key: cord-0685679-9y2kkmby
authors: Pessi, Antonello
title: Cholesterol‐conjugated peptide antivirals: a path to a rapid response to emerging viral diseases
date: 2014-10-20
journal: J Pept Sci
DOI: 10.1002/psc.2706
sha: 2fd91091adfac64207867f73c47247a1da6b2e9d
doc_id: 685679
cord_uid: 9y2kkmby

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. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The spectrum of infectious diseases that threaten human health is rapidly broadening: The number of previously unknown conditions that have emerged since 1970 exceeds 40, with a new disease discovered on average more than once a year [1, 2] . According to a 2008 World Health Organization (WHO) report, between the years 2002 and 2007, a record of 1100 worldwide epidemic events occurred [3] . A comprehensive examination of episodes of emerging infectious disease outbreaks between 1940 and 2004, after controlling for reporting bias, came to the conclusion that the incidence of the events has increased significantly over time [4] .

For viral diseases in particular, in addition to the constant fear of a new wave of pandemic influenza, the list of emerging or reemerging viruses includes HIV [5] . At the time when this manuscript was being finalized, the largest and longest outbreak of Ebola virus was still spreading [6] . The latter virus, together with Nipah virus, hantavirus, Lassa virus, and Marburg virus, is on the Centers for Disease Control and Prevention list of bioterrorism agents. For most of these viruses, the therapeutic options are limited if not completely absent.

The best description of the features of an outbreak of a novel virus comes from the SARS pandemic of 2002/2003the first major emerging disease threat of the 21st century. In the short period between the WHO global alert (March 2003) and the WHO announcement that the pandemic was over (5 July 2003) , SARS affected 8098 people, caused 774 deaths, disrupted international travel, and cost huge business losses. SARS presented many of the features of the most feared pandemic diseases: a respiratory disease that is transmitted from person to person, with a long asymptomatic incubation period, and symptoms very similar to other commonplace illnesses.

At the time of its first appearance, its causative agent (SARS-associated coronavirus) was unknown, and there was neither a diagnostic test nor a specific treatment. The international response to the new threat was extremely efficacious, leading to the isolation of SARS-CoV [7] and sequencing of its genome in the record time of just 2 weeks [8, 9] .

The measures that proved efficacious in containing the disease were isolation, quarantine, travel surveillance, increased personal hygiene, and personal protection equipment (masks, gloves, and eyeglasses). As to pharmacological treatments, however, a WHO expert panel review concluded that it was not possible to determine whether any of the treatments used for SARS-infected patients did some benefit and suggested that some treatments might have actually been harmful [10] .

As the SARS case vividly illustrates, our ability to quickly isolate and genetically fingerprint the causative agent of new viral diseases is not matched by an ability to develop suitable treatments. This is not surprising, because drug discovery typically requires additional information to that simply deriving from knowledge of the viral genome.

Against this background, peptides may represent a special case, because for enveloped viruses, it is generally possible to identify the sequence of candidate peptide inhibitors directly from genomic information.

For all enveloped viruses, fusion between the viral and the target cell membrane represents an obligatory step of the life cycle. Interfering with this process is a well-established therapeutic strategy [11] that has led to the development of the peptide fusion inhibitor enfuvirtide (Fuzeon ® , also known as T20, Roche, Basel, Switzerland), which is in clinical use for HIV [12] . Viral fusion is mediated by specialized proteins, which harbor a structural motif that consists of a repeating pattern of seven amino acids: the heptad repeat (HR). The most prevalent class (class I) of fusion proteins typically has two HR regions: the first one close to the N-terminus (HRN), adjacent to the fusion peptide, and the second one close to the C-terminus (HRC), immediately preceding the transmembrane domain. It is generally accepted that in the key intermediate of viral fusion, the so-called prehairpin intermediate, bridging the viral and cell membranes, the HRN and HRC regions are separated, and the HRN forms a trimeric coiled coil ( Figure 1 ). Folding of the HRC onto the HRN trimer leads to the formation of a six-helical bundle (6HB), and in this process, the two membranes are brought in close apposition, which leads to their fusion [11] (Figure 1 ). Peptides corresponding to the sequence of the HR regions can bind to the prehairpin intermediate, prevent its transition to the 6HB, and block fusion (Figure 2(A) ). This is the mechanism of inhibition of enfuvirtide [12] , which applies to several other peptides derived from the HR regions of many viruses [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] .

