the evaluation of these compounds as new anti-cancer drugs.

A new class of antimicrobial agents with lower rates of resistance and different targets is urgently needed because of the rapidly increasing resistance to classical antibiotics. Amphipathic cationic α-helical antimicrobial peptides (AMPs) represent such a class of compounds. In our previous studies, using a 26-residue de novo designed antimicrobial peptide, we proposed the concept of “specificity determinant(s)”: positively charged residue(s) in the center of the non-polar face of AMPs that could decrease hemolytic activity/toxicity but increase or maintain the same level of antimicrobial activity to increase dramatically the therapeutic index. In the current study, we used d-enantiomers of two AMPs, Piscidin 1 isolated from fish and dermaseptin S4 isolated from frog. We substituted different positions in the center of the hydrophobic face with one or two lysine residue(s) (one or two “specificity determinant(s)”). This simple modification not only maintained or improved antimicrobial activity against Gram-negative pathogens Acinetobacter baumannii (11 strains) and Pseudomonas aeruginosa (6 strains), but also dramatically decreased hemolytic activity of human red blood cells, as predicted. Therapeutic indices improved by 55-fold and 730-fold for piscidin 1 (I9K) and dermaseptin S4 (L7K, A14K), respectively, against A. baumannii. Similarly, the therapeutic indices improved 32-fold and 980-fold for piscidin 1 (I9K) and dermaseptin S4 (L7K, A14K), respectively, against P. aeruginosa.