Báo cáo khoa học: Apolipoprotein E-derived antimicrobial peptide analogues with altered membrane affinity and increased potency and breadth of activity pdf

We report that basic residues and peptide length of around 18 residues were required for activity; however, the Leu residues can be substituted by several other residues without loss of

with altered membrane affinity and increased potency and breadth of activity

Bridie A Kelly1, Stuart J Neil2,*, A´ ine McKnight2,

†, Joana M Santos3,‡, Photini Sinnis3, Edward R Jack1,§, David A Middleton1,– and Curtis B Dobson1

1 Faculty of Life Sciences, The Mill, The University of Manchester, UK

2 Wohl Virion Centre, Windeyer Building, University College London, UK

3 Department of Medical and Molecular Parasitology, New York University School of Medicine, NY, USA

Keywords

antimicrobial peptides; apolipoprotein E;

HIV; Plasmodium; membrane perturbation

Correspondence

C B Dobson, Faculty of Life Sciences,

The Mill, The University of Manchester,

PO Box 88, Sackville Street, Manchester

M60 1QD, UK

Fax: +44 (0)161 306 4433

Tel: +44 (0)161 306 8765

E-mail: curtis.dobson@manchester.ac.uk

Present address

*Aaron Diamond AIDS Research Center,

New York, NY, USA

†Centre for Infectious Disease, Institute of

Cell and Molecular Science, Barts and The

London, Queen Mary’s School of Medicine

and Dentistry, London, UK

‡Department of Microbiology and Molecular

Medicine, CMU, University of Geneva,

Switzerland

§Division of Structural Biology, Department

of Biological sciences, University of

Warwick, Coventry, UK

–School of Biological Sciences, University of

Liverpool, UK

(Received 4 May 2007, revised 22 June

2007, accepted 6 July 2007)

doi:10.1111/j.1742-4658.2007.05981.x

Host-derived anti-infective proteins represent an important source of sequences for designing antimicrobial peptides (AMPs) However such sequences are often long and comprise diverse amino acids with uncertain contribution to biological effects Previously, we identified a simple highly cationic peptide derivative of human apolipoprotein E (apoEdp) that inhib-ited a range of microorganisms Here, we have dissected the protein chem-istry underlying this activity We report that basic residues and peptide length of around 18 residues were required for activity; however, the Leu residues can be substituted by several other residues without loss of activity and, when substituted with Phe or Trp, resulted in peptides with increased potency These apoEdp-derived AMPs (apoE-AMPs) showed no cytotoxi-city and minimal haemolytic activity, and were active against HIV and Plasmodiumvia an extracellular target CXCR4 and CCR5 strains of HIV were inhibited though an early stage in viral infection upstream of fusion, and a lack of inhibition of vesicular stomatitis virus G protein pseudotyped HIV-1 suggested the anti-HIV activity was relatively selective Inhibition of Plasmodium invasion of hepatocytes was observed without a direct action

on Plasmodium integrity or attachment to cells The Trp-substituted apoE-AMP adhered to mammalian cells irreversibly, explaining its increased potency; NMR experiments confirmed that the aromatic peptides also showed stronger perturbation of membrane lipids (relative to apoEdp) Our data highlight the contribution of specific amino acids to the activity

of apoEdp (and also potentially unrelated AMPs) and suggest that apoE-AMPs may be useful as lead agents for preventing the early stages of HIV and Plasmodium cellular entry

Abbreviations

AMP, antimicrobial peptide; apoE, apolipoprotein E; apoE-AMP, apoE-derived AMP; apoEdp, cationic peptide derivative of human

apolipoprotein E; DMPC-d4, 1,2-dimyristoylphosphatidyl-1,1,2,2- 2 H4-choline; DOPG, dioleoylphosphatidylglycerol; FFU, focus forming unit; HSPG, heparan sulfate proteoglycan; HSV1, herpes simplex virus type 1; HSV2, herpes simplex virus type 2; LDLR, low density lipoprotein receptor; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; VSV-G, vesicular stomatitis virus G protein.