The characteristic sequence pattern of the HR provides an important advantage for the development of peptide-based antivirals, because HR can be easily identified through computer programs (e.g. LearnCoil [30] or MultiCoil [31, 32] ). This was the case for SARS-CoV, where HR regions were immediately identified [33] and led to the development of peptide inhibitors [27, 29] , in the absence of any structural information on the fusion protein. This came later and largely confirmed the predictions [34] [35] [36] [37] .

This pathway to drug development offers the opportunity to develop a specific antiviral in a very short timeframe. However, not all HR-derived peptides display the same antiviral potency as the HIV fusion inhibitor enfuvirtide. A major factor at play is the difference in fusion kinetics: viruses that unlike HIV transition through the hairpin intermediate very rapidly offer a shorter window of opportunity for peptide inhibitors that, accordingly, show reduced potency [38] [39] [40] .

If the peptide lead derived from genomic information has insufficient potency, it is necessary to modify it through peptide engineering strategies that, although often successful [18, [41] [42] [43] [44] [45] [46] [47] [48] [49] , may be time consuming. An alternative approach, which does not require any change in the native HR sequence, exploits the role of cholesterol in viral fusion.

Cholesterol plays a key role in the fusion and budding of enveloped viruses [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] , with HIV as the most studied case. It has been shown that cholesterol depletion from virus-infected cells suppresses virus production [60] and that depletion of cholesterol from either virions or target cells inhibits virus-cell fusion [61] [62] [63] [64] . HIV is able to influence the cholesterol metabolism of the host cell [65] [66] [67] [68] , and its lipid membrane is highly enriched in cholesterol [69] . Moreover, a link has been established between low levels of cholesterol in antigen-presenting cells and the status of long-term nonprogressor, a rare infected individual who controls disease progression in the absence of antiretroviral therapy [70, 71] .

The role of cholesterol in viral fusion is related to its role in membrane fluidity and in particular in the formation of lipid rafts [60, [72] [73] [74] [75] [76] . Accordingly, the composition of the lipid membrane of HIV is highly enriched in cholesterol and sphingomyelin [69, 74, 75] . Moreover, CD4, the primary receptor for HIV, is enriched in lipid rafts [77] .

In 2009, Ingallinella et al. proposed that addition of a cholesterol group to an HR-derived peptide would augment its affinity for membranes and in particular for the cholesterol-rich lipid rafts. The resulting increase in local concentration at the site of action would translate into enhanced antiviral potency (Figure 2(B) ). Application of this concept to the HR-derived peptide C34 yielded C34-Chol, where the cholesterol group was attached, via a thioether bond, to the side chain of a Cys residue added to the C-terminus, with an intervening Gly-Ser-Gly spacer. Cholesterol conjugation brought a dramatic improvement of antiviral potency: Depending on the viral strain tested, C34-Chol was 25-to 100-fold more potent than unconjugated C34 and 50-to 400-fold more potent than enfuvirtide [78] . No such improvement was apparent when cholesterol was substituted with palmitic acid [78] , a lipid that is both a poorer membrane anchor and is not enriched in lipid rafts [79] . Numerous examples have since accumulated that cholesterol conjugation can improve the antiviral potency of HR-derived peptides from many enveloped virusesthese include in vitro and in vivo studies (Table 1) . Cholesterol conjugation has also been successfully applied to a covalent inhibitor derived from C34, where an isothiocyanate group on the side chain of Asp 632 targets Lys 574 in HRN [80] .

