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Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wilbon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly ( p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS ( p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability., Competing Interests: Author GD was employed by the company Shanghai Jiyu Pharmaceutical Technology Co., Ltd. Of note, the drugs 057, 52A1, and NPSP512 have been licenced by BCN of which the co-author AN is a director. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Micewicz, Damoiseaux, Deng, Gomez, Iwamoto, Jung, Nguyen, Norris, Ratikan, Ruchala, Sayre, Schaue, Whitelegge and McBride.)

Iron disorders are associated with adverse pregnancy outcomes, yet iron homeostatic mechanisms during pregnancy are poorly understood. In humans and rodents, the iron-regulatory hormone hepcidin is profoundly decreased in pregnant mothers, which is thought to ensure adequate iron availability for transfer across placenta. However, the fetal liver also produces hepcidin, which may regulate fetal iron endowment by controlling placental iron export. To determine the relative contribution of maternal vs embryo hepcidin to the control of embryo iron endowment in iron-sufficient or iron-overloaded mice, we generated combinations of mothers and embryos that had or lacked hepcidin. We found that maternal, but not embryonic, hepcidin determined embryo and placental iron endowment in a healthy pregnancy. We further determined that inflammation can counteract pregnancy-dependent suppression of maternal hepcidin. To establish how essential maternal hepcidin suppression is for embryo iron homeostasis, we mimicked the range of maternal hepcidin activity by administering a hepcidin peptide mimetic to pregnant mice. This also allowed us to determine the effect of isolated maternal hepcidin excess on pregnancy, in the absence of other confounding effects of inflammation. Higher doses of hepcidin agonist caused maternal iron restriction and anemia, lower placenta and embryo weight, embryo anemia, and increased embryo mortality. Low agonist doses did not cause maternal anemia but still adversely affected the embryo, causing anemia, tissue iron deficiency (including in the brain), and decreased weight. Our studies demonstrate that suppression of maternal hepcidin during pregnancy is essential for maternal and embryo iron homeostasis and health., (© 2020 by The American Society of Hematology.)