Antimicrobial peptides (AMPs) represent a promising

source of novel molecules with potential as therapeutic

leads against bacteria, viruses and parasites A large

number of AMPs have been reported previously with

many being derived from nonhuman naturally

occur-ring sequences, especially from invertebrates or

amphi-bians [1] In addition, AMPs have been developed from

entirely synthetic sequences, often comprising cationic

residues (usually Arg or Lys, though sometimes His)

separated by Ala, Leu, Met, Phe, Pro, Tyr or Trp

resi-dues Peptides derived from human or mammalian

sources, such as lactoferrin [2], defensins [3] and

LL-37 [4], are often long and complex, making rational

modification and testing of their entire sequences

difficult, and meaning that such peptides may be too

costly for clinical use

APOE, the gene coding for human

apolipopro-tein E (apoE), influences the outcome of infection [5]

The APOE-e4 allele is associated with high

preva-lence of cold sores and with an increased risk of

Alz-heimer’s disease in elderly people possessing latent

herpes simplex virus type 1 (HSV1) in the brain [6],

and protection from liver damage in those infected

with hepatitis C virus [7] In addition, APOE

geno-type influences risk of developing herpes simplex

encephalitis [8], malaria in children [9] and shingles

and postherpetic neuralgia in females caused by

vari-cella zoster virus infection [10] Many infectious

agents, including viruses and intracellular parasites

such as Plasmodium, use heparan sulfate

proteogly-cans (HSPGs) as their initial attachment sites for

cells and, furthermore, many also bind to low density

lipoprotein receptor (LDLR) family receptors to enter

cells, or interact directly with lipoproteins, with the

latter sometimes being mediated through

apolipopro-teins [11] ApoE also uses both HSPGs and LDLR

family receptors in its entry pathway We recently

showed that the region of apoE responsible for entry

(i.e the HSPG⁄ LDLR receptor binding region) has

direct and broad anti-infective activity [12], when

sta-bilized by constructing a tandem repeat peptide of

apoE141-149 (an approach widely used to study the

biology of this region) [13], with this octadecamer

peptide being referred to as apoEdp ApoEdp also

shows antibacterial action, possibly due to its highly

cationic nature, although it is not amphipathic like

many AMPs In addition, apoEdp also inhibits the

attachment of HSV1 to cells, possibly due to

block-ade of cellular HSPG sites, reflecting its derivation

from the HSPG binding domain of apoE The human

origin of this sequence, coupled with its broad

anti-infective activity and its lack of haemolytic

action, suggests that it might be modified to obtain

safe and potent AMPs targeted towards serious infec-tions

The strategy of developing peptide-based therapies against HIV or other serious infections is strongly sup-ported by the clinical success of the 36-residue peptide Enfuvirtide (T20) HIV-fusion inhibitor Moreover, this approach has validated entry blockade as a viable anti-HIV strategy, and a number of other agents are now being developed as inhibitors of HIV fusion [14] Peptides targeting even earlier events in HIV-entry (i.e upstream of fusion) could offer new approaches for developing antiviral compounds [15] HIV, like many viruses, has been shown to attach to cellular HSPGs prior to CD4 attachment [16], potentially offering a further target for development of HIV therapeutics [17] This possibility is strengthened by the finding that HPSGs on the surface of nonpermissive endothelial cells may act as a reservoir for the virus and mediate its transfer to T lymphocytes [16]; similarly, HSPGs have been implicated in brain invasion by HIV [18] Plasmodiumspp., the parasites responsible for malaria, also enter (liver) cells after first adhering to HSPGs [19] Furthermore, Plasmodium sporozoites bind to a subset of HSPGs used by apoE-containing lipoproteins for uptake by the liver [20] Thus, blockade of HSPGs may offer an attractive target to prevent cellular inva-sion by a range of pathogenic organisms

Here, we dissect those features of the apoEdp pep-tide involved in its activity, and examine the activity and mechanism of action of variants of this sequence

We demonstrate the minimal length for activity, the contribution of individual residues, and show that sub-stitutions for aromatic residues increase potency and breadth of activity, giving rise to further apoE-derived antimicrobial peptides (apoE-AMPs) In addition, we show for the first time that the most effective substi-tuted peptides prevent initial attachment of both CXCR4- and CCR5-HIV strains and of Plasmodium berghei to cells, with this most likely involving increased membrane perturbation associated with their aromatic groups

Results

We examined the influence of peptide length on activ-ity using a series of dodecamer and pentadecamer pep-tides derived from the octadecamer apoEdp sequence (we previously found nonomer peptides to be inactive) (Table 1) Figure 1 shows that the greatest antiviral and antibacterial action was found in the full length tandem repeat peptide apoEdp (P < 0.001) The eico-sameric tandem repeat peptide of apoE141-150 (i.e including the additional Arg residue found in position

