Publications by authors named "Antonello Pessi"

57 Publications

A novel smaller β-defensin-derived peptide is active against multidrug-resistant bacterial strains.

FASEB J 2021 12;35(12):e22026

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human β-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the β-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human β-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.
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http://dx.doi.org/10.1096/fj.202002330RRDOI Listing
December 2021

Cholesterol-conjugated stapled peptides inhibit Ebola and Marburg viruses in vitro and in vivo.

Antiviral Res 2019 11 29;171:104592. Epub 2019 Aug 29.

United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Frederick, MD, 21702, USA. Electronic address:

Filoviridae currently includes five official and one proposed genera. Genus Ebolavirus includes five established and one proposed ebolavirus species for Bombali virus (BOMV), Bundibugyo virus (BDBV), Ebola virus (EBOV), Reston virus (RESTV), Sudan virus (SUDV) and Taï Forest virus (TAFV), and genus Marburgvirus includes a single species for Marburg virus (MARV) and Ravn virus (RAVV). Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 Ebola Virus Disease outbreak in Western Africa. Currently, there are no therapeutics approved and the need for Ebola-specific therapeutics remains a gap. In search for anti-Ebola therapies we tested the idea of using inhibitory properties of peptides corresponding to the C-terminal heptad-repeat (HR2) domains of class I fusion proteins against EBOV infection. The fusion protein GP of EBOV belongs to class I, suggesting that a similar strategy to HIV may be applied to inhibit EBOV infection. The serum half-life of peptides was expanded by cholesterol conjugation to allow daily dosing. The peptides were further constrained to stabilize a helical structure to increase the potency of inhibition. The ECs of lead peptides were in low micromolar range, as determined by a high-content imaging test of EBOV-infected cells. Lead peptides were tested in an EBOV lethal mouse model and efficacy of the peptides were determined following twice-daily administration of peptides for 9 days. The most potent peptide was able to protect mice from lethal challenge of mouse-adapted Ebola virus. These data show that engineered peptides coupled with cholesterol can inhibit viral production, protect mice against lethal EBOV infection, and may be used to build novel therapeutics against EBOV.
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http://dx.doi.org/10.1016/j.antiviral.2019.104592DOI Listing
November 2019

Structure-function relationship of an Urokinase Receptor-derived peptide which inhibits the Formyl Peptide Receptor type 1 activity.

Sci Rep 2019 08 21;9(1):12169. Epub 2019 Aug 21.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Naples, Italy.

The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.
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http://dx.doi.org/10.1038/s41598-019-47900-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704176PMC
August 2019

Polypharmacy through Phage Display: Selection of Glucagon and GLP-1 Receptor Co-agonists from a Phage-Displayed Peptide Library.

Sci Rep 2018 01 12;8(1):585. Epub 2018 Jan 12.

PeptiPharma, Viale Città D'Europa 679, 00144, Roma, Italy.

A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells. After two or three rounds of selection, 7.5% of randomly picked clones were GLP1R/GCGR co-agonists, and a further 1.53% were agonists of a single receptor. The phages were sequenced and 35 corresponding peptides were synthesized. 18 peptides were potent co-agonists, 8 of whom showed EC ≤ 30 pM on each receptor, comparable to the best rationally designed co-agonists reported in the literature. Based on literature examples, two sequences were engineered to stabilize against dipeptidyl peptidase IV cleavage and prolong the in vivo half-life: the engineered peptides were comparably potent to the parent peptides on both receptors, highlighting the potential use of phage-derived peptides as therapeutic agents. The strategy described here appears of general value for the discovery of optimized polypharmacology paradigms across several metabolically-related hormones.
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http://dx.doi.org/10.1038/s41598-017-18494-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766609PMC
January 2018

Targeting the cross-talk between Urokinase receptor and Formyl peptide receptor type 1 to prevent invasion and trans-endothelial migration of melanoma cells.

J Exp Clin Cancer Res 2017 Dec 8;36(1):180. Epub 2017 Dec 8.

IRCCS Istituto Nazionale Tumori 'Fondazione G. Pascale', Naples, Italy.

Background: Accumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated.

Methods: Expression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D-organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons.

Results: We found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR/FPR1 interaction.

Conclusions: Collectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR/FPR1 cross-talk may be useful for the treatment of metastatic melanoma.
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http://dx.doi.org/10.1186/s13046-017-0650-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721612PMC
December 2017

A first-in-human study of the novel HIV-fusion inhibitor C34-PEG-Chol.

Sci Rep 2017 08 25;7(1):9447. Epub 2017 Aug 25.

Department of Medicine, Imperial College London, London, W2 1NY, UK.

Long-acting injectable antiretroviral (LA-ARV) drugs with low toxicity profiles and propensity for drug-drug interactions are a goal for future ARV regimens. C34-PEG-Chol is a novel cholesterol tagged LA HIV-fusion-inhibitor (FI). We assessed pre-clinical toxicology and first-in-human administration of C34-PEG-Chol. Pre-clinical toxicology was conducted in 2 species. HIV-positive men were randomised to a single subcutaneous dose of C34-PEG-Chol at incrementing doses or placebo. Detailed clinical (including injection site reaction (ISR) grading), plasma pharmacokinetic (time-to-minimum-effective-concentration (MEC, 25 ng/mL) and pharmacodynamic (plasma HIV RNA) parameters were assessed. In both mice and dogs, no-observed-adverse effect level (NOAEL) was observed at a 12 mg/kg/dose after two weeks. Of 5 men enrolled, 3 received active drug (10 mg, 10 mg and 20 mg). In 2 individuals grade 3 ISR occurred and the study was halted. Both ISR emerged within 12 hours of active drug dosing. No systemic toxicities were observed. The time-to-MEC was >72 and >96 hours after 10 and 20 mg dose, respectively, and mean change in HIV RNA was -0.9 log10 copies/mL. These human pharmacodynamic and pharmacokinetic data, although limited to 3 subjects, of C34-PEG-4-Chol suggest continuing evaluation of this agent as a LA-ARV. However, alternative administration routes must be explored.
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http://dx.doi.org/10.1038/s41598-017-09230-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572697PMC
August 2017

Retro-inverso Urokinase Receptor Antagonists for the Treatment of Metastatic Sarcomas.

Sci Rep 2017 05 2;7(1):1312. Epub 2017 May 2.

Peptipharma, Viale Città D'Europa 679, 00144, Rome, Italy.

The development of metastases is a multistep process that requires the activation of physiological and biochemical processes that govern migration, invasion and entry of metastatic cells into blood vessels. The urokinase receptor (uPAR) promotes cell migration by interacting with the Formyl Peptide Receptors (FPRs). Since both uPAR and FPR1 are involved in tumor progression, the uPAR-FPR1 interaction is an attractive therapeutic target. We previously described peptide antagonists of the uPAR-FPR1 interaction that inhibited cell migration and angiogenesis. To develop enzyme-resistant analogues, we applied here the Retro-Inverso (RI) approach, whereby the topology of the side chains is maintained by inverting the sequence of the peptide and the chirality of all residues. Molecular dynamics suggests that peptide RI-3 adopts the turn structure typical of uPAR-FPR1 antagonists. Accordingly, RI-3 is a nanomolar competitor of N-formyl-Met-Leu-Phe for binding to FPR1 and inhibits migration, invasion, trans-endothelial migration of sarcoma cells and VEGF-triggered endothelial tube formation. When sarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density, circulating tumor cells and pulmonary metastases were significantly reduced in animals treated daily with 6 mg/Kg RI-3 as compared to animals treated with vehicle only. Thus, RI-3 represents a promising lead for anti-metastatic drugs.
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http://dx.doi.org/10.1038/s41598-017-01425-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430962PMC
May 2017

Lipophilicity is a key factor to increase the antiviral activity of HIV neutralizing antibodies.

Colloids Surf B Biointerfaces 2017 Apr 19;152:311-316. Epub 2017 Jan 19.

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

The HIV broadly neutralizing antibody 2F5 targets the transiently exposed epitope in the membrane proximal external region (MPER) of HIV-1 gp41, by a two-step mechanism involving the viral membrane and this viral glycoprotein. It was recently shown that 2F5 conjugation with a cholesterol moiety outside of the antibody paratope substantially increases its antiviral activity. Additionally, the antiviral activity of D5, a human antibody that binds to the N-terminal heptad repeat (NHR) of gp41 and lacks membrane binding, was boosted by the same cholesterol conjugation. In this work, we evaluated the membrane affinity of both antibodies towards membranes of different compositions, using surface plasmon resonance. A correlation was found between membrane affinity and antiviral activity against HIV-1. We propose that the conjugation of cholesterol to 2F5 or D5 allows a higher degree of antibody pre-concentration at the viral membrane. This way, the antibodies become more available to bind efficiently to the gp41 epitope, blocking viral fusion faster than the unconjugated antibody. These results set up a relevant strategy to improve the rational design of therapeutic antibodies against HIV.
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http://dx.doi.org/10.1016/j.colsurfb.2017.01.032DOI Listing
April 2017

Host defense peptide-derived privileged scaffolds for anti-infective drug discovery.

