The Design, Synthesis and Bioassay of Novel Modified Peptides as Inhibitors of the Apical

23 Pages·2009·0.91 MB·English

by  Andrew Hughes

JBC Papers in Press. Published on January 21, 2009 as Manuscript M808762200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M808762200
Anti-malarial N-methylated peptides 1
RAPID OPTIMIZATION OF A PEPTIDE INHIBITOR OF MALARIA PARASITE
INVASION BY COMPREHENSIVE N-METHYL SCANNING.
Karen S. Harris1, Joanne L. Casey1,2, Andrew M. Coley1, John A. Karas3, Jennifer K. Sabo4, Yen
Yee Tan4, Olan Dolezal5, Raymond S. Norton4, Andrew B. Hughes6 Denis Scanlon3, and Michael
,
Foley1,2
From the Department of Biochemistry, La Trobe University, VIC., 3086, Australia1, the company AdAlta
Pty. Ltd., 15/2 Park Drive, Bundoora, VIC., 3083, Australia2, BIO21 Molecular Science and
Biotechnology Institute, 30 Flemington Road, The University of Melbourne, Parkville, VIC., 3010,
Australia3, the Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC.,
3050, Australia4, CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, VIC., 3052,
Australia5, and the Department of Chemistry, La Trobe University, VIC., 3086, Australia6.
Running Title: Anti-malarial N-Methylated Peptides
Address correspondence to: Michael Foley, Department of Biochemistry, La Trobe University, VIC.,
3086, Australia; E-Mail: [email protected] or Andrew B. Hughes, Department of Chemistry, La
Trobe University, VIC., 3086, Melbourne, Australia, Tel. [phone]; Fax. [phone]; E-Mail:
[email protected]

Apical membrane antigen 1 (AMA1) of According to World Health Organization D
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the malaria parasite Plasmodium falciparum has reports, almost half of the world human population w
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been implicated in the invasion of host is at risk of contracting malaria and approximately loa
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erythrocytes and is an important vaccine one million people are killed by this disease each d
candidate. We have previously described a 20 year, most of which are young children. No fro
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residue peptide, R1, that binds to AMA1 and reliable vaccine currently exists against http
subsequently blocks parasite invasion. Since Plasmodium falciparum, the parasite that causes ://w
this peptide appears to target a site critical for the most severe form of malaria, and parasites are w
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AMA1 function, it represents an important lead becoming increasingly resistant to previously .jb
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compound for anti-malarial drug development. effective pharmaceuticals (1). Thus, novel .org
However, the effectiveness of this peptide strategies to combat this disease are urgently b/
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inhibitor was limited to a subset of parasite required. gu
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isolates, indicating a requirement for broader Proteins involved in the erythrocytic stage t o
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strain-specificity. Furthermore, a barrier to the of the parasite life cycle represent attractive targets A
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utility of any peptide as a potential therapeutic for anti-malarial therapeutics since this stage is ril 2
is its susceptibility to rapid proteolytic responsible for the clinical symptoms of malaria. , 2
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degradation. In this study, we sought to Apical membrane antigen 1 (AMA1) is an integral 19
improve the proteolytic stability and AMA1 membrane protein common to all Plasmodium
binding properties of the R1 peptide by species that is thought to be important for parasite
systematic methylation of backbone amides (N- invasion of host red blood cells and hepatocytes
methylation). The inclusion of a single N-methyl (2,3). The molecular mechanisms of the role of
group in the R1 peptide backbone dramatically AMA1 in parasite invasion are unknown, but
increased AMA1 affinity, bioactivity and recent evidence suggests that it is involved in
proteolytic stability without introducing global stabilizing the binding of the invasive form of the
structural alterations. In addition, N- parasite to the erythrocyte (3,4). In animal models,
methylation of multiple R1 residues further immunization with recombinant AMA1 induces
improved these properties. Therefore, we have protection from subsequent parasite challenge (5,6)
shown that modifications to a biologically active and it appears to be an essential protein in the
peptide can dramatically enhance activity. This parasite life-cycle as AMA1 genetic knock-outs
approach could be applied to many lead are not viable (3,7). AMA1 has therefore received
peptides or peptide therapeutics to much attention as a potential vaccine candidate or
simultaneously optimize a number of drug target. Although lacking the major structural
parameters. diversity of some other P. falciparum antigens,
AMA1 exhibits allelic variation that is a result of
Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
Anti-malarial N-methylated peptides 2
point mutations. Since immunization with AMA1 amino acid containing peptides with a range of
induces inhibitory antibodies that are less effective bioactivities, including antibiotic (18), anti-viral
against heterologous parasite lines (8-10), (19), anti-cancer (20) and immunosuppressant
overcoming this diversity is a key hurdle in the activities (21). Importantly, modification of
development of AMA1-based anti-malarials. An biologically active peptides by the inclusion of N-
immune response or drug would therefore ideally methyl amino acids into an original sequence has
target a conserved region that is required for been shown to enhance the potency (22), change
function. the receptor subtype selectivity (23), and stabilize
In addition to polyclonal and monoclonal a peptide toward proteolytic degradation in a
antibodies that bind to AMA1 and block parasite biological system (24). This approach therefore
invasion in vitro (9,11,12), peptides that offers the potential to optimize multiple parameters
specifically recognize this antigen and demonstrate simultaneously.