150 of apoE) had almost identical antiviral and

anti-bacterial activity to apoEdp ApoEdp therefore

appears to provide the highest activity relative to

pep-tide length amongst the peppep-tides derived from the

apoE HSPG receptor binding region we tested

The apoEdp sequence comprises a simple pattern of

three amino acid residues The contribution of

individ-ual residues to its activity can thus be studied by

gen-eration of a relatively small number of variant peptides

(unlike many host-derived AMPs) We initially tested

whether the cationic residues were critical for its

activ-ity Replacing either all the Lys or all the Arg residues

with a hydrophobic residue (Trp), or replacement of

both Lys and Arg with His or the acidic residues Asp

or Glu, resulted in peptides with no measurable

anti-viral or antibacterial action (data not shown)

The anti-infective activity of apoEdp depends on the

ability of the peptide to form an a-helical structure,

although the basic residues are not distributed in an

amphipathic pattern [12] as is often the case for

a-heli-cal AMPs We therefore examined whether the eight

bulky Leu residues separating the peptide’s basic resi-dues mediated its activity We prepared peptides in which all eight Leu residues were substituted by another amino acid (but maintaining the naturally occurring distribution of cationic Arg and Lys residues within the sequence) Substitutions were made with one of each of the other 16 nonbasic amino acid resi-dues, and also with His (which is only cationic under acidic conditions) (Table 1) The ability to inhibit HSV1 infection was retained by many of these peptides, with more potent activity found after Cys-substitution and especially after Trp-substitution (Fig 2A,B) Very similar data were found for herpes simplex virus type 2 (HSV2) (data not shown) We also examined antibacterial activity in this series of substi-tuted peptides, and found this to be less prevalent Peptides in which the Leu of apoEdp had been substi-tuted by Ile, Trp and Phe also inhibited Plasmodium aeruginosa, but none were as potent as apoEdp itself against this resistant bacterium Interestingly

Table 1 Amino acid sequences of apoE-derived peptides

Se-quences of peptides derived from the apoEdp sequence are

shown, including peptides both of altered length and in which

leucine residues had been substituted for another residue (shown

in bold).

Truncated peptides

ApoEdp C-3 L R K L R K R L L L R K L R K

ApoEdp C-6 L R K L R K R L L L R K

ApoE141–149 L R K L R K R L L

Elongated peptides

ApoE141–150 dp L R K L R K R L L R L R K L R K R L L R

Substituted peptides

ApoEdpL-E E R K E R K R E E E R K E R K R E E

ApoEdpL-A A R K A R K R A A A R K A R K R A A

ApoEdpL-D D R K D R K R D D D R K D R K R D D

ApoEdpL-W W R K W R K R W W W R K W R K R W W

ApoEdpL-M M R K M R K R M M M R K M R K R M M

ApoEdpL-Y Y R K Y R K R Y Y Y R K Y R K R Y Y

ApoEdpL-F F R K F R K R F F F R K F R K R F F

ApoEdpL-I I R K I R K R I I I R K I R K R I I

ApoEdpL-Q Q R K Q R K R Q Q Q R K Q R K R Q Q

ApoEdpL-N N R K N R K R N N N R K N R K R N N

ApoEdpL-C C R K C R K R C C C R K C R K R C C

ApoEdpL-S S R K S R K R S S S R K S R K R S S

ApoEdpL-V V R K V R K R V V V R K V R K R V V

ApoEdpL-T T R K T R K R T T T R K T R K R T T

ApoEdpL-G G R K G R K R G G G R K G R K R G G

ApoEdpL-H H R K H R K R H H H R K H R K R H H

ApoEdpL-P P R K P R K R P P P R K P R K R P P

A

B

Fig 1 Anti-infective activity of peptides constructed from trunca-tions or elongatrunca-tions of the apoEdp seqeunce (A) Infectivity of HSV1 as shown by plaque reduction assay in Vero cells, after treat-ment of virus with various concentrations of apoE-AMPs, showing apoEdp has optimum activity Typical data are shown; bars indicate standard errors (B) Growth of P aeruginosa, in the presence of various concentrations of ApoE-AMPs, again showing that apoEdp has optimum activity Typical data are shown; bars indicate stan-dard errors.