Zika virus (ZIKV) has become a public health concern worldwide due to its association with congenital abnormalities and neurological diseases. To date, no effective vaccines or antiviral drugs have been approved for the treatment of ZIKV infection, and new inexpensive therapeutic options are urgently needed. In this study, we have used an in vitro plaque assay to assess an antiviral activity of the second generation of anti-ZIKV compounds, based on 1,3-disubstituted (thio)urea scaffold. Several compounds in the library were found to possess excellent activity against Zika virus with IC 50 values <200 pM. The most active analog, A5 exhibited an exceptional IC 50  = 85.1 ± 1.7 pM. Further analysis delineated structural requirements necessary for potent antiviral effects of this class of compounds. Collectively, our findings suggest that 1,3-disubstituted (thio)urea derivatives are excellent preclinical candidates for the development of anti-ZIKV therapeutics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A small group of lipid-conjugated Smac mimetics was synthesized to probe the influence of the position of lipidation on overall anti-cancer activity. Specifically, new compounds were modified with lipid(s) in position 3 and C-terminus. Previously described position 2 lipidated analog M11 was also synthesized. The resulting mini library of Smacs lipidated in positions 2, 3 and C-terminus was screened extensively in vitro against a total number of 50 diverse cancer cell lines revealing that both the position of lipidation as well as the type of lipid, influence their anti-cancer activity and cancer type specificity. Moreover, when used in combination therapy with inhibitor of menin-MLL1 protein interactions, position 2 modified analog SM2 showed strong synergistic anti-cancer properties. The most promising lipid-conjugated analogs SM2 and SM6, showed favorable pharmacokinetics and in vivo activity while administered subcutaneously in the preclinical mouse model. Collectively, our findings suggest that lipid modification of Smacs may be a viable approach in the development of anti-cancer therapeutic leads., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity. In artificial bilayer membranes, this peptide exhibited abundant β-structure. Electron microscopy revealed that this peptide also promoted the stacking of liposome membranes. Together these results suggest that the fatty acylated region of synaptotagmin-1 is likely to adopt β-structure in biological membranes. This preference for β-structure versus α-helix has functional implications for the role of synaptotagmin-1 in synaptic vesicle exocytosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Loss of intestinal barrier integrity plays a fundamental role in the pathogenesis of various gastrointestinal diseases and is implicated in the onset of sepsis and multiple organ failure. An array of methods to assess different aspects of intestinal barrier function suffers from lack of sensitivity, prolonged periods of specimen collection, or high expense. We have developed a technique to measure the concentration of the food dye FD&C Blue #1 from blood and sought to assess its utility in measuring intestinal barrier function in humans., Materials and Methods: Four healthy volunteers and 10 critically ill subjects in the intensive care unit were recruited in accordance with an institutional review board approved protocol. Subjects were given 0.5 mg/kg Blue #1 enterally as an aqueous solution of diluted food coloring. Five blood specimens were drawn per subject: 0 h (before dose), 1, 2, 4, and 8 h. After plasma isolation, organic extracts were analyzed by high-performance liquid chromatography/mass spectrometry detecting the presence of unmodified dye., Results: We found no baseline detectable absorption in healthy volunteers. After including the subjects in the intensive care unit, we compared dye absorption in the six subjects who met criteria for septic shock with the eight who did not. Septic patients demonstrated significantly greater absorption of Blue #1 after 2 h., Conclusions: We have developed a novel, easy-to-use method to measure intestinal barrier integrity using a food grade dye detectable by mass spectrometry analysis of patient blood following oral administration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Iron is an essential micronutrient for most microbes and their hosts. Mammalian hosts respond to infection by inducing the iron-regulatory hormone hepcidin, which causes iron sequestration and a rapid decrease in the plasma and extracellular iron concentration (hypoferremia). Previous studies showed that hepcidin regulation of iron is essential for protection from infection-associated mortality with the siderophilic pathogens Yersinia enterocolitica and Vibrio vulnificus However, the evolutionary conservation of the hypoferremic response to infection suggests that not only rare siderophilic bacteria but also common pathogens may be targeted by this mechanism. We tested 10 clinical isolates of Escherichia coli from children with sepsis and found that both genetic iron overload (by hepcidin-1 knockout [HKO]) and iatrogenic iron overload (by intravenous iron) potentiated infection with 8 out of the 10 studied isolates: after peritoneal injection of E. coli , iron-loaded mice developed sepsis with 60% to 100% mortality within 24 h, while control wild-type mice suffered 0% mortality. Using one strain for more detailed study, we show that iron overload allows rapid bacterial multiplication and dissemination. We further found that the presence of non-transferrin-bound iron (NTBI) in the circulation is more important than total plasma or tissue iron in rendering mice susceptible to infection and mortality. Postinfection treatment of HKO mice with just two doses of the hepcidin agonist PR73 abolished NTBI and completely prevented sepsis-associated mortality. We demonstrate that the siderophilic phenotype extends to clinically common pathogens. The use of hepcidin agonists promises to be an effective early intervention in patients with infections and dysregulated iron metabolism., (Copyright © 2018 American Society for Microbiology.)

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13 C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using 19 F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2-8.4Å for L493F and L504F and 10.5Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background & Aims: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca 2+ , mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats., Methods: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence., Results: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca 2+ overload or through a Ca 2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas., Conclusions: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The recent re-emergence of Zika virus (ZIKV), a member of the Flaviviridae family, has become a global emergency and a serious public health threat worldwide. ZIKV infection causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women causing various developmental abnormalities. Currently, there are no effective methods of preventing or treating ZIKV infection, and new treatment options are urgently needed. Therefore, we have used an in vitro plaque assay to screen a limited proprietary library of small organic compounds and identified highly bioactive leads, with the most active analogs showing activity in low picomolar range. Identified "hits" possess certain common structural features that can be used in the design of the next generation(s) of ZIKV inhibitors. Collectively, our findings suggest that identified compounds represent excellent template(s) for the development of inexpensive and orally available anti-Zika drugs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens ( Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections ( Mycobacterium tuberculosis ), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections., (© 2017 by The American Society of Hematology.)