similar inhibitory activity have been isolated from In this study, we systematically N-
random peptide libraries displayed on the surface methylate residues within the previously identified
of phage (13,14). The most potent peptide inhibitor AMA1 binding peptide, R1, and describe the
of invasion was the recently identified 20-residue synthesis and altered properties of these mono-, di-
peptide, R1 (13). Peptides that recognize and tri-N-methylated R1 derivatives. To introduce
functional sites and act as biological agonists or an N-methyl group into residues for which an N-
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antagonists can be useful for the design of small methyl analogue is not commercially available, we w
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molecule mimics, or as therapeutics per se. The R1 used on-resin N-methylation (25), facilitating a lo
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peptide is an important lead compound for drug comprehensive N-methyl scan of the R1 peptide. ed
development since its ability to block parasite Analysis of each of these R1 derivatives revealed fro
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growth indicates that it targets a site critical for that inclusion of a single N-methyl group was h
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AMA1 function. However, of the four parasite sufficient to dramatically improve AMA1 affinity, ://w
lines tested, the R1 peptide was only effective bioactivity and peptide stability without altering w
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against the 3D7 and D10 strains, and not HB3 or the global peptide structure. Moreover, the .jb
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W2mef, limiting its utility as a therapeutic agent in introduction of multiple N-methyl groups further .o
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its current form. A further barrier to the general increased AMA1 affinity and slightly improved b/
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use of peptides as pharmaceuticals is that they are recognition of AMA1 derived from different g
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rapidly degraded by peptidases and proteases in parasite lines. This represents a substantial step st o
biological fluids, leading to poor bioavailability forward in the design of an effective anti-malarial n A
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(p1e5p)t.i dIet twoa ism tphreorvefeo irtes pohf airnmtearceestu ttioc aml poodtiefyn ttihale aRn1d cmoamlaproiauln dp araabsliete tgor obwinthd. Ftou rtAheMrmAo1r e,a ntdh isi nwhoibrikt ril 2, 2
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AMA1 binding properties. may contribute to the development of a rational 1
9
The methylation of backbone amides (N- strategy for modulating and optimizing the activity
methylation) is one strategy of peptide of bioactive peptides.
modification that has been employed to confer
superior characteristics on a lead peptide. This EXPERIMENTAL PROCEDURES
involves the replacement of residues within a Fmoc solid-phase peptide synthesis
peptide sequence with the corresponding N-methyl Materials - Chlorotrityl resin, DIPEA, piperidine,
amino acid, subtly changing the peptide backbone. hydroxybenzotriazole (HOBt), HBTU were all
The introduction of a methyl group into a peptide obtained from GLBiochem, Shanghai, China.
has the advantage of substantially improving a DMF, DCM, diethyl ether and 2-mercaptoethanol
number of pharmacokinetically useful parameters, were obtained from Ajax Chemicals, Melbourne
including membrane permeability, conformational Australia. MTBD, DBU, Methyl triflate, o-
flexibility and proteolytic stability (16). nitrobenzenesulfonyl chloride, chloranil and TNBS
Additionally, it results in a loss of hydrogen were obtained from Sigma-Aldrich, Sydney,
bonding potential at the affected site, reducing the Australia.
role of main chain hydrogen bonds at a binding The N-methyl amino acids Fmoc-NMe-
interface and potentially altering binding properties Val-OH, Fmoc-NMe-Phe-OH, Fmoc-NMe-Ala-
(17). There are a number of examples of N-methyl OH, Fmoc-NMe-Leu-OH, Fmoc-NMe-Ile-OH,
Anti-malarial N-methylated peptides 3
Fmoc-NMe-Met-OH and Fmoc-Ser-OH were 2 h. The N-terminus was then methylated by
obtained from Peptide Solutions, Melbourne, treatment with a solution of methyl triflate (0.75
Australia. Fmoc-NMe-Arg(Mtr)-OH was obtained mmol, 3 equivalents) and the base MTBD (7-
from ChemImpex, USA. Fmoc-Lys(Boc)-OH, methyl-1,5,7-triazabicyclo-[4.4.0]-dec-5-ene, 2
Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc- equivalents, 77 mg) in DMF (4 ml) and the
Glu(OtBu)-OH, were obtained from GLBiochem, mixture was stirred for 30 min. After this period
Shanghai, China and incorporated into the the resin was drained, washed with DMF (4 × 10
sequence as N-methyl residues by the ‘on-resin’ ml) and then treated repeatedly with a solution of
method of Miller and Scanlan (25). 2-mercaptoethanol/DBU/DMF (200 μl/200 μl/4
Derivatisation of the resin - Chlorotrityl resin (1.0 ml). Usually this procedure involved 3 × 30 min
g, 1 mmol) was suspended in dry DCM (10 ml). treatments until there was no more yellow color
Fmoc-Lys(Boc)-OH (1 mmol) was dissolved in evident in the solution (due to the o-
dry DCM (10 ml) and the solution was added to nitrobenzenesulfonyl group). The resin was then
the resin. DIPEA (4 mmol, 680 μl) was added and tested for the presence of a secondary amine due to
the solution was stirred for 2 h. After this time the N-methyl group on the N-terminus. The next
methanol (1 ml) was added and the solution was amino acid was then activated and coupled in the
stirred for another 30 min. The resin was washed normal manner (HBTU). Note that the TNBS test
sequentially with DCM and ether and then air- could not be used to detect the secondary amine of D
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dried. The loading of FmocLys(Boc) onto the resin proline and the N-methyl amino acids: in this case w
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was estimated by the increase in resin weight and the chloranil test (27) was used. loa
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was typically 0.5-0.6 mmol/g Peptide cleavage from resin and purification - On ed
Solid-phase synthesis protocol - The scale for completion of the required peptide synthesis cycles from
synthesis was typically 0.5 g, 0.25 mmol. Resin the N-terminal amino group was deprotected and http
(0.5 g) was washed with DMF (4 × 10 ml) and the resin washed with DCM (5 × 10 ml) and lastly ://w
then treated with a solution of 25% ether. The resin (typically 0.8-1.0 g) was air-dried w
w
piperidine/DMF (containing 0.1 M HOBt) for 10 overnight and then treated with a solution of .jb
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min. After this time the resin was washed with TFA/DTT/TIPS/H2O (90/5/2.2/2.5, 10 ml) for 2 h. .org
DCM (4 × 10 ml), then DMF (4 × 10 ml), and then The mixture was then filtered and cold ether (40 b/
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a small sample (~5mg) was subjected to a TNBS ml) was added to the solution, which resulted in gu
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test (26) for the presence of primary amine. The the precipitation of the peptide. The peptide slurry st o
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Fmoc-amino acid (1 mmol, 4-fold excess) was was centrifuged (3500 rpm, 3 min) and the crude A
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activated by dissolution in DMF (2 ml) followed peptide was isolated by decanting the ethereal ril 2
by the addition of a solution of HBTU in DMF (2 supernatant. The peptide was then dissolved in , 2
0
ml, 0.5 M) to which was added DIPEA (340 μl, 2 30% acetonitrile/water and the solution 19
mmol). This solution was sonicated for 2 min and lyophilized.