apoEdpL-A, which resembles the previously reported

[21] Ala⁄ Arg ⁄ Lys antimicrobial peptides derived from

human heparan binding sequences, was inactive as

were the other 13 substituted peptides (Fig 2A) Even

fewer peptides inhibited the Gram-positive bacterium

Staphylococcus aureus, with the Ala substituted

pep-tides again proving inactive along with the other

12 substituted peptides (Fig 2A) Tyr and Ile

substi-tuted peptides slightly inhibited S aureus, whereas Trp

and Phe substituted peptides had IC50 concentrations

lower than that of apoEdp

The nontandem-repeat apoE141-149 sequence

(previ-ously found to be inactive, possibly due to its inability

to form a-helix) inhibited HSV1 after substitution of

its four Leu residues with Trp, although this activity

was low (IC50 concentration¼ 125 lgÆmL)1 (95%

confidence interval¼ 48–160 lgÆmL)1) compared with

the tandem repeat of this sequence (apoEdpL-W),

which had an IC50concentration of 9.3 lgÆmL)1 (95%

confidence interval, 8.5–10.5 lgÆmL)1) or 3.1 lm

(Fig 2C) Furthermore, CD measurements revealed

that neither apoEdpL-W or apoEdpL-F have a-helical

structure even in 50% 2,2,2-trifluoroethanol (data not

shown), suggesting that such structure was not required for their activity, unlike apoEdp [12]

Although the biological effects of apoEdp on mam-malian cell cultures have been previously reported, these occur only under unusual physiological condi-tions; for example, in the absence of serum [22] or the absence of full length apoE [23] Indeed, we previously found that apoEdp had minimal activity in standard cytotoxicity and haemolytic assays Here, we examined whether the substituted peptides showed similar appar-ent biocompatibility ApoEdpL-W had no effect

on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction in Vero cells, even after

2 days of incubation at 40 lm (Fig 3A) We previ-ously found that apoEdp had no haemolytic action, and thus examined this for apoEdpL-W, finding only mild haemolytic action at high concentrations (15% red blood cell lysis at 35 lm) (Fig 3B), with this being much less than that for other cationic antimicrobial peptides tested Indeed, Fig 3C shows that the related Trp-substituted peptide apoE 141–150dpL-W shows only 30% haemolysis even when tested at 160 lm By contrast, we found similar levels of haemolysis with

C

Fig 2 Anti-infective activity of Leu-substituted apoEdp-derived peptides, whereby all eight Leu residues were substituted for another resi-due (A) Relative antiviral and antibacterial activity of Leu-substituted forms of apoEdp (unsubstituted apoEdp is indicated by the letter ‘L’) Values shown are average IC50concentrations (bacteria) or IC25 concentrations (HSV1) in lM, after turbidity or plaque reduction assay (B) Anti-HSV1 activity of apoEdp and apoEdpL-W, measured by a plaque reduction assay Typical data are shown; bars indicate standard errors (C) Anti-HSV1 activity of apoEdpL-W relative to that for the nonomer apoE141–149-L-W Concentrations are plotted in weight per vol-ume rather than moles per volvol-ume, thus allowing a direct comparison of the 18mer apoEdpL-W with activity of its two 9mer components when not joined together Activity was measured by plaque reduction assay Typical data are shown; bars indicate standard errors.

the previously described lytic antimicrobial peptides

RLLR5 and N-RLLR3 when these were used at 5 lm

and 31 lm, respectively In conclusion, we found no

significant cytotoxic or haemolytic action of these

pep-tides at concentrations of apoEdp, apoEdpL-W, and

apoE141–150dpL-W found to be efficacious against

microorganisms

We also compared the activities of other previously

described antimicrobial peptides that were constructed

from Trp, Arg, Leu or Lys residues alone, like the

apoE-AMPs described here (Table 2) Few of these

peptides showed the broad anti-infective activity of

apoEdp-derived peptides, although constructing

tandem repeats of some of these previously described

shorter peptides did increase activity, as had been

found for the apoE141–149 sequence, suggesting that

peptides of length 14–18 consisting of at least two

charged regions separated by hydrophobic residues

may be an optimum feature for AMPs The strongest

antiviral activity was found in the buforin-related

peptide RLLR5 [24,25]; however, the antibacterial

effects of this peptide were limited, perhaps due to the

presence of physiological levels of salt in our assays,

known to reduce the anti-infective activity of this peptide [25]

ApoEdp inhibits HIV replication and, for herpes viruses, blocks the early stages of replication [12] Accordingly, we next examined whether the more potent peptide apoEdpL-W might inhibit the early stages of HIV replication In initial experiments, we found that apoEdpL-W had strong inhibitory action against HIV-IIIB, under conditions in which the pep-tide was added with viral inoculum, and then washed from the system, suggesting that an early stage in repli-cation was targeted Interestingly, apoEdp itself did not show activity in this assay system, possibly reflect-ing either the low concentrations of peptide tested or the different strain of virus used ApoE141–149 and the control peptide apoE263–286 also had no activity,

as expected (Fig 4A) To test whether the action might involve selectively targeting either CXCR4 or CCR5 coreceptors during viral fusion, we tested activity against HIV strains that use one or other or both these coreceptors Similar strong activity was found against strains 2028 (CXCR4⁄ CCR5), 2044 (CXCR4) as well

as SF162 and BaL (CCR5) (Fig 4B), suggesting that

A

C

B

Fig 3 Lack of toxicity of AMPs against mammalian cells (A) MTT reduction in Vero cells treated for extended periods with AMPs Values shown are expressed relative to untreated control wells; bars indicate standard errors (B) Haemolytic assessment of apoE-AMPs ApoEdp and other apoE-AMPs were introduced to red blood cells at various concentrations, Values shown indicate A 540 relative to that for red blood cells completely lysed in 0.45% ammonium hydroxide; bars indicate standard errors (C) Relative haemolytic activity of apoE-AMPs compared with that for the lytic peptides N-[RLLR]3and RLLR5 [24,25] Values shown indicate A540relative to that for red blood cells completely lysed in 0.45% ammonium hydroxide; bars indicate standard errors.

the peptide does not operate through blockage of one

or other coreceptor

To test whether activity may relate to blockage of a

step prior to viral fusion (e.g initial attachment of virus

particles to the cell surface), assays were repeated at

4C, and 10 lm peptide was introduced either before

the virus or after initial viral preadsorption to cells In

neither case was antiviral activity abolished or

signifi-cantly diminished relative to that found in earlier

exper-iments (Fig 4C), suggesting that a step prior to viral

fusion was targeted involving disruption of reversible

attachment of virus to cells, or a direct lytic action on

virus occurring whether or not the virus was cell-bound

To confirm this, we tested whether apoEdpL-W

could also inhibit the entry of vesicular stomatitis virus

G protein (VSV-G) pseudotyped HIV-1 This virus is

identical to HIV-1, but has a VSV envelope, with its

entry mediated by clathrin-mediated endocytosis

lead-ing to pH dependent fusion [26,27] We found that this

virus was not inhibited (Fig 4D), demonstrating that

the action of apoEdpL-W against HIV was relatively

selective, either involving blockage of attachment of

virus to the cell or by interfering with early

envelope-mediated events although without compromising

mem-brane integrity

The Plasmodium sporozoite (i.e the infective stage

of the malaria parasite) enters hepatocytes via

attach-ment to HSPGs, and apoE-containing lipoproteins can

inhibit sporozoite infectivity both in vitro and in vivo

[20] Using a rodent model of the disease, we therefore

examined whether apoEdp, apoEdpL-F and⁄ or

apo-EdpL-W could also disrupt this process Figure 5A shows that both apoEdp and apoEdpL-W inhibit entry

of P berghei into Hepa 1-6 cells; the effect of apo-EdpL-F at this level was not significant We also con-firmed that the active peptides had no cytotoxic action against Hepa 1-6 cells (as this might account for the apparent Plasmodium inhibition): MTT reduction was not inhibited after treatment of these cells with 5–25 lm apoEdp or apoEdpL-W for 2 days (i.e 48 times the length of incubation in the Plasmodium inhi-bition assays) (Fig 5B)

In further assays in which nonbound peptide was washed away prior to introducing the sporozoites, apo-EdpL-W (but not apoEdp) retained its ability to block

P berghei entry (Fig 5C) These data suggest apo-EdpL-W is likely binding to the cell surface irreversibly, thus preventing entry of the parasite Consistent with this, we found that apoEdpL-W had no action on the attachment of sporozoites to cells, but in fact blocks invasion of the parasite after attachment (data not shown) Additionally apoEdpL-W had no direct action against the parasite itself, unlike apoEdp, which caused extensive lysis of the sporozoites (Fig 5D) Thus, apoEdp appears to directly inactivate the sporozoite, whereas apoEdpL-W likely acts through interactions with the mammalian cell membrane

We next examined the relative ability of apoE-AMPs

to perturb model membrane systems, to indicate their relative propensity for their biological activity to be mediated through a direct effect on membrane lipid Wideline deuterium NMR spectra were collected to

Table 2 Antiviral and antibacterial activities of various non-apoE-derived antimicrobial peptides comprised predominantly of Leu or Trp and Arg Anti-HSV1 activity was assessed by plaque reduction assay, antibacterial assays were carried out using turbidity assessment of serial dilutions of peptide inoculated with bacteria In both cases, concentrations yielding 50% inhibition of growth were obtained from the plots (averages for several experiments are shown) Peptides had either been used previously in other studies, were tandem repeats of those peptides (indicated by the subscript ‘dp’), or were devised for the present study (MU103 and MU104).

IC 50 concentration (l M )

HSV1 Pseudomonas aeruginosa

Staphylococcus aureus

a Sequences described in Strom et al [30] b Sequences described in Park et al [24,25].

detect interactions of the apoEdp, apoEdpL-W and

apoEdpL-F derived peptides with the surface of

multilamellar vesicles composed of

1,2-dimyristoyl-phosphatidyl-1,1,2,2-2H4-choline (DMPC-d4) and

di-oleoylphosphatidylglycerol (DOPG) mixed in a 2 : 1

molar ratio The NMR spectrum arises from the

repor-ter molecule, choline methylene-deurepor-terated DMPC, and

is sensitive to any interactions between peptides and the

membrane surface that alter the mean orientation of

the choline moiety [28] Such orientational changes are

manifest in the spectrum as changes in the quadrupole

splittings, which is the frequency separation between

the two pairs of Pake doublets for the a- and

b-deute-rons The anionic DOPG headgroups provide an

over-all negative surface charge and this lipid membrane

system has previously been used in NMR studies of this

type to provide a model membrane for studying the

behaviour of amphipathic peptides [29]

The peptides were titrated into the sample to a lipid⁄ peptide molar ratio of 50 : 1 or 20 : 1 The dashed lines in Fig 6A indicate the scaled changes in splittings (an increase for the b-deuterons and a decrease for the a-deuterons) consistent with the pep-tide interacting with lipid head groups at increasing concentration ApoEdp appears to interact relatively weakly with the mixed lipid bilayers of the multila-mellar vesicles because it induces only minor changes

in the splitting values, yet aromatic substitution greatly enhances the effect on the splittings The marked effects of apoEdpL-W and apoEdpL-F on the spec-trum may reflect a high affinity of these peptides for the lipid head groups or a greater destabilizing effect

on the membrane surface, perhaps as a result of the aromatic groups inserting deeper into the lipid bilayer

By contrast, the spectra in the presence of apoEdp are consistent with the peptide being situated away from

Fig 4 Anti-HIV activity of the apoE-AMP apoEdpL-W (A) Potent anti-HIV action of apoEdpL-W relative to apoEdp, apoE141–149 and apoE263–286 Experiments were performed by treating NP2 cells with peptide prior to introducing HIV IIIB Values show level of HIV infec-tion (in FFU) relative to untreated control; bars indicate standard errors (B) Anti-HIV-1 activity of apoEdpL-W against CXCR4 using HIV-1 strain 2044, CXCR4 ⁄ CCR5 using HIV-1 strain 2028, and CCR5 using HIV strains SF162 and BaL Values show the level of HIV infection (in FFU) relative to untreated control; bars indicate standard errors (C) Anti-HIV-1 activity of apoEdpL-W after incubation of virus with NP2 cells

at 4 C, either before or after addition of peptide Values show the level of HIV infection (in FFU) relative to untreated control; bars indicate standard errors (D) Lack of effect of apoEdpL-W on VSV-G infection of NP cells Virus was added to cells at 4 C, either in the presence or absence of apoEdpL-W at 10 l M (a concentration that strongly inhibits HIV) Infectivity was assessed by enumerating green fluorescence protein-positive cells using fluorescence activated cell sorting (Becton Dickinson) 48 h after transduction.

the membrane surface and participating in a weak

elec-trostatic interaction In all three cases, the degree of

interaction scales with increasing peptide concentration

confirming that the observed effects are

peptide-medi-ated (Fig 6B) but suggesting that the binding sites for

the peptides are not fully saturated at lipid⁄ peptide

ratios of greater than 20 : 1

Discussion

We previously reported that a highly cationic sequence

within human apoE has broad anti-infective properties

when presented as a tandem repeat peptide [12] Here,

we have shown that a large family of similarly active

peptides can be obtained by systematic modification of

this highly cationic human sequence The activity of

apoEdp-AMPs was greatest for tandem repeat

octa-decamer sequences and, as expected, was abolished by

replacing cationic residues We found that substitution

of some or all of the Leu residues with the aromatic residue Trp increased the potency of activity for most species, although unsubstituted apoEdp itself was most active against the Gram negative bacterium P aerugi-nosa Additionally, Phe substitutions increased antibac-terial activity against S aureus, and Cys substitutions maintained anti-HSV1 activity Other substitutions for Leu reduce or abolished antiviral or antibacterial activ-ity In general, anti-infective activity was associated with peptides where bulkier residues separated the cationic amino acids, although exceptions occurred (e.g apoEdpL-Y had little antibacterial activity) Furthermore, our data show that the increased inhibitory action of apoEdpL-W and apoEdpL-F against certain bacteria and viruses, including HIV, and the stronger association of apoEdpL-W peptide with mammalian cell membranes in the Plasmodium assays, may be caused by stronger membrane interactions of these aromatic substituted peptides This may directly

Fig 5 Inhibition of Plasmodium invasion by apoE-AMPs (A) Plasmodium berghei infection of Hepa 1-6 cells after incubation of sporozoites and cells with various apoE-AMPs at 50 lgÆmL)1 Values shown are expressed relative to untreated control wells; bars indicate standard errors (B) Confirmation that growth of Hepa 1-6 cells was not inhibited by apoE-AMPs Hepa 1-6 cells were grown in the presence of up to

25 l M (> 60 lgÆmL)1) apoEdp or apoEdpL-W for 2 days, before assessment of cell viability by MTT reduction Values shown are expressed relative to untreated control wells; bars indicate standard errors (C) Effect of pretreatment of Hepa 1-6 cells with apoEdp and apoEdpL-W at

50 lgÆmL)1on entry of P berghei, after washing peptide-treated cells prior to introduction of Plasmodium Values shown are expressed rela-tive to untreated control wells (D) Effect of apoEdp and apoEdpL-W treatment (50 lgÆmL)1) on P berghei membrane integrity, assessed by propidium iodide exclusion relative to organisms treated with heat (positive control) or untreated (negative control) Values show proportion

of organisms taking up stain; bars indicate standard errors.

damage bacterial membranes, or anchor peptides to

mammalian membranes, allowing later influence on

HIV or Plasmodium invasion of cells

The activity of apoE-AMPs appears to be broader

and more potent than that of comparable short

(pent-amer to undec(pent-amer) Trp⁄ Arg ⁄ Tyr ⁄ Ala peptides

previ-ously described [30], which showed only modest

antibacterial activity in our assays, and no antiviral

activity (Table 2) We found that activities were lower

than those for apoE-AMPs; however, antiviral activity

was found in tandem repeat peptides constructed from

the latter (paralleling the increase in activity obtained

by presenting apoE141–149 as a tandem repeat) These

data suggest that only cationic peptides of a certain

length have potent antiviral activity Nonetheless, unlike apoE141–149, constructing tandem repeats of the short Trp⁄ Arg ⁄ Tyr ⁄ Ala peptides did not increase antibacterial activity; indeed, in the case of Hepta1dp, activity was abolished

The antibacterial effects of the most potent apoE-AMPs compares favourably with published activities for commercially available peptidic antimicrobials For example, the minimum inhibitory concentration (MIC)

of nisin against Pseudomonas aeruginosa is reported to

be 32 lgÆmL)1 [31], whereas the same value for apoEdp is 7.3 lgÆmL)1 or 3 lm (Fig 1B) Similarly magainin II and cecropin P1, both commercially avail-able antimicrobial peptides, have reported MICs

A

B

Fig 6 Wideline NMR spectra (at 303 K) and corresponding quadrupole splitting values for multilamellar vesicles composed of 2 : 1

DMPC-d 4 ⁄ DOPG, before and after introduction of apoEdp, apoEdpL-F and apoEdpL-W (A) Spectra are shown with dashed lines indicating the changes in a- and b-splittings observed for untreated lipid (bottom) and after introduction of each of the three peptides to a lipid ⁄ peptide molar ratio of 50 : 1 (middle) and 20 : 1 (top) The distance between outer pair of dotted lines represents the splittings for the choline a-deu-terons and the distance between the inner pair represents the splitting for the choline b-deua-deu-terons (B) Peptide concentration-dependent changes in the values of the measured quadrupole splittings for the choline a- and b-deuterons after addition of apoEdpL-W, apoEdpL-F and apoEdp.

against (nonresistant) S aureus strains in the range

16–128 lgÆmL)1 [32], whereas we found apoEdpL-F to

have an MIC against S aureus of 9.5 lm or

25.5 lgÆmL)1(data not shown)

Cationic AMPs may act against bacteria by

disrupt-ing the negatively charged inner membrane, to which

such peptides are electrostatically attracted [33] At

least one function of the positive charge associated

with such peptides is to promote selective adherence of

the peptide to the bacterial membrane The ‘carpet’

model is one of several proposed to explain cell death,

in which peptides accumulate on the membrane

sur-face and cover (carpet) the bilayer, ultimately

destroy-ing the membrane by a detergent-like action [34]

Alternately monomers may form either ‘barrel-stave’

[35] or ‘toroidal’ [36] pores in the membrane, with loss

of cellular contents or membrane potentially killing

the bacterial cell A further model proposes the

forma-tion of pores formed by peptide aggregates, allowing

entry of further peptides into the cell and interference

with either protein synthesis or DNA replication [37]

It is unlikely that the octadecamer apoEdp-AMPs

would form barrel stave pores because the membrane

spanning barrel stave mechanism is linked to peptides

greater than 22 amino acids in length [36], and

because they are not amphipathic [12] However, both

the ‘carpet’ and ‘aggregate pore’ mechanisms do

appear possible

One structural motif previously considered to

medi-ate binding to bacterial lipopolysaccharide (LPS) is

two positive amino acid residues separated from a

third by several hydrophobic residues Such motifs are

found in a number of antimicrobial peptides, including

bovine lactoferrin (RRWQWR) [38], polyphemusin 1

(RRWCFR) and tachylpepsin (KWCFR) [39] and the

synthetic hexapeptide RRWWCR [40] ApoEdp and

apoEdpL-W contain similar motifs (‘KRLLLR’ and

‘KRWWWR’, respectively), which might therefore be

suitable to mediate such interactions with LPS

How-ever our finding that the pentadecamer and dodecamer

apoEdp-related peptides (which also contain the

‘KRLLLR’ motif) showed relatively little antibacterial

activity suggests this is not the case Nonetheless, it

would be interesting to examine whether apoE-AMPs

might inactivate immunostimulatory LPS released

from bacterial cells

The anti-HSV1 activity of apoEdp involves inhibition

of virus particle attachment to cells, with this likely

being related to the derivation of this peptide from the

apoE HSPG⁄ LDLR binding region [12] Inhibition of

viral (or Plasmodium) attachment to cells may relate to

the blockade of either HSPGs on the eukaryotic cell

sur-face or blockade of LDLR family receptors An

alterna-tive would be direct lysis of the virus or parasite, which appears to occur with apoEdp itself and P berghei, thereby indirectly preventing their attachment to cells Our finding that VSV-pseudotyped HIV was not inhibited by apoEdpL-W suggests that the effect of the latter does at least in part involve disruption specific to the HIV viral membrane This is consistent with our finding that apoEdpL-W had a far greater propensity to perturb model membrane systems than apoEdp Such membrane interactions did not appear to directly medi-ate the activity of apoEdpL-W against Plasmodium, which appeared to remain viable after exposure to apoEdpL-W In conclusion, the mechanism of action

of apoEdpL-W against HIV may involve a selective biophysical detergent-like action or attachment inhibi-tion, whereas its activity against Plasmodium sporozoites involves inhibition of parasite invasion of cells, medi-ated through attachment of the peptide to mammalian cells These findings are consistent with those of a recent study which demonstrated that the membrane effects of Leu- and Trp-containing cationic peptides varies consi-derably, depending on the distribution of cationic residues (and resulting degree of amphipathicity), the hydrophobic residues and the nature of the membrane interacting with the peptide [41]

Only a subset of AMPs have antiviral activity For example, indolicidin, which superficially resembles apoEdpL-W, is relatively inactive against HIV, with a reported IC50 concentration of 35–50 lm [42] A recent study surveyed potential anti-HIV activity in a range of amphibian-derived AMPs, and found only three peptides with suitable activities [15] ApoE-AMPs are active against HIV in the single digit lM concentration range, unlike the low concentration (nm) activity range of many small molecule anti-HIV lead compounds However, unlike many such com-pounds, apoE-AMPs also appear nontoxic in the lM range ApoE-AMPs offer a means to interfere with a very early stage in the attachment of HIV (and herpes-viruses) to cells, and with peptides based on a human cationic sequence The HIV strains inhibited included those using both CCR5 and CXCR4 coreceptors (2028), those using CXCR4 alone (2044 and IIIB) or those using CCR5 alone (SF162 and BaL) With the exception of IIIB, all can infect both macrophages as well as CD4+ T cells [43] These data support a tar-get upstream of viral fusion events (or upstream of those fusion events involving HIV coreceptors) In addition, the clear inhibition of HIV when peptide was introduced to cells both before and after the virus adsorbs at 4C suggests that any action involves blockade of the reversible attachment of the virus to cells