Iron is both an essential and a potentially toxic element, and its systemic homeostasis is controlled by the iron hormone hepcidin. Hepcidin binds to the cellular iron exporter ferroportin, causes its degradation, and thereby diminishes iron uptake from the intestine and the release of iron from macrophages. Given that hepcidin-resistant ferroportin mutant mice show exocrine pancreas dysfunction, we analysed pancreata of aging hepcidin knockout (KO) mice. Hepcidin and Hfe KO mice were compared with wild-type (WT) mice kept on standard or iron-rich diets. Twelve-month-old hepcidin KO mice were subjected to daily minihepcidin PR73 treatment for 1 week. Six-month-old hepcidin KO mice showed cytoplasmic acinar iron overload and mild pancreatitis, together with elevated expression of the iron uptake mediators DMT1 and Zip14. Acinar atrophy, massive macrophage infiltration, fatty changes and pancreas fibrosis were noted in 1-year-old hepcidin KO mice. As an underlying mechanism, 6-month-old hepcidin KO mice showed increased pancreatic oxidative stress, with elevated DNA damage, apoptosis and activated nuclear factor-κB (NF-κB) signalling. Neither iron overload nor pancreatic damage was observed in WT mice fed iron-rich diet or in Hfe KO mice. Minihepcidin application to hepcidin KO mice led to an improvement in general health status and to iron redistribution from acinar cells to macrophages. It also resulted in decreased NF-κB activation and reduced DNA damage. In conclusion, loss of hepcidin signalling in mice leads to iron overload-induced chronic pancreatitis that is not seen in situations with less severe iron accumulation. The observed tissue injury can be reversed by hepcidin supplementation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The human molecular chaperones heat shock protein 70 (Hsp70) and heat shock cognate protein 70 (Hsc70) bind to the hepatitis C viral nonstructural protein 5A (NS5A) and regulate its activity. Specifically, Hsp70 is involved in NS5A-augmented internal ribosomal entry site (IRES)-mediated translation of the viral genome, whilst Hsc70 appears to be primarily important for intracellular infectious virion assembly. To better understand the importance of these two chaperones in the viral life cycle, infected human cells were treated with allosteric Hsp70/Hsc70 inhibitors (AHIs). Treatment with AHIs significantly reduced the production of intracellular virus at concentrations that were non-toxic to human hepatoma Huh7.5 cells. The supernatant of treated cultures was then used to infect naïve cells, revealing that AHIs also lowered levels of secreted virus. In contrast to their effects on virion assembly, AHIs did not impact the stability of NS5A or viral protein translation in IRES assays. These results suggest that Hsc70 plays a particularly important and sensitive role in virion assembly. Indeed, it was found that combination of AHIs with a peptide-based viral translation inhibitor exhibited additive antiviral activity. Together these results suggest that the host Hsc70 is a new antiviral target and that its inhibitors utilise a new mechanism of action., (Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

A small library of anticancer, cell-permeating, stapled peptides based on potent dual-specific antagonist of p53-MDM2/MDMX interactions, PMI-N8A, was synthesized, characterized and screened for anticancer activity against human colorectal cancer cell line, HCT-116. Employed synthetic modifications included: S-alkylation-based stapling, point mutations increasing hydrophobicity in key residues as well as improvement of cell-permeability by introduction of polycationic sequence(s) that were woven into the sequence of parental peptide. Selected analogue, ArB14Co, was also tested in vivo and exhibited potent anticancer bioactivity at the low dose (3.0 mg/kg). Collectively, our findings suggest that application of stapling in combination with rational design of polycationic short analogues may be a suitable approach in the development of physiologically active p53-MDM2/MDMX peptide inhibitors.

The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify β-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Minihepcidins are in vitro and in vivo active mimetics of iron-regulatory hormone hepcidin. They contain various unusual amino acids including: N-substituted, β-homo-, and d-amino acids with their combination depending on particular minihepcidin. In the current study, we sought to limit the use of unusual/more expensive amino acids derivatives by peptide cyclization. Novel cyclic mimetics of hepcidin were synthesized and tested in vitro and showed activity at low nanomolar concentration. Nonetheless, the most active cyclic compound (mHS17) is approximately ten times less active than the parental minihepcidin PR73. Collectively, our findings suggest that cyclization is viable approach in the synthesis of hepcidin mimetics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A small library of monovalent and bivalent Smac mimics was synthesized based on 2 types of monomers, with general structure NMeAla-Xaa-Pro-BHA (Xaa=Cys or Lys). Position 2 of the compounds was utilized to dimerize both types of monomers employing various bis-reactive linkers, as well as to modify selected compounds with lipids. The resulting library was screened in vitro against metastatic human breast cancer cell line MDA-MB-231, and the two most active compounds selected for in vivo studies. The most active lipid-conjugated analogue M11, showed in vivo activity while administered both subcutaneously and orally. Collectively, our findings suggest that lipidation may be a viable approach in the development of new Smac-based therapeutic leads., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A small library of truncated/lipid-conjugated neuromedin U (NmU) analogs was synthesized and tested in vitro using an intracellular calcium signaling assay. The selected, most active analogs were then tested in vivo, and showed potent anorexigenic effects in a diet-induced obese (DIO) mouse model. The most promising compound, NM4-C16 was effective in a once-weekly-dose regimen. Collectively, our findings suggest that short, lipidated analogs of NmU are suitable leads for the development of novel anti-obesity therapeutics., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Minihepcidins are small peptides that mimic biological activity of the iron-regulatory hormone hepcidin. Structurally, they contain thiol-free-cysteine residue in position 7 which is crucial for their bioactivity. Nonetheless, free sulfhydryl group is not desirable in pharmaceutical entities as it may lead to dermatological side effects. Moreover free thiol moiety is quite reactive and depending on conditions/reagents may be alkylated and/or oxidized giving various Cys-derivatives: S-alkyl cysteines, sulfoxides, sulfones, disulfides, cysteinesulfinic and cysteic acids. To limit such reactivity and maintain bioactivity of minihepcidin(s) we used thiol-protection strategy based on activated vinyl thioethers. Novel S-protected analogs of physiologically active minihepcidin PR73 were synthesized and tested in vitro showing activity comparable to parental molecule. The most active compound, PR73SH was also tested in vivo showing activity profile analogous to PR73. Collectively, our findings suggest that S-vinyl-derivatization of minihepcidin(s) may be a suitable approach in the development of physiologically active agonists of hepcidin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We previously identified the NS5A/HSP70 binding site to be a hairpin moiety at C-terminus of NS5A domain I and showed a corresponding cyclized polyarginine-tagged synthetic peptide (HCV4) significantly blocks virus production. Here, sequence comparison confirmed five residues to be conserved. Based on NS5A domain I crystal structure, Phe171, Val173, and Tyr178 were predicted to form the binding interface. Substitution of Phe171 and Val173 with more hydrophobic unusual amino acids improved peptide antiviral activity and HSP70 binding, while similar substitutions at Tyr178 had a negative effect. Substitution of non-conserved residues with arginines maintained antiviral activity and HSP70 binding and dispensed with polyarginine tag for cellular entry. Peptide cyclization improved antiviral activity and HSP70 binding. The cyclic retro-inverso analog displayed the best antiviral properties. FTIR spectroscopy confirmed a secondary structure consisting of an N-terminal beta-sheet followed by a turn and a C-terminal beta-sheet. These peptides constitute a new class of anti-HCV compounds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hereditary hemochromatosis, an iron overload disease caused by a deficiency in the iron-regulatory hormone hepcidin, is associated with lethal infections by siderophilic bacteria. To elucidate the mechanisms of this susceptibility, we infected wild-type and hepcidin-deficient mice with the siderophilic bacterium Vibrio vulnificus and found that hepcidin deficiency results in increased bacteremia and decreased survival of infected mice, which can be partially ameliorated by dietary iron depletion. Additionally, timely administration of hepcidin agonists to hepcidin-deficient mice induces hypoferremia that decreases bacterial loads and rescues these mice from death, regardless of initial iron levels. Studies of Vibrio vulnificus growth ex vivo show that high iron sera from hepcidin-deficient mice support extraordinarily rapid bacterial growth and that this is inhibited in hypoferremic sera. Our findings demonstrate that hepcidin-mediated hypoferremia is a host defense mechanism against siderophilic pathogens and suggest that hepcidin agonists may improve infection outcomes in patients with hereditary hemochromatosis or thalassemia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as 'helical adjuvants' to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges ("ion-locks") promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu(-)-Lys(+) into the parent SP-C's. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with 'amyloid-like' properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard (12)C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu(-)-Lys(+) insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu(-)-Lys(+) ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.