then added to the resin. This resin coupling The lyophilized peptides were purified on
reaction mixture was agitated for 1 h and then a Gemini C18 5 μM semi-prep HPLC column
drained and washed with DMF (4 × 10 ml). A resin (Phenomenex, Dimension: 50 × 21.2 mm, Flow: 6
sample was then tested for the presence of residual ml/min). Collected fractions were analysed on an
amine using the TNBS test (26). If the test was Absorbosphere HD C18 5 μM analytical HPLC
positive then another aliquot of the Fmoc-amino column (Alltech, Dimension: 150 × 3.2 mm, Flow:
acid was activated and the coupling procedure 0.7 ml/min). Pure fractions were pooled,
repeated. lyophilized and the molecular weight was
Where an N-methyl was to be incorporated confirmed by electrospray mass spectrometry.
into the peptide sequence following the piperidine
treatment, o-nitrobenzenesulfonyl chloride (0.75 Phage display - Production of M13 bacteriophage
mmol, 3 equivalents) was dissolved in dry DCM (4 displaying the R1 peptide was carried out as
ml) and the solution was added to the resin described elsewhere (28). Briefly, K91 Escherichia
together with 2,4,6-collidine (1.25 mmol, 5 coli cells harboring the modified phage genome
equivalents). This reaction mixture was stirred for fUSE5, which included DNA encoding the R1
Anti-malarial N-methylated peptides 4
peptide, were incubated in broth culture to produce visualized using tetramethylbenzidine (TMB;
phage. Phage were harvested by precipitation with Sigma) and the absorbance was read at 450 nm.
PEG/NaCl (30% [w/v] polyethylene glycol 8000
[Sigma], 2.6 M NaCl) followed by centrifugation Immobilization of AMA1 proteins to surface
and resuspension of the phage pellet in phosphate plasmon resonance (SPR) chip - A Biacore T100
buffered saline (PBS). Phage concentration was biosensor instrument (32) was used to measure the
determined in colony forming units per milliliter kinetics of the interaction of R1 peptides and their
(CFU/ml) as previously described (28). N-methylated derivatives with AMA1 proteins
from different strains of P. falciparum. All
Invasion inhibition assay - The 3D7 parasite line immobilization and binding experiments were
was cultured essentially as described previously performed at 25oC using HBS-EP+ buffer (10 mM
(29,30) and parasites were synchronized by HEPES, 150 mM NaCl, 3.4 mM EDTA and 0.05%
sorbitol lysis of all but ring stage parasites (31). R1 surfactant P20, pH 7.4) as the running buffer.
peptide and methylated R1 analogues were assayed AMA1 was immobilized on CM5 sensor chips
for their ability to inhibit parasite growth as using standard amine-coupling chemistry. The
previously described (13). Briefly, peptides were carboxymethyl dextran surface was activated with
diluted in PBS and 50 µl was added in triplicate to a 7 min injection at 10 µl/min of a 1:1 ratio of 0.4
sterile flat-bottomed microtiter wells (Nunc). M 1-ethy-3-(3-dimethylaminopropyl) carbodiimide
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Complete culture media (50 µl) and synchronized hydrochloride (EDC)/0.1 M N-hydroxy w
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schizont-stage parasites (100 µl) were added to succinimide (NHS). Coupling of AMA1 to the lo
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each well (4% hematocrit; 0.3% parasitemia) and chip surface was achieved using a manual control ed
plates were incubated for 40-42 h at 37oC in a approach whereby small volumes (1-15 μl) of fro
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moist atmosphere of 94% N , 1% O , 5% CO . AMA1 diluted to 12.5 μg/ml in 10 mM sodium h
2 2 2 ttp
After washing with ice-cold PBS, parasites were acetate pH 4.5 were injected over the activated ://w
frozen, then thawed, and relative parasitemia levels surface at 5 μl/min until immobilization of w
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were determined by assaying for parasite lactate approximately 1500 - 2500 RU was reached (1 RU .jb
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dehydrogenase (LDH) activity (10). Absorbance = 1 pg protein/mm2). The immobilization .o
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was measured at 650 nm (A650 nm) and the procedure was completed by a 7 min injection (10 b/
y
percent inhibition of invasion was calculated as: μl/min) of 1 M ethanolamine (pH 8.5) to deactivate g
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100 – [(A650 nm peptide sample – A650 nm red residual reactive sites. Final immobilized AMA1 st o
blood cells only)/(A650 nm no peptide control – levels achieved were as follows: 3D7 = 1530, HB3 n A
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A 650 nm red blood cells only) × 100]. =ex p2e7r0im5 eRntU a ta nleda sWt 2omnee fo f= th2e4 9f9lo wR-Uce. ll sI no ne atchhe ril 2, 2
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Enzyme-linked immunosorbent assay (ELISA) - 96- chip was used as “mock surface” for referencing 1
9
well microtiter plates (Maxisorp, Nunc) were purposes. This was prepared by subjecting a
coated with 2 µg/ml of recombinant AMA1. The surface to the amine coupling procedure with no
antigen was diluted in coating buffer (15 mM protein present.
Na CO , 34 mM NaHCO , pH 9.6) and plates were
2 3 3
incubated overnight at 4oC. Unbound protein was SPR binding analysis - All biosensor binding
removed by washing with PBS and any unbound experiments were performed in triplicate in HBS-
surfaces were blocked with 10% skim milk powder EP+ running buffer at 25oC. To generate the
in PBS. R1 peptide or N-methylated R1 analogues binding data, a series of peptide concentrations,
were applied to the wells in duplicate in the typically ranging from 5 to 320 nM for 3D7 and
presence of a constant concentration of R1 phage from 0.32 to 40.96 µM for W2mef and HB3, were
(~2 × 109 CFU/ml). Following incubation for 1 h at injected over immobilized AMA1 at a constant
room temperature, with shaking, unbound phage flow rate of 60 µl/min for 1 min. Peptide
was removed by washing with PBS 0.05% Tween dissociation was then monitored by injecting HBS-
20 (Sigma). Bound phage was detected with EP+ running buffer for 5 min, after which time all
horseradish peroxidase conjugated anti-M13 of the peptide completely dissociated from the
antibodies (1:5000; GE Healthcare). Binding was AMA1 surface and thus no further regeneration
was required.
Anti-malarial N-methylated peptides 5
(NOESY) spectra with a mixing time of 250 ms
SPR data Analysis - All Biacore sensorgrams were were also acquired at 600 MHz. For N-Me-Leu8
processed using Scrubber software (Version 2; and N-Me-Val1/N-Me-Leu8/N-Me-Ser14, NOESY
available from Biologic Software, Australia; spectra with a mixing time of 250 ms were also
www.biologic.au). Sensograms were first zeroed acquired at 800 MHz on a Bruker Avance 800.
on the y axis and then x-aligned at the beginning of Water was suppressed using the WATERGATE
the injection. Bulk refractive index changes were pulse sequence (36). All spectra were collected at
removed by subtracting the reference flow cell (Fc) 5°C unless otherwise stated and were referenced to
responses. The average response of all blank an impurity peak at 0.15 ppm or to the water
injections was subtracted from all analyte resonance.
injections and blank sensorgrams to remove Translational diffusion measurements were
systematic artefacts in the experimental and performed for N-Me-Leu8, N-Me-Leu8/N-Me-
reference flow cells. Scrubber analysis software Ser14, and N-Me-Val1/N-Me-Leu8/N-Me-Ser14
was used to determine ka and kd from the using a pulsed field gradient longitudinal eddy-
processed data sets by globally fitting to a 1:1 current delay pulse sequence (37,38) as
biomolecular binding model that included the mass implemented by Yao et al. (39). Spectra were
transport term (33). The K was calculated from processed using XWINNMR (Version 3.5, Bruker
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the quotient kd/ka. Alternatively, for rapidly Biospin) and TOPSPIN (Version 1.3, Bruker D
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dissociating interactions, affinity (K ) estimates Biospin) and analyzed using XEASY (Version w
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were derived using steady state affinity algorithm 1.3.13) (40). Chemical shift assignments for the lo
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available within Scrubber. major conformation of the peptides are tabulated in ed
the Supplementary Material and been deposited in fro
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Nuclear magnetic resonance (NMR) spectroscopy - the BioMagResBank (41). h
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Samples were prepared for NMR analysis by ://w
dissolving peptides in 500 µL of 95 % H O / 5 % Structural constraints - 3J coupling constants w
2 HNHA w
2H2O containing 10 mM sodium acetate to a final were measured from DQF-COSY spectra at 600 .jb
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concentration of approximately 1.7 mM. The pH MHz and converted to dihedral restraints as .o
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was adjusted to 4.6-4.7. 1H NMR spectra of most follows: 3J > 8 Hz, φ = -120±40º; 3J < 6 b/
HNHα HNHα y
peptides showed a single major set of resonances, Hz, φ = -60±30°. If a positive φ angle could be gu
e
indicating that any impurities were non-peptidic excluded on the basis of NOE data (42), φ angles st o
(the amide and aromatic regions of one- were restricted to the range -180 to 0°. No χ1 angle n A
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dSiumppenlesmioennatla ry spMecattrear ial,a reF ig. shSo1w).n Adind itiotnhael oMr eh-Lydeuro8g en abnodn d rNes-tMraein-Vtsa wl1e/Nre- Minec-lLudeued8 /Nfo-rM Ne-- ril 2, 2
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peaks with intensities < 10 % of the main peak 1
Ser14. 9
were present in spectra of N-Me-Leu8, N-Me-

Leu8/N-Me-Ser14, and N-Me-Val1/N-Me-Leu8/N-
Structure calculations - Intensities of NOE cross
Me-Ser14. As these peptides were > 90% pure,
peaks were measured in XEASY and calibrated
these additional peaks must have arisen from
using the CALIBA macro of the program CYANA
minor conformers. The most likely origin of this
(version 1.0.6) (43). NOEs providing no restraint
conformational heterogeneity is cis-trans
or representing fixed distances were removed. For
isomerism around the peptide bond involving the
structure calculations of N-Me-Leu8 and N-Me-
N-methyl group at Leu8; this has been observed
Val1/N-Me-Leu8/N-Me-Ser14, the constraint list
previously in other N-methylated peptides (34,35).
resulting from the CALIBA macro of CYANA was
Two different preparations of N-Me-Leu8 showed
used directly in XPLOR-NIH (44) to calculate a
identical ratios of major to minor species.
family of 100 structures using the simulated
Two-dimensional homonuclear total
annealing script. The library files supplied with
correlation (TOCSY) spectra with a spin-lock time XPLOR-NIH were modified to include a methyl at
of 60 ms and double quantum filtered correlation
the backbone amide of the corresponding residue.
(DQF-COSY) NMR spectra were acquired at 600
The 70 lowest energy structures were selected for
MHz on a Bruker DRX-600 spectrometer. Two-
each peptide. A box of water with a periodic
dimensional nuclear Overhauser enhancement
Anti-malarial N-methylated peptides 6
boundary of 18.856 Ǻ was built around the peptide because of its close proximity to the solid support.
structure, which was then energy minimized based Therefore, 18 R1 derivatives were individually
on NOE and dihedral restraints and the geometry synthesized by Fmoc solid phase peptide chemistry
of the bonds, angles and impropers. From this set as part of the initial ‘N-methyl scan’. The mode of
of structures, final families of 20 lowest energy incorporation of particular N-methyl residues
structures for each peptide were chosen for depended on the nature of the amino acid.
analysis. In all cases, the final structures had no Relatively few N-Fmoc-N-methyl amino acids are
experimental distance violations greater than 0.2 Å commercially available and ready for the peptide
or dihedral angle violations greater than 5°. All coupling reaction: these include Fmoc-NMe-Val-
structural figures were prepared using the OH, Fmoc-NMe-Phe-OH, Fmoc-NMe-Ala-OH,
programs MOLMOL (45) and PyMOL (46) Fmoc-NMe-Leu-OH, Fmoc-NMe-Ile-OH, and
(www.pymol.org). Fmoc-NMe-Met-OH. These compounds were
readily synthesized by the general method of
Peptide stability in mouse plasma - Mouse plasma Aurelio et al. (47) and incorporated into the
samples were prepared by adding 1 ml Milli Q peptide using standard HBTU-mediated peptide
water to the Sigma P-9275 mouse plasma product coupling chemistry. When N-methylation of other
and aliquoting the solution into 5 x 400 μl lots. residues was required the ‘on-resin’ method of
Peptides (0.4 mg) were dissolved in PBS (400 μl Miller and Scanlan (25) was used. Briefly, the D
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containing 0.1% [v/v] benzyl alcohol as internal Fmoc group was removed from the amino acid to w
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standard). Equal volumes of the plasma and be N-methylated and re-protected with the ortho- loa
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peptides solutions were combined (total volume nitrobenzenesulfonyl group (o-nosyl). This moiety ed
800 μL) and incubated at 37oC. Samples were rendered the amine hydrogen slightly acidic and from
taken in triplicate (3 x 50μL) at 0, 10, 20, 40, and vulnerable to alkylation by a methylating agent http
80 min and placed on ice. To each sample, 20 μl of such as methyl triflate (25). Lastly, the nosyl group ://w
was removed by treatment with base and w
0.5 M lysine monohydrochloride was added, w
followed by 65 μl acetonitrile. The samples were mercaptoethanol generating a free secondary .jbc
amine ready for the next coupling reaction. Using .o
then cooled on ice for 10 min and centrifuged for rg
10 min at 3000 x g resulting in a bi-phasic these two techniques the 18 R1 analogues were by/
prepared. g
solution. A 50 μl sample was taken from the ue
supernatant, diluted with 100 μl Milli Q water and st o
analysed by RP-HPLC on a C18 reversed-phase AMA1 binding ability of N-methylated R1 n Ap
caroelau mton t. hTeh bee rnaztyiol aolfc othheo ld ipseaapkp eaarerian gw apse pctaildceu lpaeteadk dmeertihvyaltaivteeds R- 1 Tdherei vraetilvateisv efo ra AffiMniAty1 wofa st hasessees sNed- ril 2, 20
by determining the ability of each peptide to 1
for each time point and normalized against the t = 9
inhibit the interaction of phage displaying the R1
0 time point (representing 100% peptide
peptide with immobilized AMA1 by ELISA (Fig.
remaining) as a percentage peptide remaining.
2 A-C). The peptides could be classed into four

broad categories according to the impact of
RESULTS
methylation. Binding was significantly improved

by the presence of a methyl group at Leu8.
Synthesis of N-methylated R1 derivatives - The R1
Methylation of residues Lys11, Phe12, Gly13 and
peptide consists of 20 residues and its sequence is
Arg15 resulted in a striking reduction in AMA1
shown in Table 1. R1 analogues incorporating a
binding affinity, as judged by the reduced ability of
methyl group at the backbone amide of each
these peptides to prevent phage-displayed R1
residue, except Pro7 and Lys20, have been
peptide from binding to AMA1. Inclusion of an N-
synthesized (Table 1 entries 2-19 and Fig. 1).
methyl group at residues Ala3, Phe5, Phe9, Ser10
Proline contains a secondary amine and N-
and Met16 slightly decreased AMA1 binding. The
methylation at Pro7 would effectively terminate
remaining methylations (Val1, Phe2, Glu4, Leu6,
the peptide sequence. Difficulties were
Ser14, His17, Iso18, Leu19) exhibited similar
experienced in forming the N-methyl derivative of
binding to native R1 in this assay.
Lys at the C-terminus of the peptide, presumably
Anti-malarial N-methylated peptides 7
Ability of N-methylated R1 derivatives to inhibit precisely measure the affinity of these peptides for
parasite invasion - To determine the effect of N- the 3D7 form of AMA1, kinetic and steady state
methyl substitution on bioactivity, each R1 binding to immobilized AMA1 proteins from three
analogue was tested for its ability to inhibit P. falciparum strains were examined by SPR
parasite invasion in vitro (Fig. 3 A). At 50 μg/ml (Table 2 and Supplementary Material, Fig. S7 and
of peptide, inhibition correlated well with the S8). The unmodified R1 peptide bound to 3D7
binding data, which are summarized in Fig. 3 B. AMA1 with a K of ~80 nM. Progressive
D
For example, R1 with N-methylation of Lys11, methylation of the R1 peptide resulted in an
which was unable to bind AMA1, showed little increased affinity for 3D7 although the difference
effect on parasite growth, whereas the N-Me-Val1 between doubly and triply methylated R1 was not
analogue both retained its AMA1 binding capacity significant. The primary reason for this increased
and prevented parasite growth in vitro. However, affinity appears to be slower dissociation of the
since native R1 peptide completely inhibited peptide from AMA1 (slower kd rate constant)
parasite growth at this concentration, any suggesting that the presence of N-methyl groups in
improvements in inhibition conferred by the R1 peptide backbone increases the overall
methylation could not be assessed. Those peptides stability of the peptide-AMA1 complex.
exhibiting maximal levels of invasion inhibition Interactions between native and N-methylated R1
were therefore selected for titration in growth peptides and immobilized AMA1 derived from D
o
inhibition assays (Fig. 3 C). These titrations W2mef and HB3 strains of parasite were analyzed w
n
revealed that the N-Me-Leu8 peptide was a more by equilibrium analysis. Although binding of all lo
a
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potent invasion inhibitor than native R1, R1 peptides to these isoforms of AMA1 was ed
correlating with the enhanced affinity observed for weaker than to 3D7 AMA1, the inclusion of fro
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this analogue. Interestingly, N-Me-Val1 and N-Me- multiple N-methyl groups in the R1 peptide h
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Ser14 analogues were approximately 2-fold more resulted in increased affinities: ~ 17 to ~6 μM and ://w
effective inhibitors than the native peptide despite ~70 to ~25 μM for W2mef and HB3 AMA1 w
w
exhibiting similar AMA1 binding to native R1. respectively. .jb
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This suggests that the binding assay may not be .org
sufficiently sensitive to distinguish small changes Solution structure of N-methylated R1 derivatives - b/
y
in affinity. To determine the impact of N-methylation on the gu
e
solution structure of R1, one- and two-dimensional st o
Double and triple N-methylated R1 derivatives - 1H NMR spectra were recorded on the following n A
p
Since the addition of a single methyl group at R1 analogues: N-Me-Phe5, N-Me-Leu8, N-Me- ril 2
Val1, Leu8 and Ser14 enhanced the ability of R1 Lys11, N-Me-Phe12, N-Me-Gly13, N-Me-Ser14, , 2
0
to block parasite invasion, it was of interest to and N-Me-Leu8/N-Me-Ser14 and N-Me-Val1/N- 1
9
determine if combining these methylations could Me-Leu8/N-Me-Ser14. The deviations of the
further improve R1 activity. Therefore, two backbone NH and CαH chemical shifts from
analogues incorporating multiple N-methylations random coil values are plotted in Fig. S2
were synthesized: a doubly N-methylated peptide (Supplementary Material). The differences
with N-methyl groups at Leu8 and Ser14, and a between these backbone chemical shifts for each of
triply N-methylated peptide with N-methyl groups the peptides and corresponding values for native
at Val1, Leu8 and Ser14. These peptides competed R1 are plotted in Fig. 5. A striking feature of these
with phage-displayed R1 peptide for binding to plots is that the backbone chemical shift
immobilized AMA1 to a similar degree to the N- differences caused by N-methylation are
Me-Leu8 peptide (Fig. 4 A). When tested in predominantly local, mostly within 2-3 residues
growth inhibition assays, these peptides reduced either side of the N-methyl substitution, which
parasite growth approximately 10-fold more implies that N-methylation does not cause any
effectively than native R1 (Fig. 4 B). long-range structural changes in R1. The structures
are consistent with the previously described
Strain-specificity of N-methylated R1 derivatives - solution structure of R1, which contains two
To investigate strain-specificity of R1 analogues structured regions, both involving turns, the first of
with improved inhibitory characteristics and to which, encompassing residues 5-10, is
Anti-malarial N-methylated peptides 8
hydrophobic and the second, involving residues (Val1 or Leu8) or multiple N-methyl groups were
13-17, is more polar (13). The i+3 residues in these tested for their stability in serum (Fig. 6). It was
turns are Ser10 and Met16, respectively, and N- evident that the native R1 peptide and the N-Me-
methylation at these positions would be expected Val1 peptide were rapidly degraded and after 10
to interfere with formation of i+3 to i hydrogen min only ~20% of the starting peptide remained.
bonds and thus destabilize the turns. The However, addition of a single methyl group at
observation that N-Me-Ser10 and N-Me-Met16 had Leu8, or the inclusion of three methyl groups
only slightly decreased binding affinities for (Val1, Leu8 and Ser14) resulted in a dramatic
AMA1 indicates that the turns present in solution improvement in peptide stability. Strikingly, even
may not persist in the AMA1-bound peptide, at after 80 min in serum more than 75% of the
least not in standard turn conformations. Even in peptide incorporating N-methyl groups at Val1,
solution these turns are unlikely to represent the Leu8 and Ser14 remained intact.
only conformations sampled by the peptide, as
linear peptides lacking any covalent cross-links, as DISCUSSION
in the case of R1, are known to sample a range of
rapidly interconverting conformations in aqueous In this study we have shown that a
solution. comprehensive N-methyl scan of a 20-residue
It was of interest to explore whether the N- inhibitory peptide can facilitate optimization of
D
o
methyl groups nucleated local structure (as potency, both in the peptides’ affinity for the target w
n
opposed to long-range structure, which is protein and its bioactivity. Some N-methyl lo
a
d
effectively ruled out by the lack of extensive modifications also resulted in a dramatic ed
chemical shift changes). A confounding factor here improvement in the proteolytic stability of the fro
m
is that the N-methyl groups give sharp, strong peptide. h
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resonances which are likely to detect inter-proton Methylation of backbone amides at ://w
NOEs over longer distances than the parent different positions can introduce a number of w
w
backbone amide proton. For this reason we possible effects on a peptide. The introduction of .jb
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calculated structures for various N-methyl the methyl group imposes conformational .o
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analogues in the presence and absence of NOEs to constraints on both the modified residue and that b/
y
the N-methyl groups. The various families of preceding it, increases peptide hydrophobicity and g
u
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calculated structures are shown in the ablates hydrogen bonding potential at the affected st o
Supplementary Material (Fig. S3-S6). In essence, site (48). Furthermore, the reduction in amide n A
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twhiet hosturtu ctNu-rmese tohfy l N-NmOeEthsy l waenrael ogsuimesi lacra lctou lattehde breosnisdtsa nchea st o thpero tepooltyesnitsi.a lT ot oi nvimesptirgoavtee hpoewp tiNde- ril 2, 2
0
structure of R1, but inclusion of N-methyl NOEs methylation affects the interaction of the R1 1
9
and two long-range NOEs in the structure peptide with AMA1, a panel of 18 R1 analogues
calculations for N-Me-Leu8 caused an apparent with a methyl group added to the backbone amide
stabilization of structure in the vicinity of the N- at different positions was synthesized. Several of
methyl group, resulting in a more compact global the N-methyl amino acids were not commercially
structure. However, as corresponding long-range available and the particular residues were
NOEs were not observed in N-Me-Val1/N-Me- consequently N-methylated on solid phase. It is not
Leu8/N-Me-Ser14, its structure was similar to that possible to N-methylate the R1 peptide at Pro7
of R1. and, as previously described, there were problems
when attempting to synthesise the terminal residue,
Proteolytic stability of N-methylated R1 Lys20, version using the method of Miller and
derivatives - An important property of bioactive Scanlan (25). This problem would not arise if the
peptides is their resistance to proteolytic C-terminal N-methyl amino acid was commercially
degradation. In order to determine whether the N- available and could be introduced by standard
methyl substitutions that increase affinity and peptide synthesis procedures.
bioactivity can also augment resistance of the Assessing the ability of all 18 R1 peptide
peptide to serum proteolysis, peptides analogues to interact with AMA1 (Fig. 2 and Fig. 3
incorporating either a single N-methyl amino acid B) revealed that N-methylation at four positions,
Anti-malarial N-methylated peptides 9
Lys11, Phe12, Gly13 or Arg15, dramatically AMA1 has recently been determined (49). One
reduced binding to AMA1. Inspection of the important feature of this structure was the presence
previously determined R1 peptide structure (13) of a conserved hydrophobic pocket surrounded by
indicated that three of these residues (Lys11, loops incorporating a high density of polymorphic
Phe12 and Gly13) are located either within, or residues. It has been postulated that this groove
close to, a region of undefined structure. Since it is may represent an important functional site that is
known that the introduction of a methyl group into shielded from the immune system by the
a peptide backbone largely restricts local surrounding polymorphic residues. Interestingly,
conformation, it is possible that decreased the crystal structure of AMA1 in complex with the
flexibility within this region is responsible for the inhibitory MAb 1F9 has indicated that the MAb
loss of activity observed. Perhaps a flexible core is 1F9 epitope is located at one end of the
required for the two regions of well-defined hydrophobic pocket whilst mutational analysis
structure (residues 5-10 and 13-17) to interact upon suggests that residues involved in MAb 4G2dc1
binding to AMA1. Indeed, amide exchange data recognition of AMA1 may be located near the
has indicated that this may be the case (13), opposite end of this conserved trough (50-53).
lending support to this theory. It should also be Since R1 peptide competes with both of these
noted that the introduction of a methyl group MAbs for binding to AMA1, it is conceivable that
removes hydrogen bonding potential at a given the peptide is targeting the conserved pocket
D
o
position, and this may also contribute to a loss of between these two sites and is thus able to interfere w
n
activity. Nevertheless, it is clear that methylation at with the binding of each MAb, despite their lo
a
d
a single site within R1 can dramatically alter the apparently distinct binding sites. Such a scenario is ed
peptide’s affinity for AMA1. in general agreement with the findings of other fro
m
The R1 peptide has been reported to be an published binding studies indicating that small h
ttp
effective inhibitor of malaria parasite invasion and molecule binders are better able to access grooves ://w
the impact of N-methylation on invasion inhibition than antibodies, which often exert their effects by w
w
was examined. In general, the AMA1 binding occluding, rather than directly interacting with, .jb
c
capacity of N-methylated analogues correlated well binding pockets (54,55). Furthermore, NMR .o
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with their inhibitory properties (Fig. 3). For studies have revealed a common feature of the R1 b/
y
example, N-Me-Lys11 did not inhibit parasite and F1 peptides is the clustering of hydrophobic g
u
e
growth, whereas N-Me-Leu8 showed increased residues within turn-like conformations consistent st o
inhibition. Thus, improved AMA1 binding ability with these peptides’ ability to probe a hydrophobic n A
tinravnessltaigteadte toif sthuep eerifofer ctisn hoifb itNo-rmy etphryolpaetriotine s.w eTroe suinted er(w1a3y,5 6)to. Minuvteastitoignaatle stthuidsi esh yaproet hecsuisrr enatnlyd pril 2, 2
0
additive, peptides with a methyl group added to the confirm that the hydrophobic trough is the target of 1
9
backbone amide at multiple positions were inhibitory peptides. This highly conserved trough
synthesized. Both double and triple methyl represents an important target for the design of
substitutions resulted in an increase in AMA1 anti-malarial therapeutics since it is probable that
affinity, as determined by SPR (Table 2), and the fitness of escape mutants will be limited by a
growth inhibitory ability (Fig. 4B) as compared to requirement to maintain function.
the N-Me-Leu8 peptide indicating that combining The polymorphic nature of AMA1 is
single methylations with the desired properties can another factor requiring consideration in the
further improve peptide characteristics. investigation of this antigen as a therapeutic target.
Although the binding site recognized by The native R1 peptide has been shown previously
native R1 has not yet been described, the peptide is to interact with the 3D7 and D10 strains of AMA1,
known to compete with the inhibitory MAbs 1F9 but no binding to AMA1 derived from the HB3 or
and 4G2dc1, as well as with the inhibitory peptide W2mef parasite lines was observed. When we
F1 (13). This suggests that these molecules all examined the strain-specificity of R1 analogues,
interact with a similar AMA1 binding site, thus the peptides incorporating multiple N–methyl
identifying a hot-spot on the antigen that appears groups exhibited a slightly increased affinity for
to be the target of inhibitory molecules. The crystal the HB3 and W2mef allelic forms of AMA1, as
structure of domains I and II of P. falciparum compared to the native peptide (Table 2). This
Anti-malarial N-methylated peptides 10
suggests that, with further modification, it may be infection (58). However, in human trials high
possible to circumvent AMA1 allelic variation doses of T-20 (almost 200 mg/day) were required
altogether by optimizing access to the conserved to achieve anti-viral effects comparable to those
region of the putative binding site. observed in vitro, despite much lower doses being
Since R1 adopts an extended structure effective in cell culture, contributing to the high
with only two regions of local structure, it is likely cost of this drug and clearly indicating a
that N-methyl substitution causes some local requirement for improved bioavailability (59).
changes in the mix of interconverting Development of strategies to rapidly improve
conformations, but no global structural peptide stability will therefore be of value to a
rearrangement. Given that the surface trough on wide range of areas within peptide research.
AMA1 likely to be targeted by R1 is non-polar, the This work has illustrated that the
effect of N-methylation on affinity may reflect a properties of a peptide (R1) that inhibits malaria
balance between the enhanced hydrophobicity and parasite invasion of erythrocytes can be enhanced
the ability of the introduced N-methyl group to fit by systematic methylation of backbone amides.
into the trough without steric clashes. It is feasible Importantly, N-methyl substitution of a single
then that the introduction of methyl groups at some residue improved target binding affinity and
positions improves the ability of the R1 peptide to bioactivity and dramatically enhanced peptide
interact with the hydrophobic pocket, and that the stability in mouse plasma. Furthermore, the
D
o
resulting increase in affinity reduces the reliance incorporation of multiple N-methyl groups further w
n
upon surrounding polymorphic residues, increased target binding affinity and bioactivity. lo
a
d
facilitating improved binding to AMA1 derived These data demonstrate that comprehensive N- ed
from different parasite lines. Data on the site- methyl scanning of peptides offers the advantage fro
m
specific effects of N-methylation will be valuable of enabling bioactivity and proteolytic stability to h
ttp
in assessing any model of how R1 might occupy be enhanced simultaneously. With the renewed ://w
this trough. interest in peptide therapeutics, this approach could w
w
Previous work has indicated that be applied to many lead peptides to rapidly .jb
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methylation can also result in resistance to optimize a number of parameters relevant to .o
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proteolytic degradation. For example, methylation development of an effective pharmaceutical. b/
y
of a peptide that prevents dimerization of an HIV-1 g
u
e
protease has been shown previously to result in a st o
dramatic improvement in resistance to pepsin (57). ACKNOWLEDGMENTS n A
Wanea lothgeureesf ofroer ttehsetierd staa bniulimtyb einr osef rumme th(Fyilgat. e6d) .R I1t This work was supported in part by grants from the pril 2, 2
0
was clear that the presence of a single methyl National Health and Medical Research Council of 1
9
group at Leu8, as well as multiple methylations (at Australia and the National Institutes of Health
Val1, Leu8 and Ser14), were able to dramatically (NIH RO1AI59229). The authors thank Robin
improve the amount of intact peptide available Anders and Vince Murphy for provision of
after 80 min in serum. This is a significant finding recombinant AMA1 and for stimulating
since poor bioavailability represents a limitation to discussions.
the use of peptides as therapeutics. Although there
are some peptides in clinical use, rapid breakdown COMPETING INTERESTS STATEMENT
often necessitates administration of high doses. For
example, T-20, or enfuvirtide, is a 36-residue The authors declare that they have no competing
peptide that is currently in clinical for use in HIV-1 financial interests.