J Pept Sci 2017 Apr 12;23(4):303-310. Epub 2017 Jan 12.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145, Naples, Italy.

'Privileged scaffolds' are molecular frameworks which have been successfully exploited for small molecule drug discovery. Peptide privileged scaffolds, featuring a strictly conserved multiple-disulfide framework and high variability in the rest of the sequence, display a broad range of biological effects, including antimicrobial and antiviral activity. Unlike small molecules, however, the cost of manufacturing these peptides is high, and their synthesis challenging. We previously described a simplified privileged scaffold corresponding to the γ-core of human β-defensin-3 (HBD3). The γ-core is a common structural signature found in virtually all host defense peptides (HDPs) stabilized by multiple disulfides, and we showed that for HBD3, it represents the evolutionary starting point of the full-length molecule and, thus, is itself a primordial HDP. Accordingly, we showed that the peptide folded rapidly and was stable in human serum, and displayed many of the biological activities of HBD3. We report here that in addition to the previously reported antibacterial activity on planktonic bacteria, the γ-core peptide is active against biofilm formation and maturation. We also show that it is readily cell penetrant, like HBD3, although with a different mechanism, which is independent from CD98. Overall, the potency of the single-disulfide, 23-amino acid γ-core is comparable with the full-length peptide across the whole spectrum of examined properties, and the peptide is not toxic to human cells. The HBD3 γ-core peptide may therefore represent the first example of an economically viable lead peptide derived from a HDP privileged scaffold. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.2962DOI Listing
April 2017

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule.

Sci Rep 2015 Dec 21;5:18450. Epub 2015 Dec 21.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy.

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated "γ-core motif". We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents.
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http://dx.doi.org/10.1038/srep18450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685272PMC
December 2015

Design and activity of a cyclic mini-β-defensin analog: a novel antimicrobial tool.

Int J Nanomedicine 2015 15;10:6523-39. Epub 2015 Oct 15.

Dipartimento di Farmacia, Università di Napoli Federico II, Naples, Italy ; Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy.

We have designed a cyclic 17-amino acid β-defensin analog featuring a single disulfide bond. This analog, designated "AMC" (ie, antimicrobial cyclic peptide), combines the internal hydrophobic domain of hBD1 and the C-terminal charged region of hBD3. The novel peptide was synthesized and characterized by nuclear magnetic resonance spectroscopy. The antimicrobial activities against gram-positive and gram-negative bacteria as well as against herpes simplex virus type 1 were analyzed. The cytotoxicity and serum stability were assessed. Nuclear magnetic resonance of AMC in aqueous solution suggests that the structure of the hBD1 region, although not identical, is preserved. Like the parent defensins, AMC is not cytotoxic for CaCo-2 cells. Interestingly, AMC retains the antibacterial activity of the parent hBD1 and hBD3 against Pseudomonas aeruginosa, Enterococcus faecalis, and Escherichia coli, and exerts dose-dependent activity against herpes simplex virus type 1. Moreover, while the antibacterial and antiviral activities of the oxidized and reduced forms of the parent defensins are similar, those of AMC are significantly different, and oxidized AMC is also considerably more stable in human serum. Taken together, our data also suggest that this novel peptide may be added to the arsenal of tools available to combat antibiotic-resistant infectious diseases, particularly because of its potential for encapsulation in a nanomedicine vector.
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http://dx.doi.org/10.2147/IJN.S89610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610797PMC
August 2016

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin.

J Pept Sci 2015 Sep;21(9):743-9

CEINGE, Via Gaetano Salvatore 486, 80145, Napoli, Italy.

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand-binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand-binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol-conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41-Fc-chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV-1 strains, it was considerably more potent than the unconjugated C41-Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol-conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat-derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses.
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http://dx.doi.org/10.1002/psc.2802DOI Listing
September 2015

Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human β-Defensin 3 in epithelial cells.

Chem Biol 2015 Feb 29;22(2):217-28. Epub 2015 Jan 29.

CEINGE-Biotecnologie Avanzate S.c.a r.l., Via Gaetano Salvatore 486, 80145 Naples, Italy; IRCCS-Fondazione SDN, Via Emanuele Gianturco 113, 80142 Naples, Italy. Electronic address:

Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.
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http://dx.doi.org/10.1016/j.chembiol.2014.11.020DOI Listing
February 2015

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.

J Biol Chem 2014 Dec 23;289(50):35015-28. Epub 2014 Oct 23.

From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy, JV Bio, Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.
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http://dx.doi.org/10.1074/jbc.M114.591826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263897PMC
December 2014

Cholesterol-conjugated peptide antivirals: a path to a rapid response to emerging viral diseases.

Authors:
Antonello Pessi

J Pept Sci 2015 May 20;21(5):379-86. Epub 2014 Oct 20.

PeptiPharma, Viale Città D'Europa 679, 00141, Roma, Italy; JV Bio, Via Gaetano Salvatore 486, 80145, Napoli, Italy; CEINGE, Via Gaetano Salvatore 486, 80145, Napoli, Italy.

While it is now possible to identify and genetically fingerprint the causative agents of emerging viral diseases, often with extraordinary speed, suitable therapies cannot be developed with equivalent speed, because drug discovery requires information that goes beyond knowledge of the viral genome. Peptides, however, may represent a special opportunity. For all enveloped viruses, fusion between the viral and the target cell membrane is an obligatory step of the life cycle. Class I fusion proteins harbor regions with a repeating pattern of amino acids, the heptad repeats (HRs), that play a key role in fusion, and HR-derived peptides such as enfuvirtide, in clinical use for HIV, can block the process. Because of their characteristic sequence pattern, HRs are easily identified in the genome by means of computer programs, providing the sequence of candidate peptide inhibitors directly from genomic information. Moreover, a simple chemical modification, the attachment of a cholesterol group, can dramatically increase the antiviral potency of HR-derived inhibitors and simultaneously improve their pharmacokinetics. Further enhancement can be provided by dimerization of the cholesterol-conjugated peptide. The examples reported so far include inhibitors of retroviruses, paramyxoviruses, orthomyxoviruses, henipaviruses, coronaviruses, and filoviruses. For some of these viruses, in vivo efficacy has been demonstrated in suitable animal models. The combination of bioinformatic lead identification and potency/pharmacokinetics improvement provided by cholesterol conjugation may form the basis for a rapid response strategy, where development of an emergency cholesterol-conjugated therapeutic would immediately follow the availability of the genetic information of a new enveloped virus.
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http://dx.doi.org/10.1002/psc.2706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167725PMC
May 2015

Improvement of HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure.

J Antimicrob Chemother 2014 May 23;69(5):1286-97. Epub 2014 Jan 23.

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.

Objectives: The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)₄-cholesterol] and HIVP4 [(C34-PEG₄)₂-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy.

Methods: Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays.

Results: Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol.

Conclusions: The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.
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http://dx.doi.org/10.1093/jac/dkt529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977611PMC
May 2014

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

J Pept Sci 2014 Jan 13;20(1):7-19. Epub 2013 Nov 13.

IRBM P. Angeletti, 00040, Pomezia, RM, Italy; NIBR WSJ-507.1.02, Novartis Pharma AG, Campus, CH-4056, Basel, Switzerland.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.
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http://dx.doi.org/10.1002/psc.2582DOI Listing
January 2014

Fatal measles virus infection prevented by brain-penetrant fusion inhibitors.

J Virol 2013 Dec 9;87(24):13785-94. Epub 2013 Oct 9.

International Center for Research in Infectious Diseases-CIRI, INSERM U1111, CNRS UMR5308, ENS-Lyon, University of Lyon 1, Lyon, France.

Measles virus (MV) infection causes an acute childhood disease that can include infection of the central nervous system and can rarely progress to severe neurological disease for which there is no specific treatment. We generated potent antiviral peptide inhibitors of MV entry and spreading and MV-induced cell fusion. Dimers of MV-specific peptides derived from the C-terminal heptad repeat region of the MV fusion protein, conjugated to cholesterol, efficiently protect SLAM transgenic mice from fatal MV infection. Fusion inhibitors hold promise for the prophylaxis of MV infection in unvaccinated and immunocompromised people, as well as potential for the treatment of grave neurological complications of measles.
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http://dx.doi.org/10.1128/JVI.02436-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838268PMC
December 2013

A PEGylated analog of the gut hormone oxyntomodulin with long-lasting antihyperglycemic, insulinotropic and anorexigenic activity.

Bioorg Med Chem 2013 Nov 19;21(22):7064-73. Epub 2013 Sep 19.

IRBM P. Angeletti, 00040 Pomezia, Rome, Italy.

Peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor (GLP1R) are rapidly gaining favor as antidiabetic agents, since in addition to increasing glucose-dependent insulin secretion, they also cause weight loss. Oxyntomodulin (OXM), a natural peptide with sequence homology to both glucagon and GLP-1, has glucose-lowering activity in rodents and anorectic activity in rodents and humans, but its clinical utility is limited by a short circulatory half-life due to rapid renal clearance and degradation by dipeptidyl peptidase IV (DPP-IV). Here, we describe the development of a novel DPP-IV-resistant, long-acting GLP1R agonist, based on derivatization of a suitably chosen OXM analog with high molecular weight polyethylene glycol (PEG) ('PEGylation'). PEG-OXM exerts an anti-hyperglycemic effect in diet-induced obese (DIO) mice in a glucose-dependent manner, with a maximally efficacious dose of 0.1mg/kg, and reduces food intake and body weight with a minimally efficacious dose of 1mg/kg. If this pharmacology is recapitulated in patients with type 2 diabetes, these results indicate PEG-OXM as a potential novel once-weekly GLP-1 mimetic with both glucose-lowering activity and weight loss efficacy.
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http://dx.doi.org/10.1016/j.bmc.2013.09.016DOI Listing
November 2013

PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes.

Bioorg Med Chem 2012 Aug 9;20(15):4751-9. Epub 2012 Jun 9.

IRBM P. Angeletti, 00040 Pomezia (RM), Italy.

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.
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http://dx.doi.org/10.1016/j.bmc.2012.06.003DOI Listing
August 2012

A general strategy to endow natural fusion-protein-derived peptides with potent antiviral activity.

PLoS One 2012 16;7(5):e36833. Epub 2012 May 16.

PeptiPharma, Pomezia, Rome, Italy.

Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy.Viral fusion is driven by specialized proteins which, although specific to each virus, act through a common mechanism, the formation of a complex between two heptad repeat (HR) regions. The HR regions are initially separated in an intermediate termed "prehairpin", which bridges the viral and cell membranes, and then fold onto each other to form a 6-helical bundle (6HB), driving the two membranes to fuse. HR-derived peptides can inhibit viral infectivity by binding to the prehairpin intermediate and preventing its transition to the 6HB.The antiviral activity of HR-derived peptides differs considerably among enveloped viruses. For weak inhibitors, potency can be increased by peptide engineering strategies, but sequence-specific optimization is time-consuming. In seeking ways to increase potency without changing the native sequence, we previously reported that attachment to the HR peptide of a cholesterol group ("cholesterol-tagging") dramatically increases its antiviral potency, and simultaneously increases its half-life in vivo. We show here that antiviral potency may be increased by combining cholesterol-tagging with dimerization of the HR-derived sequence, using as examples human parainfluenza virus, Nipah virus, and HIV-1. Together, cholesterol-tagging and dimerization may represent strategies to boost HR peptide potency to levels that in some cases may be compatible with in vivo use, possibly contributing to emergency responses to outbreaks of existing or novel viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036833PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353973PMC
October 2012

The glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin.

Obesity (Silver Spring) 2012 Aug 16;20(8):1566-71. Epub 2012 Mar 16.

Merck Research Laboratories, Rahway, NJ, USA.

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L-cells of the gut that has a weak affinity for both the glucagon-like peptide-1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight-lowering effects of OXM. We identified an equipotent GLP1R-selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ~100-fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid-lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R-selective agonist. Studies in Glp1r(-/-) mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight-lowering action of OXM.
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http://dx.doi.org/10.1038/oby.2012.67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408645PMC
August 2012

Rapid screening for entry inhibitors of highly pathogenic viruses under low-level biocontainment.

PLoS One 2012 2;7(3):e30538. Epub 2012 Mar 2.

Department of Pediatrics, Weill Medical College, Cornell University, New York, New York, United States of America.

Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions. Universal methods for rapidly identifying and screening candidate antivirals are urgently needed. We have developed a modular antiviral platform strategy that relies on simple bioinformatic and genetic information about each pathogen. Central to this platform is the use of envelope glycoprotein cDNAs to establish multi-cycle replication systems under BSL2 conditions for viral pathogens that normally require BSL3 and BSL4 facilities. We generated monoclonal antibodies against Nipah G by cDNA immunization in rats, and we showed that these antibodies neutralize both Nipah and Hendra live viruses. We then used these effective Henipavirus inhibitors to validate our screening strategy. Our proposed strategy should contribute to the response capability for emerging infectious diseases, providing a way to initiate antiviral development immediately upon identifying novel viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030538PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292545PMC
July 2012

Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus.

J Biol Chem 2011 Dec 12;286(49):42141-42149. Epub 2011 Oct 12.

Departments of Pediatrics and of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021. Electronic address:

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.
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http://dx.doi.org/10.1074/jbc.M111.254243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234914PMC
December 2011

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

J Virol 2011 Dec 5;85(24):12867-80. Epub 2011 Oct 5.

Departments of Pediatrics and of Microbiology and Immunology, Weill Medical College of Cornell University, 515 East 71st Street, 6th Floor, Box 309, New York, NY 10021, USA.

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.
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http://dx.doi.org/10.1128/JVI.05873-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233114PMC
December 2011

Infection of primary neurons mediated by nipah virus envelope proteins: role of host target cells in antiviral action.

J Virol 2011 Aug 8;85(16):8422-6. Epub 2011 Jun 8.

Department of Pediatrics and Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA.

We have previously described heterotypic peptides from parainfluenza virus that potently inhibit Nipah virus in vitro but are not efficacious in vivo. In contrast, our second-generation inhibitors, featuring a cholesterol moiety, are also efficacious in vivo. The difference between in vitro and in vivo results led us to investigate the basis for this discrepancy. Here, we compare the activities of the compounds in standard laboratory cells and in cells relevant to the natural tropism of Nipah virus, i.e., primary neurons, and show that while our first-generation inhibitors are poorly active in primary neurons, the cholesterol-conjugated compounds are highly potent. These results highlight the advantage of evaluating antiviral potency in cells relevant to natural host target tissue.
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http://dx.doi.org/10.1128/JVI.00452-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147962PMC
August 2011

DPP-IV-resistant, long-acting oxyntomodulin derivatives.

J Pept Sci 2011 Apr 3;17(4):270-80. Epub 2011 Feb 3.

Istituto di Ricerche di Biologia Molecolare P. Angeletti, 00040 Pomezia, Rome, Italy.

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon-like peptide 1 (GLP-1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose-dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37-amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP-1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment-associated nausea than GLP-1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half-life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP-IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP-IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258-2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use.
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http://dx.doi.org/10.1002/psc.1328DOI Listing
April 2011

ApoA-I mimetic peptides promote pre-β HDL formation in vivo causing remodeling of HDL and triglyceride accumulation at higher dose.

Bioorg Med Chem 2010 Dec 4;18(24):8669-78. Epub 2010 Nov 4.

Merck Research Laboratories, 126 East Lincoln Avenue, RY80Y-140, Rahway, NJ 07065, USA.

Reverse cholesterol transport promoted by HDL-apoA-I is an important mechanism of protection against atherosclerosis. We have previously identified apoA-I mimetic peptides by synthesizing analogs of the 22 amino acid apoA-I consensus sequence (apoA-I(cons)) containing non-natural aliphatic amino acids. Here we examined the effect of different aliphatic non-natural amino acids on the structure-activity relationship (SAR) of apoA-I mimetic peptides. These novel apoA-I mimetics, with long hydrocarbon chain (C(5-8)) amino acids incorporated in the amphipathic α helix of the apoA-I(cons), have the following properties: (i) they stimulate in vitro cholesterol efflux from macrophages via ABCA1; (ii) they associate with HDL and cause formation of pre-β HDL particles when incubated with human and mouse plasma; (iii) they associate with HDL and induce pre-β HDL formation in vivo, with a corresponding increase in ABCA1-dependent cholesterol efflux capacity ex vivo; (iv) at high dose they associate with VLDL and induce hypertriglyceridemia in mice. These results suggest our peptide design confers activities that are potentially anti-atherogenic. However a dosing regimen which maximizes their therapeutic properties while minimizing adverse effects needs to be established.
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http://dx.doi.org/10.1016/j.bmc.2010.09.074DOI Listing
December 2010

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry.

PLoS Pathog 2010 Oct 28;6(10):e1001168. Epub 2010 Oct 28.

Departments of Pediatrics and of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.
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http://dx.doi.org/10.1371/journal.ppat.1001168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965769PMC
October 2010

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Proc Natl Acad Sci U S A 2010 Jun 18;107(23):10655-60. Epub 2010 May 18.

Department of Peptide Chemistry, Merck Research Laboratories, Pomezia, 00040 Rome, Italy.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.
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http://dx.doi.org/10.1073/pnas.1004261107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890830PMC
June 2010
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