A small library of monovalent Smac mimics with general structure NMeAla-Tle-(4R)-4-Benzyl-Pro-Xaa-cysteamide, was synthesized (Xaa=hydrophobic residue). The library was screened in vitro against human breast cancer cell lines MCF-7 and MDA-MB-231, and two most active compounds oligomerized via S-alkylation giving bivalent and trivalent derivatives. The most active bivalent analogue SMAC17-2X was tested in vivo and in physiological conditions (mouse model) it exerted a potent anticancer effect resulting in ∼23.4days of tumor growth delay at 7.5mg/kg dose. Collectively, our findings suggest that bivalent Smac analogs obtained via S-alkylation protocol may be a suitable platform for the development of new anticancer therapeutics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Inappropriate production of the iron-regulatory hormone hepcidin contributes to the pathogenesis of common iron disorders. Absolute or relative deficiency of hepcidin causes iron overload in hereditary hemochromatosis and iron-loading anemias. Elevated hepcidin causes iron restriction in inflammatory conditions including autoimmune disease, critical illness, some cancers, and chronic kidney disease. Multiple agents targeting hepcidin and its regulators are under development as novel therapeutics for iron disorders. This review summarizes hepcidin biology and discusses the current landscape for hepcidin-targeting therapeutic strategies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

Since the discovery of human immunodeficiency virus (HIV) as a causative agent of acquired immune deficiency syndrome (AIDS) various strategies were employed to counter its devastating actions. One such concept relies on the prevention of HIV entry into host's "competent" cells by means of compounds known as entry inhibitors. HIV entry inhibitors comprise a group of immensely diverse compounds ranging from proteins/antibodies to small organic molecules and capable of targeting various stages of viral entry. Although already in clinical use, this approach to HIV therapy is still being investigated to produce new promising antiviral compounds. Here, we review the latest advances in this area.

The deficiency of hepcidin, the hormone that controls iron absorption and its tissue distribution, is the cause of iron overload in nearly all forms of hereditary hemochromatosis and in untransfused iron-loading anemias. In a recent study, we reported the development of minihepcidins, small drug-like hepcidin agonists. Here we explore the feasibility of using minihepcidins for the prevention and treatment of iron overload in hepcidin-deficient mice. An optimized minihepcidin (PR65) was developed that had superior potency and duration of action compared with natural hepcidin or other minihepcidins, and favorable cost of synthesis. PR65 was administered by subcutaneous injection daily for 2 weeks to iron-depleted or iron-loaded hepcidin knockout mice. PR65 administration to iron-depleted mice prevented liver iron loading, decreased heart iron levels, and caused the expected iron retention in the spleen and duodenum. At high doses, PR65 treatment also caused anemia because of profound iron restriction. PR65 administration to hepcidin knockout mice with pre-existing iron overload had a more moderate effect and caused partial redistribution of iron from the liver to the spleen. Our study demonstrates that minihepcidins could be beneficial in iron overload disorders either used alone for prevention or possibly as adjunctive therapy with phlebotomy or chelation.

We developed a new peptide, natural phytochelatin (PC), which tightly binds to CdSe/ZnS quantum dots' (QDs) surfaces and renders them water-soluble. Coating QDs with this flexible and all-hydrophilic peptide offers high colloidal stability, adds only 0.8-0.9 nm to the radius of the particles (as compared to their original inorganic radius), preserves very high quantum yield (QY) in water, and affords facile bioconjugation with various functional groups. We demonstrate specific targeting (with minimal nonspecific binding) of such fluorescein-conjugated QDs to ScFv-fused mouse prion protein expressed in live N2A cells. We also demonstrated homogeneous in vivo biodistribution with no significant toxicity in live zebrafish.

Unlabelled: NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)-mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same-size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, whereas controls did not. Triple-alanine scan mutagenesis determined that an exposed beta-sheet hairpin in C34 was primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10-amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity., Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A-mediated IRES activity, sequence-specific HSP recognition, and rational drug design. (HEPATOLOGY 2012;55:1662-1672)., (Copyright © 2012 American Association for the Study of Liver Diseases.)

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds)., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

The structural basis for the gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both gram-positive and gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains gram-positive activity but is 30-fold less active against gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of gram-positive bacteria. (31)P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. (13)C chemical shifts and (13)C-(1)H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. Indeed, (1)H spin diffusion from lipid chains to the peptides shows that PG-1 fully inserts into the LPS-rich membrane whereas the mutant does not. The (13)C-(31)P distances between the most hydrophobically embedded Arg of PG-1 and the lipid (31)P are significantly longer in the LPS membrane than in the POPE/POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against gram-negative bacteria. The data provide a rational basis for controlling gram selectivity of AMPs by adjusting the charge densities.

Structure-mechanism relationships are key determinants of host defense peptide efficacy. These relationships are influenced by anatomic, physiologic and microbiologic contexts. Structure-mechanism correlates were assessed for the synthetic peptide RP-1, modeled on microbicidal domains of platelet kinocidins. Antimicrobial efficacies and mechanisms of action against susceptible ((S)) or resistant ((R)) Salmonella typhimurium (ST), Staphylococcus aureus (SA), and Candida albicans (CA) strain pairs were studied at pH 7.5 and 5.5. Although RP-1 was active against all study organisms, it exhibited greater efficacy against bacteria at pH 7.5, but greater efficacy against CA at pH 5.5. RP-1 de-energized SA and CA, but caused hyperpolarization of ST in both pH conditions. However, RP-1 permeabilized ST(S) and CA strains at both pH, whereas permeabilization was modest for ST(R) or SA strain at either pH. Biochemical analysis, molecular modeling, and FTIR spectroscopy data revealed that RP-1 has indistinguishable net charge and backbone trajectories at pH 5.5 and 7.5. Yet, concordant with organism-specific efficacy, surface plasmon resonance, and FTIR, molecular dynamics revealed modest helical order increases but greater RP-1 avidity and penetration of bacterial than eukaryotic lipid systems, particularly at pH 7.5. The present findings suggest that pH- and target-cell lipid contexts influence selective antimicrobial efficacy and mechanisms of RP-1 action. These findings offer new insights into selective antimicrobial efficacy and context-specificity of antimicrobial peptides in host defense, and support design strategies for potent anti-infective peptides with minimal concomitant cytotoxicity.

Background: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties., Methodology/results: The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC(50) of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface., Conclusion: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.

Background: RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques., Methodology/principal Findings: RC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals., Conclusions/significance: Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.

The protein transduction domain of HIV-1 TAT, TAT(48-60), is an efficient cell-penetrating peptide (CPP) that diffuses across the lipid membranes of cells despite eight cationic Arg and Lys residues. To understand its mechanism of membrane translocation against the free energy barrier, we have conducted solid-state NMR experiments to determine the site-specific conformation, dynamics, and lipid interaction of the TAT peptide in anionic lipid bilayers. We found that TAT(48-60) is a highly dynamic and nearly random coil peptide in the lipid bilayer and inserts into the membrane-water interface near the glycerol backbone region. Arg-phosphate salt bridge interaction was revealed by short guanidinium-phosphate distances and restricted dynamics of the guanidinium. Together with the observation of strong peptide-water cross-peaks in (1)H spin diffusion spectra, these results indicate that TAT binding to the membrane-water interface is stabilized not only by electrostatic attraction to the anionic lipids but also by intermolecular hydrogen bonding with the lipid phosphates and water, which may take the role of intramolecular hydrogen bonds in canonical secondary structures. The random coil structure of TAT and another CPP, penetratin, suggests that the lack of amphipathic structure is essential for rapid translocation of these Arg-rich CPPs across the lipid membrane without causing permanent damages to the membrane integrity.

Background: Many gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids., Methodology/results: Several of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable alpha-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features., Conclusion: Given the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and other Oxpholipins may have therapeutic potential.

Lung surfactant protein B (SP-B) is required for proper surface activity of pulmonary surfactant. In model lung surfactant lipid systems composed of saturated and unsaturated lipids, the unsaturated lipids are removed from the film at high compression. It is thought that SP-B helps anchor these lipids closely to the monolayer in three-dimensional cylindrical structures termed "nanosilos" seen by atomic force microscopy imaging of deposited monolayers at high surface pressures. Here we explore the role of the SP-B NH(2) terminus in the formation and stability of these cylindrical structures, specifically the distribution of lipid stack height, width, and density with four SP-B truncation peptides: SP-B 1-25, SP-B 9-25, SP-B 11-25, and SP-B 1-25Nflex (prolines 2 and 4 substituted with alanine). The first nine amino acids, termed the insertion sequence and the interface seeking tryptophan residue 9, are shown to stabilize the formation of nanosilos while an increase in the insertion sequence flexibility (SP-B 1-25Nflex) may improve peptide functionality. This provides a functional understanding of the insertion sequence beyond anchoring the protein to the two-dimensional membrane lining the lung, as it also stabilizes formation of nanosilos, creating reversible repositories for fluid lipids at high compression. In lavaged, surfactant-deficient rats, instillation of a mixture of SP-B 1-25 (as a monomer or dimer) and synthetic lung lavage lipids quickly improved oxygenation and dynamic compliance, whereas SP-B 11-25 surfactants showed oxygenation and dynamic compliance values similar to that of lipids alone, demonstrating a positive correlation between formation of stable, but reversible, nanosilos and in vivo efficacy.

Background: Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity., Methodology/results: FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B., Conclusion: Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Amyloid beta-protein (Abeta) self-assembly is linked strongly to Alzheimer's disease. We found that PP-Leu, a tridecapeptide analogue of broad-spectrum antiviral peptides termed theta-defensins, potently inhibits Abeta oligomer and fibril formation. This effect appeared to be mediated through sequestration of the amyloidogenic Abeta peptide in colloid-like assemblies. PP-Leu comprises a turn formed by a d-Pro-l-Pro amino acid dyad and stabilized by a disulfide bond, a motif that was exceptionally resistant to endoproteinase K digestion. This combination of assembly inhibitory activity and protease resistance suggests that PP-Leu may have potential therapeutic value.

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.

Administered subcutaneously, D-4F or L-4F are equally efficacious, but only D-4F is orally efficacious because of digestion of L-4F by gut proteases. Orally administering niclosamide (a chlorinated salicylanilide used as a molluscicide, antihelminthic, and lampricide) in temporal proximity to oral L-4F (but not niclosamide alone) in apoE null mice resulted in significant improvement (P < 0.001) in the HDL-inflammatory index (HII), which measures the ability of HDL to inhibit LDL-induced monocyte chemotactic activity in endothelial cell cultures. Oral administration of L-[113-122]apoJ with niclosamide also resulted in significant improvement (P < 0.001) in HII. Oral administration of niclosamide and L-4F together with pravastatin to female apoE null mice at 9.5 months of age for six months significantly reduced aortic sinus lesion area (P = 0.02), en face lesion area (P = 0.033), and macrophage lesion area (P = 0.02) compared with pretreatment, indicating lesion regression. In contrast, lesions were significantly larger in mice receiving only niclosamide and pravastatin or L-4F and pravastatin (P < 0.001). In vitro niclosamide and L-4F tightly associated rendering the peptide resistant to trypsin digestion. Niclosamide itself did not inhibit trypsin activity. The combination of niclosamide with apolipoprotein mimetic peptides appears to be a promising method for oral delivery of these peptides.

Four of the six human alpha-defensins (human neutrophil peptides 1-3 and human alpha-defensin 5; HD5) have a lectin-like ability to bind glycosylated proteins. Using HD5 as a model, we applied surface plasmon resonance techniques to gain insights into this property. HD5 bound natural glycoproteins > neoglycoproteins based on BSA > nonglycosylated BSA >> free sugars. The affinity of HD5 for simple sugars covalently bound to BSA was orders of magnitude greater than its affinity for the same sugars in solution. The affinity of HD5 for protein-bound carbohydrates resulted from multivalent interactions which may also involve noncarbohydrate residues of the proteins. HD5 showed concentration-dependent self-association that began at submicromolar concentrations and proceeded to dimer and tetramer formation at concentrations below 5 microM. The (R9A, R28A) and (R13A, R32A) analogs of HD5 showed greatly reduced self-association as well as minimal binding to BSA and to BSA-affixed sugars. From this and other evidence, we conclude that the extensive binding of HD5 to (neo)glycoproteins results from multivalent nonspecific interactions of individual HD5 molecules with carbohydrate and noncarbohydrate moieties of the target molecule and that the primary binding events are magnified and enhanced by subsequent in situ assembly and oligomerization of HD5. Self-association and multivalent binding may play integral roles in the ability of HD5 to protect against infections caused by viruses and other infectious agents.

Antimicrobial host defense peptides (AHDPs) are effective against a wide range of microbes, including viruses. The arteriviral infection caused by porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pandemic that causes the most economically significant disease of swine. We sought to determine if the expression of AHDPs was influenced by infection with PRRSV, and if porcine AHDPs have direct antiviral activity against PRRSV. Because pulmonary alveolar macrophages (PAMs) are primary targets of PRRSV infection, gene expression of porcine AHDPs was evaluated in lungs from fetal and 2-wk-old congenitally infected pigs. In PRRSV-positive lungs and PAMs, gene expression of most porcine AHDPs showed little upregulation. However, gene expression of porcine beta-defensin-1 (pBD-1), pBD-4, pBD-104, pBD-123, and pBD-125 were downregulated more than threefold in 2-wk-old congenitally infected pig lungs. Incubation of PRRSV with pBD-3 or PG-4 significantly inhibited viral infectivity in MARC-145 cells. Using nine protegrin or protegrin-derived peptides, we determined that a cyclic analog of PG-4 increased anti-PRRSV activity, and that substitution of phenylalanine with valine eliminated most PG-4 antiviral activity. In PAMs, pBD-3 and PG-4 at 5-40 microg/mL consistently suppressed PRRSV titers. Collectively, these findings suggest a potential role for some porcine AHDPs as innate antiviral effectors in PRRSV infection. Moreover, modulation of porcine innate immune mechanisms with AHDPs may be one means of limiting the impact of this costly pandemic viral disease.

We have reported that the alpha-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These alpha-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral theta-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1-3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human beta-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1-3. Like HNP-1-3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1-3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.

Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg(10) and Lys(13), in the CPP penetratin. (13)C chemical shifts indicate that Arg(10) adopts a rigid beta-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic beta-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature (13)C-(31)P distances between the peptide and the lipid phosphates indicate that both the Arg(10) guanidinium Czeta atom and the Lys(13) Cepsilon atom are close to the lipid (31)P (4.0-4.2 A), proving the existence of charge-charge interaction for both Arg(10) and Lys(13) in the gel-phase membrane. However, since lysine substitution in CPPs is known to weaken their translocation ability, we propose that the low temperature stabilizes interactions of both lysine and arginine with the phosphates, whereas at high temperatures, the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg(10) side chain and its beta-strand conformation at high temperatures. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium side chains. (19)F and (13)C spin diffusion experiments indicate that penetratin is oligomerized into beta-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes.