Apolipoprotein E-derived antimicrobial peptide analogues with altered membrane affinity and increased potency and breadth of activity ´ Bridie A Kelly1, Stuart J Neil2,*, Aine McKnight2,†, Joana M Santos3,‡, Photini Sinnis3, Edward R Jack1,§, David A Middleton1,– and Curtis B Dobson1 Faculty of Life Sciences, The Mill, The University of Manchester, UK Wohl Virion Centre, Windeyer Building, University College London, UK 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 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 inhibited a range of microorganisms Here, we have dissected the protein chemistry 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 cytotoxicity and minimal haemolytic activity, and were active against HIV and Plasmodium via 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 apoEAMP 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 apoEAMPs may be useful as lead agents for preventing the early stages of HIV and Plasmodium cellular entry (Received May 2007, revised 22 June 2007, accepted July 2007) doi:10.1111/j.1742-4658.2007.05981.x 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-1,1,2,2-2H4-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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4511 Aromatic substitution of ApoE-derived AMPs B A Kelly et al 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 occurring sequences, especially from invertebrates or amphibians [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 residues 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 apolipoprotein E (apoE), influences the outcome of infection [5] The APOE-e4 allele is associated with high prevalence of cold sores and with an increased risk of Alzheimer’s disease in elderly people possessing latent herpes simplex virus type (HSV1) in the brain [6], and protection from liver damage in those infected with hepatitis C virus [7] In addition, APOE genotype influences risk of developing herpes simplex encephalitis [8], malaria in children [9] and shingles and postherpetic neuralgia in females caused by varicella zoster virus infection [10] Many infectious agents, including viruses and intracellular parasites such as Plasmodium, use heparan sulfate proteoglycans (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 apolipoproteins [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 stabilized 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 blockade 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 4512 safe and potent AMPs targeted towards serious infections The strategy of developing peptide-based therapies against HIV or other serious infections is strongly supported 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] Plasmodium spp., 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 invasion by a range of pathogenic organisms Here, we dissect those features of the apoEdp peptide 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 substitutions 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 substituted 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 activity using a series of dodecamer and pentadecamer peptides derived from the octadecamer apoEdp sequence (we previously found nonomer peptides to be inactive) (Table 1) Figure shows that the greatest antiviral and antibacterial action was found in the full length tandem repeat peptide apoEdp (P < 0.001) The eicosameric tandem repeat peptide of apoE141-150 (i.e including the additional Arg residue found in position FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al Aromatic substitution of ApoE-derived AMPs Table Amino acid sequences of apoE-derived peptides Sequences 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) Peptide A Amino acid sequence ApoEdp L Truncated peptides ApoEdp N-3 ApoEdp N-6 ApoEdp C-3 L ApoEdp C-6 L ApoE141–149 L Elongated peptides ApoE141–150 dp L Substituted peptides ApoEdpL-E E ApoEdpL-A A ApoEdpL-D D ApoEdpL-W W ApoEdpL-M M ApoEdpL-Y Y ApoEdpL-F F ApoEdpL-I I ApoEdpL-Q Q ApoEdpL-N N ApoEdpL-C C ApoEdpL-S S ApoEdpL-V V ApoEdpL-T T ApoEdpL-G G ApoEdpL-H H ApoEdpL-P P RKL RKRL L L RKL RKRL L L RKR R RKL RKR RKL RKR RKL RKR L L L L L L L L L L L L L L R R R R KL RKRL L KL RKRL L KL RK K B RKL RKRL L R L RK L RKR L LR RKE RKA RKD RKW RKM RKY RKF RKI RKQ RKN RKC RKS RKV RKT RKG RKH RKP RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR RKR E E E R A A A R D D D R WWWR MMMR Y Y Y R F F F R I I I R Q Q Q R N N N R C C C R S S S R V V V R T T T R G G G R H H H R P P P R K K K K K K K K K K K K K K K K K E RK A RK D RK WRK MRK Y RK F RK I RK Q RK N RK C RK S RK V RK T RK G RK H RK P RK RE E RA A RD D RWW RMM RY Y RF F RI I RQ Q RN N RC C RS S RV V RT T RG G RH H RP P 150 of apoE) had almost identical antiviral and antibacterial activity to apoEdp ApoEdp therefore appears to provide the highest activity relative to peptide length amongst the peptides 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 individual residues to its activity can thus be studied by generation of a relatively small number of variant peptides (unlike many host-derived AMPs) We initially tested whether the cationic residues were critical for its activity 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 antiviral 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-helical AMPs We therefore examined whether the eight Fig Anti-infective activity of peptides constructed from truncations or elongations of the apoEdp seqeunce (A) Infectivity of HSV1 as shown by plaque reduction assay in Vero cells, after treatment 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 standard errors bulky Leu residues separating the peptide’s basic residues 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 residues, 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 Cyssubstitution and especially after Trp-substitution (Fig 2A,B) Very similar data were found for herpes simplex virus type (HSV2) (data not shown) We also examined antibacterial activity in this series of substituted peptides, and found this to be less prevalent Peptides in which the Leu of apoEdp had been substituted by Ile, Trp and Phe also inhibited Plasmodium aeruginosa, but none were as potent as apoEdp itself against this resistant bacterium Interestingly FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4513 Aromatic substitution of ApoE-derived AMPs B A Kelly et al A B C Fig Anti-infective activity of Leu-substituted apoEdp-derived peptides, whereby all eight Leu residues were substituted for another residue (A) Relative antiviral and antibacterial activity of Leu-substituted forms of apoEdp (unsubstituted apoEdp is indicated by the letter ‘L’) Values shown are average IC50 concentrations (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 volume rather than moles per volume, 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 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 peptides again proving inactive along with the other 12 substituted peptides (Fig 2A) Tyr and Ile substituted 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 (previously 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 concentration ¼ 125 lgặmL)1 (95% condence interval ẳ 48160 lgặmL)1) compared with the tandem repeat of this sequence (apoEdpL-W), which had an IC50 concentration of 9.3 lgỈmL)1 (95% confidence interval, 8.5–10.5 lgỈmL)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 4514 shown), suggesting that such structure was not required for their activity, unlike apoEdp [12] Although the biological effects of apoEdp on mammalian cell cultures have been previously reported, these occur only under unusual physiological conditions; 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 apparent 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 days of incubation at 40 lm (Fig 3A) We previously 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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al A Aromatic substitution of ApoE-derived AMPs B C Fig Lack of toxicity of apoE-AMPs against mammalian cells (A) MTT reduction in Vero cells treated for extended periods with apoEAMPs Values shown are expressed relative to untreated control wells; bars indicate standard errors (B) Haemolytic assessment of apoEAMPs ApoEdp and other apoE-AMPs were introduced to red blood cells at various concentrations, Values shown indicate A540 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]3 and RLLR5 [24,25] Values shown indicate A540 relative to that for red blood cells completely lysed in 0.45% ammonium hydroxide; bars indicate standard errors the previously described lytic antimicrobial peptides RLLR5 and N-RLLR3 when these were used at lm and 31 lm, respectively In conclusion, we found no significant cytotoxic or haemolytic action of these peptides 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 peptide was added with viral inoculum, and then washed from the system, suggesting that an early stage in replication was targeted Interestingly, apoEdp itself did not show activity in this assay system, possibly reflecting 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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4515 Aromatic substitution of ApoE-derived AMPs B A Kelly et al Table 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) IC50 concentration (lM) Peptide Octa 1a MU89 (Octa dp) Hepta 1a MU92 (Hepta dp) Deca 1a MU94 (Deca dp) N-[RLLR]3b RLLR5b MU103 MU104 ApoEdpL-W R R R R Y Y A R R R W Pseudomonas Staphylococcus HSV1 aeruginosa aureus Amino acid sequence a R R W W R R P L R R R W W W W W W K L R R K Y Y R R W W A R R R W R R W W R R M R R R R W W W W W W R L R R K Sequences described in Strom et al [30] b W W R R A A L L R R R R R R R W Y R R R L R W R W W R R R R W R W W W W R L W R W R W W R Y L L R R R R L R R R K W R R R R W > 20 8.3 > 20 3.3 > 20 W A R R W L R 7.3 R R L L R < 3.3 R R R > 20 W W W 9.25 R W W 3.1 W W R W R L R R R R L L R R K > > > > > > 21 30 28.5 53 53 53 53 28 53 53 42 20 14 > 53 > 53 > 53 16 26 Sequences described in Park et al [24,25] 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 °C, 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 significantly diminished relative to that found in earlier experiments (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 leading 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 envelopemediated events although without compromising membrane integrity The Plasmodium sporozoite (i.e the infective stage of the malaria parasite) enters hepatocytes via attachment 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 apo4516 R 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 apoEdpL-F at this level was not significant We also confirmed 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 days (i.e 48 times the length of incubation in the Plasmodium inhibition assays) (Fig 5B) In further assays in which nonbound peptide was washed away prior to introducing the sporozoites, apoEdpL-W (but not apoEdp) retained its ability to block P berghei entry (Fig 5C) These data suggest apoEdpL-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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al Aromatic substitution of ApoE-derived AMPs A B C D Fig 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 infection (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 °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 °C, either in the presence or absence of apoEdpL-W at 10 lM (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 detect interactions of the apoEdp, apoEdpL-W and apoEdpL-F derived peptides with the surface of multilamellar vesicles composed of 1,2-dimyristoylphosphatidyl-1,1,2,2-2H4-choline (DMPC-d4) and dioleoylphosphatidylglycerol (DOPG) mixed in a : molar ratio The NMR spectrum arises from the reporter molecule, choline methylene-deuterated 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-deuterons The anionic DOPG headgroups provide an overall 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 : or 20 : 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 peptide interacting with lipid head groups at increasing concentration ApoEdp appears to interact relatively weakly with the mixed lipid bilayers of the multilamellar 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 spectrum 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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4517 Aromatic substitution of ApoE-derived AMPs B A Kelly et al A B C D Fig 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 lgỈmL)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 lM (> 60 lgỈmL)1) apoEdp or apoEdpL-W for 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 lgỈmL)1 on entry of P berghei, after washing peptide-treated cells prior to introduction of Plasmodium Values shown are expressed relative to untreated control wells (D) Effect of apoEdp and apoEdpL-W treatment (50 lgỈmL)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 the membrane surface and participating in a weak electrostatic interaction In all three cases, the degree of interaction scales with increasing peptide concentration confirming that the observed effects are peptide-mediated (Fig 6B) but suggesting that the binding sites for the peptides are not fully saturated at lipid ⁄ peptide ratios of greater than 20 : 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 octadecamer sequences and, as expected, was abolished by replacing cationic residues We found that substitution 4518 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 aeruginosa Additionally, Phe substitutions increased antibacterial activity against S aureus, and Cys substitutions maintained anti-HSV1 activity Other substitutions for Leu reduce or abolished antiviral or antibacterial activity 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 FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al Aromatic substitution of ApoE-derived AMPs A B Fig Wideline NMR spectra (at 303 °K) and corresponding quadrupole splitting values for multilamellar vesicles composed of : DMPCd4 ⁄ 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 : (middle) and 20 : (top) The distance between outer pair of dotted lines represents the splittings for the choline a-deuterons and the distance between the inner pair represents the splitting for the choline b-deuterons (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 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 (pentamer to undecamer) Trp ⁄ Arg ⁄ Tyr ⁄ Ala peptides previously 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 apoEAMPs 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 lgỈmL)1 [31], whereas the same value for apoEdp is 7.3 lgỈmL)1 or lm (Fig 1B) Similarly magainin II and cecropin P1, both commercially available antimicrobial peptides, have reported MICs FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4519 Aromatic substitution of ApoE-derived AMPs B A Kelly et al against (nonresistant) S aureus strains in the range 16–128 lgỈmL)1 [32], whereas we found apoEdpL-F to have an MIC against S aureus of 9.5 lm or 25.5 lgỈmL)1 (data not shown) Cationic AMPs may act against bacteria by disrupting 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 surface and cover (carpet) the bilayer, ultimately destroying 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 formation 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 appear possible One structural motif previously considered to mediate 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 (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 However 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 surface or blockade of LDLR family receptors An alterna4520 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 mediate 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 inhibition, whereas its activity against Plasmodium sporozoites involves inhibition of parasite invasion of cells, mediated 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 considerably, 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] ApoEAMPs 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 compounds, 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 herpesviruses) 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 target 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 °C suggests that any action involves blockade of the reversible attachment of the virus to cells FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al ApoEdpL-W has much greater anti-HIV potency than apoEdp, suggesting apoE-AMPs may be developed as a new approach for HIV-therapy The early anti-HIV target of apoEdpL-W suggests that it may also be used as a microbicide, especially because it also inhibits HSV2, an important cofactor for the transmission and acquisition of HIV infection [44] Additionally, compounds preventing Plasmodium sporozoite entry into the liver would represent an attractive new means to prevent infection by this parasite ApoE-AMPs have unusually broad anti-infective activity, relative to wellknown AMPs such as cecropins, clavanins and LL-37, which show little antiviral activity [45], and are highly active, with IC50 concentrations against many organisms (including HIV and P aeruginosa) in the 1–4 lm region Although there is precedent for safe clinical use of peptides based on nonhuman sequences (e.g the 36 amino acid peptide T20), such peptides run the risk of an immunogenic response The human origin and relatively simple nature of the apoEdp sequence should expedite the rational design of apoE-AMPs for potential clinical use Experimental procedures Aromatic substitution of ApoE-derived AMPs 293T cells with p8.91 (HIV-1 Gag-Pol), pCSGW (Lentivector genome encoding enhanced green fluorescent protein under spleen focus-forming virus promoter control) and pMD-G (CMV-VSV-G) [48] Infectivity of stocks was measured by enumerating green fluorescence protein-positive cells using fluorescence activated cell sorting (Becton Dickinson), 48 h after transduction Bacterial stocks were grown by inoculating Luria–Bertani broth with either P aeruginosa (ATCC strain 9027) or S aureus (ATCC strain 6538P) obtained in Cultiloop format (Oxoid, Basingstoke, UK) [12] Plasmodium berghei stocks were prepared by feeding 3–5 day-old Anopheles stephensi mosquitoes on anesthetized P berghei (NK65)infected Swiss Webster mice, which had been checked by blood smear for the abundance of gametocyte-stage parasites Salivary gland sporozoites were harvested on days 18–21 postinfective blood meal The mosquitoes were rinsed in 70% ethanol and washed in DMEM before salivary gland dissection The glands were gently ground, centrifuged (80 g for using an Eppendorf microfuge, model 5417C, fixed angle rotor S45-30-11) to remove mosquito debris, and sporozoites counted in a hemocytometer Ethics approval for the use of experimental animals was provided by NYU Animal Ethics Committee (IACUC ref, Animal Study Protocol 050310-03) Cell cultures Peptides Vero cells were maintained in Eagle’s minimum essential medium supplemented with 10% (v ⁄ v) fetal bovine serum, mm l-glutamine, penicillin (100 ImL)1) and streptomycin (100 lgỈmL)1), hereafter referred to as growth medium Hepa 1-6 cells (ATCC CRL-1830, American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium containing 10% (v ⁄ v) fetal bovine serum, hereafter referred to as growth medium (DMEM) NP2 ⁄ CD4 ⁄ CXCR4 or NP2 ⁄ CD4 ⁄ CCR5 cells were maintained in growth medium (DMEM) [46] Growth medium containing only 2% or 0.5% fetal bovine serum is referred to as ‘2% medium’ or ‘0.5% medium’ Peptides were obtained commercially (Alta Bioscience, Birmingham, UK) having been synthesized using 9-fluorenylmethyl carbamate chemistry and purified by HPLC as described previously [12] For peptides apoEdp and apoEdpL-W, peptide weight was confirmed by amino acid analysis, and far-UV CD spectra obtained for peptides solubilized in either distilled water or 50% trifluoroethanol Measurements were carried out at 20 °C, using a Jasco J810 spectropolarimeter (Jasco Inc., Easton, MD, USA) Peptide stocks were solubilized in NaCl ⁄ Pi or growth medium at 400 lm, aliquoted and stored at )80 °C Herpes virus plaque reduction assays Microorganisms HSV1 stocks (strain SC16 provided by Professor Roy Jennings, Sheffield University, UK) and HSV2 stocks (clinical isolate provided by Professor Anthony Hart, Liverpool University, UK) were prepared in Vero and HEp2a cells, respectively [12] HIV-1 stocks were prepared in human peripheral blood mononuclear cells stimulated with phytohemagglutinin and interleukin-2 except strain IIIB which was prepared in H9 T cells Titres determined on NP2 ⁄ CD4 cells bearing the appropriate coreceptor by counting p24+ foci by immunostaining [47] The VSV-G pseudotype of HIV-1 based vectors were made by transient transfection of Confluent Vero or HEp2a cells in 24-well plates were inoculated with 90 plaque forming units per well HSV1 or HSV2 in 0.5% medium, containing various concentrations of peptide This was removed after h, and 1% medium containing 0.2% high viscosity carboxymethylcellulose was added After days of incubation, cells were fixed in formal saline, stained with carbol fuchsin and plaques were enumerated Concentrations of peptide that inhibited infection by 50% were calculated (IC50 concentrations) Because peptide was only present during inoculation, the assay measured inhibition of only the very early steps of infection; we have previously confirmed this using acyclovir and heparin controls [12] FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS 4521 Aromatic substitution of ApoE-derived AMPs B A Kelly et al HIV inhibition assays Dilutions of peptide were added to NP2 ⁄ CD4 ⁄ CXCR4 or NP2 ⁄ CD4 ⁄ CCR5 cells which had been plated at · 104 cells per well the previous day Peptides were allowed to associate with the cells for 30 before addition of 100 focus forming units (FFU) of the test virus (with additional peptide to maintain peptide concentration) Cells were washed h post infection and cultured for 72 h After fixation in cold acetone methanol (1 : 1), cells were immunostained for HIV p24 with secondary b-galactosidase conjugate (stained with 5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside), and the recovered titre enumerated For experiments in which the activity of peptides was examined both before and after viral attachment to cells, 100 FFU of virus was bound to NP2 ⁄ CD4 cells expressing CXCR4 or CCR5 at °C for h in the presence or absence of peptide Cells were washed and the medium replaced (with or without peptide) Cells were incubated at 37 °C for a further h and then processed as above Plasmodium assays Sporozoite development assay and viewed under a fluorescent microscope Controls included sporozoites that were heat-killed (and so the majority should have taken up the propidium iodide) and sporozoites incubated in medium without the peptide Antibacterial assays A microdilution method was used [12]: paired dilutions of compounds in Luria–Bertani broth, arranged in 96-well plates were inoculated with around · 105 colony forming units of P aeruginosa (ATCC 9027) or S aureus (ATCC 6538P) After overnight incubation at 37 °C, absorbance at 620 nm (A620) was assessed, and the IC50 concentrations were determined Haemolytic assays Fresh washed human red blood cells were added to peptides diluted in NaCl ⁄ Pi in 96-well plates (20 · 106 red blood cells were added per well) After h of incubation at 37 °C, plates were centrifuged (3000 g for using a Sorvall RT6000B centrifuge and H1000B rotor) and 80 lL of supernatant transferred to further 96-well plates containing 0.75% ammonium hydroxide in distilled water After assessment of absorbance (A540), the concentrations of peptide resulting in 5% or 50% haemolysis (referred to as EC5 and EC50 concentrations) were calculated (100% haemolysis was considered to be the average A540 for red blood cells treated directly with 0.45% ammonium hydroxide) Hepa 1-6 cells, a mouse hepatoma cell line permissive for P berghei sporozoite development, were seeded (8 · 104 cells ⁄ well) in Laboratory-Tek permanox chamber slides (Nalgene Nunc Corp., Naperville, IL, USA) and grown until confluent On the day of the experiment, sporozoites were dissected from mosquitoes and preincubated in DMEM with 1% bovine serum albumin alone or with the indicated peptide for 45 at 28 °C and plated on cells in the continued presence of the peptide in growth medium After h at 37 °C, the medium with unattached sporozoites and peptide was removed and replaced with complete medium Twenty four hours later, the cells were fixed with cold methanol and developing exoerythrocytic stages were stained with mAb 2E6 [49] followed by anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate The number of exoerythrocytic stages in each well was counted with a · 40 objective on a Nikon fluorescent microscope (Nikon Corp., Tokyo, Japan) In other experiments, Hepa 1-6 cells were pretreated with the peptides in complete medium for h, washed and then sporozoites were added in medium without peptide and the assay was continued as outlined above Vero cells growing in 96-well plates were treated with various peptide concentrations After 48 h of incubation, 25 lL MTT in 0.5% medium was added (1 mgỈmL)1 final concentration), and cells were incubated for h before removal of growth medium and solubilization of formazan crystals in 100 lL of dimethyl sulfoxide, prior to reading absorbance (A570) Sporozoite toxicity assay Solid-state NMR To check for toxicity of the inhibitors on sporozoites, we used propidium iodide Plasmodium berghei sporozoites were incubated with the peptides for 45 at 28 °C, propidium iodide was then added to a final concentration of lgỈmL)1 for 10 at 28 °C, sporozoites were washed The lipids DMPC-d4 and DOPG in chloroform were mixed in a : molar ratio, respectively, with a total lipid content of 10 mg (Avanti Polar Lipids, Inc., Alabaster, AL, USA) The sample was then dried under argon and then under 4522 Data analysis For anti-infective assays, activity was expressed as percentage reduction relative to control Standard error was calculated using a special case of Fieller’s theorem, and significance assessed using analysis of variance Cytotoxicity assessment FEBS Journal 274 (2007) 4511–4525 ª 2007 The Authors Journal compilation ª 2007 FEBS B A Kelly et al reduced pressure overnight The thin film of lipids was then resuspended in 50 lL of deuterium depleted phosphate buffer (20 mm, pH 7.4) with mm EDTA and transferred to a mm diameter zirconia magic angle spinning NMR rotor In some cases, the total lipid was reduced to attain the maximum lipid : peptide molar ratio of 20 : (maintaining the lipid concentration by decreasing the volume of resuspension buffer) The lipid mixture was then vigorously mixed to promote the formation of vesicles, followed by five cycles of freeze thawing on dry ice to integrate the peptide into the multilamellar vesicles that form A Bruker Avance 400 NMR spectrometer (Bruker, Ettlingen, Germany) equipped with a magic angle spinning triple resonance probe and cooling unit was used to make deuterium NMR measurements (at 61 MHz) on nonspinning samples The NMR experiments were carried out at 303 °K using a single deuterium p ⁄ pulse of 4.5 ls and a s recycle time Typically, °K transients were accumulated for every 10 mg lipid sample, increasing to 80 °K for the smallest lipid sample (1 mg) to maintain a satisfactory signal Acknowledgements We thank Ruth Itzhaki, Matthew Wozniak, and Keith Crutcher for review of the manuscript, and Andrew Doig for discussion of peptide CD and structural data C.B.D and B.A.K were supported by grants from UMIP Ltd, and the Manchester Technology Fund ´ A.McK and S.J.N were funded by a grant awarded ´ to Professors Robin Weiss and Aine McKnight by the Medical Research Council (UK) P.S was supported by NIH R01 AI056840 References Jenssen H, Hamill P & Hancock RE (2006) Peptide antimicrobial agents Clin Microbiol Rev 19, 491–511 Farnaud S, Spiller C, Moriarty LC, Patel A, Gant V, Odell EW & Evans RW (2004) Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity FEMS Microbiol Lett 233, 193–199 Wu Z, Li X, de Leeuw E, Ericksen B & Lu W (2005) Why is the Arg5-Glu13 salt bridge conserved 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2007 FEBS 4525 ... substitution of ApoE-derived AMPs Table Amino acid sequences of apoE-derived peptides Sequences of peptides derived from the apoEdp sequence are shown, including peptides both of altered length and in... contribution of individual residues, and show that substitutions for aromatic residues increase potency and breadth of activity, giving rise to further apoE-derived antimicrobial peptides (apoE-AMPs)... those features of the apoEdp peptide 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