Interestingly, in line with the hypothesized mechanism of action of cholesterol conjugation, another lipid that is enriched in the HIV lipidome, dihydrosphingosine [69] , also augments potency when conjugated to an HR peptide; in this case, one derived from the HRN instead of the HRC [81] .

Cholesterol-conjugated peptides show improved pharmacokinetics and are active in vivo Cholesterol conjugation also solves an outstanding problem of peptide therapeutics, the short in vivo half-life because of enzyme degradation and rapid clearance [82] . An effective way to improve peptide pharmacokinetics (PK) is conjugation to lipids [82] , which drive binding to serum proteins. The most typical derivatization is with long chain fatty acids, but derivatization with cholesterol has also been explored [83] [84] [85] .

In a comparative subcutaneous PK study in mice at the concentration of 3.5 mg/kg, C34 was undetectable in plasma after 6 h, while 130 nM of C34-Chol was still detectable 24 h after the injection: a concentration ≈300-fold higher than the IC 90 measured against multiple HIV-1 strains [78] .

In another example, a single dose of 2 mg/kg of a cholesterolconjugated inhibitor of Nipah virus, administered intraperitoneally to golden hamsters, was sufficient to achieve a plasma concentration >100-fold higher than the in vitro IC 50 for 24 h [86] .

A single intramuscular injection of 1.6 mg/kg of a cholesterolconjugated inhibitor of Newcastle disease virus (NDV) administered to chickens yielded a plasma concentration of 210 nM at 24 h and ≈120 nM after 48 h, to be compared with the in vitro IC 50 = 8.1 nM and IC 90 = 13 nM [87] .

Overall, the PK of a cholesterol-conjugated inhibitor is expected to be at least comparable withand likely better thanthe PK of enfuvirtide, which is administered twice daily by subcutaneous injection [88] .

One further possibility to potentiate the activity of HR-derived peptide inhibitors is to exploit multimerization, another modification that can be implemented on the native sequence of the peptide. Multimerization brings an avidity effect [89] , which effectively reduces the k off of the inhibitor fusion protein complex and increases the potency of fusion [90, 91] and entry [92] inhibitors. The effect of cholesterol conjugation, which increases the k on by facilitating the encounter of the peptide with the viral target protein, is complementary: The two modifications can therefore work in concert.

A reagent that can simultaneously dimerize the peptide and install a cholesterol group onto it is described in Figure 3 [93] . Trimers or higher-order multimers could be prepared by the same strategy, but the maximum benefit of multimerization is achieved at the level of a dimer [90, 91] . The reagent is stable upon prolonged storage at À20°C. Notably, the same cysteine-containing precursor peptide used to produce the monomeric cholesterol-conjugated peptide can be reacted with the cholesterol dimer-forming moiety: This enables parallel synthesis of the cholesterol-conjugated monomer and dimer (Figure 3) , which can then also be tested in parallel to establish the optimal inhibitor.

Using the reagent in Figure 3 , dimeric cholesterol-conjugated inhibitors were prepared for HIV [93] , Nipah [93] , and measles [94] virus (MV), which were more potent and effective than the corresponding cholesterol-conjugated monomers. Potentiation of a trimeric HIV peptide inhibitor [91] by cholesterol conjugation [95] has independently been reported by another laboratory.

Since its discovery in 2009, the scope of cholesterol conjugation has considerably increased, and it now includes examples for retroviruses, paramyxoviruses, coronaviruses, orthomyxoviruses, henipaviruses, and filoviruses (Table 1 ). For some of these viruses, in vivo efficacy has been demonstrated in a suitable animal model [86, 87, 94] . For example, Porotto et al. showed that once-daily intraperitoneal administration of a cholesterol-conjugated inhibitor of Nipah virus to golden hamsters, an established model of infection, effectively prevented what would otherwise be fatal Nipah virus encephalitis. Prophylactic treatment was 100% efficacious when initiated 2 days before viral challenge and 80% efficacious when initiated concurrently with the infection. Notably, in light of the highly lethal nature of this virus, treatment 2 days after infection led to survival of 40% of the animals [86] .

The same laboratory used intranasal infection of MV in suckling mice expressing the human SLAM transgene as a model to evaluate the efficacy of cholesterol-conjugated HR-derived peptides. Infection with MV causes a lethal acute neurological syndrome, which was completely prevented, in 100% of the animals, by prophylactic once-daily administration of a cholesterol-conjugated dimer derived from the sequence of the MV HRC domain [94] .

Li et al. used cholesterol conjugation to improve the potency of their previously described inhibitor of NDV [96] . Intramuscular injection of the cholesterol-conjugated peptide 1 day before virus infection, and then every 3 days, protected 70% of the chickens from different serotypes of NDV [87] . Moreover, treatment of the animals 2 days after infection resulted in 50% protection [87] .

Two interesting features of these studies deserve further comment: First, in the aforementioned three studies, the peptide was detectable in the brain 24 h after administration [86, 87, 94] , indicating that cholesterol conjugation may enable penetration of the blood-brain barrier, a difficult feat for drugs in general and for biologics in particular [97] .

Second, the modest but observable degree of protection observed at 2 days postexposure (40% in the Porotto et al. [86] and 50% in the Li et al. [87] study) is comparable with the results reported for monoclonal antibodies against Ebola virus in macaques (50% efficacy when dosed 2 days after exposure) [98, 99] , including the ZMapp antibody cocktail [99] , which is being used as an emergency therapeutic in the latest Ebola outbreak [100] .

It must be noted though that all the examples reported so far come from viruses featuring class I fusion proteins. While being the most prevalent class, it does not include major pathogens such as hepatitis C virus and dengue virus, both belonging to class II, and herpes simplex virus belonging to class III.

Class I proteins are mainly α-helical and form 6HBs as the postfusion structure, as exemplified by HIV (Figure 1 ). Class II fusion proteins are characterized by trimers of hairpins composed of β-structures [101] , with a fusion loop located at the tip of an elongated β-sheet, in place of a fusion peptide upstream of an α-helix. Class III proteins form trimers of hairpins by combining a central α-helical trimeric core, similar to class I proteins, with fusion loops structurally similar to class II proteins [101] . Despite these structural differences, all fusion proteins adopt a hairpin structure [102] that is susceptible to inhibition.

For example, although the entry of herpes viruses (featuring a class III fusion protein) is a complex process, not yet fully clarified, in which multiple glycoproteins are involved, several laboratories have reported that peptides derived from the HR regions of gB and gH of herpes simplex virus type 1, bovine herpes virus type 1, and human cytomegalovirus are able to inhibit infection [103] .

For dengue virus, helical domains have been identified in the stem region of E protein (a class II protein) that are critical for virus assembly and entry [104] . Peptide inhibitors derived from this membrane-binding region have been described [105] [106] [107] , one of which inhibits all four virus serotypes [107] . Moreover, de novo computational design of peptide inhibitors has been successful [108, 109] . The work by Xu et al. in particular [109] indicates how the same computational approach may yield candidate peptide inhibitors for fusion proteins of all classes.

Given the ubiquitous importance of cholesterol-rich lipid rafts in viral fusion, it is expected that cholesterol conjugation may enhance the antiviral potency also for peptides derived from class II and III fusion proteins. However, no data in this regard have yet been reported.

The combination of bioinformatic lead identification (lead ID) and potency/PK improvement provided by cholesterol conjugation may form the basis for a rapid response strategy to emerging viral diseases (Figure 4) . The project would begin immediately after the genome of the new pathogen is made available. The lead ID phase would consist in the bioinformatic analysis (e.g. via LearnCoil [30] or MultiCoil [31, 32] ) of the newly sequenced genome to identify the HR regions; it could last a few days. As discussed in the previous section, the computational identification of inhibitory peptides for viruses utilizing class II or III fusion proteins would be less straightforward, and the number of candidate peptides to be tested likely larger. However, given the high throughput of parallel solid-phase Figure 4 . Rapid response strategy based on cholesterol-conjugated fusion inhibitors. From the sequenced genome of the emerging virus, the HR regions are identified bioinformatically. This corresponds to the lead identification (lead ID) phase. In the lead optimization (lead opt) phase, candidate HR peptides are synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in Figure 3 . The cholesterol-conjugated monomer and dimer are tested for antiviral activity. The lead opt cycle continues until a peptide is found potent enough to be moved to large-scale production. peptide synthesis and the modularity of the cholesterol conjugation reaction, the initial number of target sequences should not represent a key limiting factor in the lead optimization (lead opt) phase. In lead opt, candidate peptides would be synthesized in the form of suitable precursors for cholesterol conjugation and reacted with the reagents shown in Figure 3 or similar ones. The cholesterol-conjugated monomers and dimers would be tested for antiviral activity. In the logic of off-the-shelf reagents for immediate use, variations of the reagents of Figure 3 to optimize the length of the linker joining the monomers might provide considerable increase in potency [89] , as shown by Kay et al. [91] . Haloacetyl or maleimide-functionalized cores with polyethylene glycol spacers of different length could all be reacted with the same precursor to rapidly select the most potent inhibitor.

The lead opt cycle would continue until a peptide was found, potent enough to be moved to large-scale production. With a relatively small number of candidate peptides, and parallel synthesis and conjugation, it is expected that the lead opt phase would be rapid, optimally a few weeks. Large-scale production of the candidate peptide therapeutic could begin immediately, because the protocol for lab-scale and large-scale solid-phase peptide synthesis are essentially the same. Preclinical evaluation (formulation, animal PK to predict the human dose) could proceed in parallel with the good manufacturing practices batch production. Abbreviated preclinical toxicology, in connection with the 'animal rule' [110] , would be justified in an emergency situation.

The strategy outlined in the previous section could provide in a very short timeframe a cholesterol-conjugated therapeutic specific for the emerging virus. Although not necessarily the optimal drug for the new disease, it would represent a key complement to preventive measures and could enable better control of the initial outbreak. Further research efforts may then lead to a substitute therapy with increased efficacy and/or better ease of administration.

One could even envisage setting up a core laboratory that routinely runs the lead ID and lead opt stages on newly identified viruses of potential concern, in advance of an actual outbreak of disease. The limited resources necessary could be part of the Emergency and Preparedness Response set out by the Centers for Disease Control and Prevention (http://emergency.cdc.gov/) or the Global Alert and response by WHO (http://www.who.int/csr/en/) for preparing for and responding to public health emergencies.

Emerging and reemerging diseases: a historical perspective

Emerging viral diseases

The World Health Report

Global trends in emerging infectious diseases

Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the coronavirus study group

World struggles to stop Ebola

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

SARS: systematic review of treatment effects

Mechanisms of viral membrane fusion and its inhibition

Design of recombinant protein-based SARS-CoV entry inhibitors targeting the heptad-repeat regions of the spike protein S2 domain

Analysis of a peptide inhibitor of paramyxovirus (NDV) fusion using biological assays, NMR, and molecular modeling

Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

Development of antiviral fusion inhibitors: short modified peptides derived from the transmembrane glycoprotein of feline immunodeficiency virus

Peptide inhibitors targeting virus-cell fusion in class I enveloped viruses

Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency

Peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection

A synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of Sendai virus-cell fusion: an emerging similarity with functional domains of other viruses

Antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein

Peptide-based inhibitors of the HIV envelope protein and other class I viral fusion proteins

Inhibition of Ebola virus entry by a C-peptide targeted to endosomes

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion

Inhibition of Hendra virus fusion

Inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides

Identification of a minimal peptide derived from heptad repeat (HR) 2 of spike protein of SARS-CoV and combination of HR1-derived peptides as fusion inhibitors

Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors

LearnCoil-VMF: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins

MultiCoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone

MultiCoil: a program for predicting two-and three-stranded coiled coils

The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39)

Structural characterization of the SARS-coronavirus spike S fusion protein core

Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein

Structural characterization of the fusion-active complex of severe acute respiratory syndrome (SARS) coronavirus

Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics

A quantitative and kinetic fusion protein-triggering assay can discern distinct steps in the Nipah virus membrane fusion cascade

Kinetic dependence of paramyxovirus entry inhibition

Broad antiviral activity and crystal structure of HIV-1 fusion inhibitor sifuvirtide

Short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant HIV-1

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus

Design of a novel HIV-1 fusion inhibitor that displays a minimal interface for binding affinity

Remodeling of gp41-C34 peptide leads to highly effective inhibitors of the fusion of HIV-1 with target cells

Inhibition of HIV type 1 infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism

Short constrained peptides that inhibit HIV-1 entry

Hydrocarbon doublestapling remedies the proteolytic instability of a lengthy peptide therapeutic

Design and evaluation of sifuvirtide, a novel HIV-1 fusion inhibitor

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Cholesterol enhances mouse hepatitis virus-mediated cell fusion

A single point mutation controls the cholesterol dependence of Semliki Forest virus entry and exit

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

The major cellular sterol regulatory pathway is required for Andes virus infection

Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle

Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1

Cholesterol dependence of varicella-zoster virion entry into target cells

Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion

Plasma membrane rafts play a critical role in HIV-1 assembly and release

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells

Human immunodeficiency virus impairs reverse cholesterol transport from macrophages

Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions

Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

The HIV lipidome: a raft with an unusual composition

Alterations in cholesterol metabolism restrict HIV-1 trans infection in nonprogressors

New clues to understanding HIV nonprogressors: low cholesterol blocks HIV trans infection

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

Lipid composition and fluidity of the human immunodeficiency virus

Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes

Role of lipid rafts in virus replication

The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation

Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency

Membrane targeting of lipid modified signal transduction proteins

A multi-functional peptide as an HIV-1 entry inhibitor based on self-concentration, recognition, and covalent attachment

Sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1

Converting peptides into drug leads by lipidation

Structure-activity and protraction relationship of longacting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity, and bulkiness

Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice

DPP-IV-resistant, long-acting oxyntomodulin derivatives

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

A cholesterol tag at the N terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo

Enfuvirtide: a fusion inhibitor for the treatment of HIV infection

Dependence of avidity on linker length for a bivalent ligand-bivalent receptor model system

Potent D-peptide inhibitors of HIV-1 entry

Design of a potent D-peptide HIV-1 entry inhibitor with a strong barrier to resistance

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

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

Fatal measles virus infection prevented by brain-penetrant fusion inhibitors

Design of a modular tetrameric scaffold for the synthesis of membrane-localized D-peptide inhibitors of HIV-1 entry

Characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of NDV and IBV fusion glycoproteins

Drug transport across the blood-brain barrier

Successful treatment of Ebola virus-infected cynomolgus macaques with monoclonal antibodies

Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail

Experimental Ebola drugs enter the limelight

Virus membrane fusion

Virus membrane-fusion proteins: more than one way to make a hairpin

Peptide inhibitors against herpes simplex virus infections

The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry

Peptide inhibitors of dengue-virus entry target a late-stage fusion intermediate

Peptide inhibitors of flavivirus entry derived from the E protein stem

Release of dengue virus genome induced by a peptide inhibitor

Structural optimization and de novo design of dengue virus entry inhibitory peptides

Computational identification of self-inhibitory peptides from envelope proteins

New drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. Final rule

Viral entry inhibitors targeted to the membrane site of action

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

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking