Selective inhibitors of plasmepsin II of Plasmodium falciparum on the basis of pepstatin

Article abstract of DOI:10.1134/S1068162008060034

A number of new inhibitors of plasmepsin II (PlmII) Plasmodium falciparum, which was one of the key factors of survival of malarial parasite, was synthesized. The inhibitors were analogues of pepstatin with different substitutions for the alanine residue.

Full text of DOI:10.1134/S1068162008060034

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 6, pp. 660–667. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © L.D. Rumsh, A.G. Mikhailova, I.V. Mikhura, I.A. Prudchenko, L.D. Chikin, I.I. Mikhaleva, E.N. Kaliberda, N.I. Dergousova, E.E. Mel’nikov, A.A. For-
manovskii, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 6, pp. 739–746.
Selective Inhibitors of Plasmepsin II of Plasmodium falciparum
on the Basis of Pepstatin
L. D. Rumsh1, A. G. Mikhailova, I. V. Mikhura, I. A. Prudchenko, L. D. Chikin, I. I. Mikhaleva,
E. N. Kaliberda, N. I. Dergousova, E. E. Mel’nikov, and A. A. Formanovskii
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received October 15, 2007; in final form, October 20, 2007
Abstract—A number of new inhibitors of plasmepsin II (PlmII) Plasmodium falciparum, which was one of the
key factors of survival of malarial parasite, was synthesized. The inhibitors were analogues of pepstatin with
different substitutions for the alanine residue. Effects of the inhibitors on human PlmII and cathepsin D were
studied. Inhibition of PlmII by the substrate was found. This discovery required modification of the Henderson
method for determination of inhibition constants. Two synthesized inhibitors were shown to exhibit a pro-
nounced selectivity to PlmII (Ki = 5.5 and 5 nM) in comparison with that of cathepsin D (Ki = 230 and 3000 nM,
respectively).
Key words: malaria, proteases, inhibitors, plasmepsin II, Plasmodium falciparum
DOI: 10.1134/S1068162008060034
INTRODUCTION
disorder of metabolism of the parasite cells and their
fast death [2].
Elaboration of treatment modes of malaria is one of
priority directions in the world. Urgency of this prob-
lem is determined by social factors and the absence of
effective treatment2.
Degradation of hemoglobin proceeds in a food vacuole,
the proteolytic compartment of P. falciparum with pH 5.0–
5.4 [3, 4]. Plasmpepsins PlmI and PlmII recognize hemo-
globin and hydrolyze initially single peptide bond between
Phe33 and Leu34 [5] that is located in the region of a loop
of the hemoglobin α-chain. Amino acid sequence of this
chain is highly conservative in all hemoglobins of verte-
brates: -EALERMF33-L34SFPTTK-. It is believed that the
ordered decomposition of hemoglobin is initiated by aspar-
tate proteases (PlmI and PlmII). The subsequent hydrolysis
proceeds by the action of other enzymes of the food vacuole
of the P. falciparum, for example by the cysteine protease,
falcipain [5–7].
PlmI and PlmII, like other aspartate proteases, are
synthesized as inactive precursors, but proareas of the
plasmodium proteases are much longer than those of
other zymogens: 123 and 124 amin acid residues,
respectively [8]. A propeptides is cleaved in vitro as a
result of autoprocessing of the precursors at acidifica-
tion to pH 4.5–5.0 with the formation of mature active
enzymes with molecular mass of 37 kDa. Their sizes
are identical to those of proteases directly isolated from
P. falciparum [9]. Activities of natural and recombinant
forms of PlmII have been shown to be identical [4].
According to World’s Public Health Organization,
malaria morbidity caused by the Plasmodium is 500
millions people every year, including 2 millions of fatal
terminations (predominantly among children) [1]. Fast
variability of the parasite and stability to the known
medicines requires a search for novel antimalarial
agents with a new mechanism of action. Four Plasmo-
dium species pathogenic for humans are known: vivax,
ovale, malariae, and falciparum. The latter is the most
dangerous.
The key factor of survival of the etiological malarial
agent is the presence of aspartyl proteases in its cells.
They are called plasmepsins (PlmI–PlmV) and partici-
pate in catabolism of hemoglobin, which is a food
resource of the parasite during intraerythrocyte devel-
opment of the plasmodium. These enzymes (first of all
PlmII, EC 3.4.23.39) are considered to be the most
promising targets for therapy of malaria, because their
specific activation by inhibitors results in the complete
1
Corresponding author; phone: +7 (495) 336-2811; e-mail:
rumsh@enzyme.siobc.ras.ru.
Specificity of the recombinant PlmII was studied
using a number of synthetic chromogenic and fluoro-
genic substrates [10–14]. These substrates were chosen
on the basis of the aforementioned amino acid sequence
2
Abbreviations: Abbreviations: CatD, cathepsin D; CS, the H-Leu-
Glu-Arg-Ile-Phe-Phe(NO )-Ser-Phe-OH chromogenic substrate;
2
PlmII–PlmV, plasmepsins II–V; Pst, pepstatin.
660

SELECTIVE INHIBITORS OF PLASMEPSIN II OF Plasmodium falciparum
661
Table 1. Hydrolysis constants of the (CS) substrate by plasmepsin II and human cathepsin D in 0.1 M sodium formate buffer
at 37°C
Enzyme
PlmII
pH
4.4
[S], µM
Km, µM
kcat, min–1
kcat/Km, µM min–1
K'S, µM
3–10
20–90
10–100
9.53
–
466
466
450
48.90
–
–
5.05
–
CatD
3.5
91.00
4.94
that surrounds the primary site of hydrolysis of the then the Michaelis constant, suggesting unusually
hemoglobine α-chain. The chromogenic substrates strong inhibition of PlmII by the CS octapeptide at
were prepared by substitution of the residue of p-nitro- [S] > Km. We did not observe the substrate inhibition in
phenylalanine, Phe(NO2) for Leu in the P1'-position.
Easily oxidable Met residue in the P2-position was
replaced by Thr [10], Ile, Val, or Nle [13]. We used the
H-Leu-Glu-Arg-Ile-Phe-Phe(NO2)-Ser-Phe-OH (CS)
chromogenic substrate [11] in this study.
The highly pure pro-PlmII preparation was prepared
by expression of the recombinant gene encoding the
last 48 amino acid residues of the propeptides and all
mature enzyme in E. coli [10]. The three-dimensional
structure of PlmII was determined. It has a typical
topography of aspartate proteases of eukaryotes [12].
It is very important to find compounds of selective
action for prevention of possible inhibition of the activ-
ity of endogenous aspartate proteases, especially rennin
and cathepsin D (EC 3.4.23.5) when inhibitors of plas-
mepsins are used. Highly selective inhibitors of PlmII,
which are derivatives of pepstatin, were prepared in this
study.
the case of cathepsin D in the studied concentration
range (10–100 µM) (Table 1).
The known inhibitor of aspartate proteases (Iva-Val-
Val-Sta-Ala-Sta) was taken as a basic structure of the
inhibitors synthesized by us. Iva was the residue of
isovaleric acid, and Sta was the residue of statin: –HN–
CH(CH2CH(CH3)2)CH(OH)CH2COOH [8]. We synthe-
sized five modified peptide inhibitors (I-1)–(I-5) of the
Y-Val-Val-Sta-X-Sta-OH general structure:
(I-1): X – –HN–CH(CH2–O–(CH2)2–CH2–OH)–CO–;
Y – Iva;
(I-2): X – –HN–CH((CH2)3–CH2–OH)–CO–; Y – Iva;
(I-3): X – –HN–CH(CH2–O–(CH2)2–CH2–OH)CO–;
Y – (CH3)2CH–CH(CH2–CH2OH)–CO–;
(I-4): X –Ala; Y – (CH3)2CH–CH(OH)–CO–;
(I-5): X – –HN–CH(CH2–O–(CH2)2–CH2–OH)–CO–;
Y – (CH3)2CH–CH(OH)–CO–.
RESULTS AND DISCUSSION
Fragments X and Y for compounds (I-1)–(I-5) were
prepared with the protective groups necessary for the
further peptide synthesis. (S)-2-tert-Butyloxycarbonyl-
Kinetic parameters of hydrolysis of the CS substrate
by plasmepsin II and human cathepsin D are given in
Table 1. Values of the kinetic constants of the hydroly-
sis of the CS substrate by PlmII determined by us
(Table 1) are rather close to the literature data
(kcat 780 min–1; Km 10 µM, kcat/Km 78 µM–1min–1 [11]).
However, we found that kinetics of hydrolysis of high
concentrations of the substrate is deviated from the
classic dependence of Michaelis–Menten (Fig. 1). Such
kinetic dependence corresponds to inhibition by the
substrate:
–1
v , OE , min
0
300
0.006
0.005
0.004
Km
kcat
E + S
ES
E + P.
'
KS
0.003
0.002
ES2
The equation of the starting reaction rate looks as fol-
lows:
0
20
40
60
80
100
[S], µM
2
'
v0 = kcat[E][S]/(Km + [S] + [S] /KS).
(1)
Fig. 1. Inhibition of plasmepsin II (17.6 nM) by the CS sub-
strate; [CS] 3–90 µM, 0.1 M sodium formate buffer, pH 4.4,
37°C.
The value of the inhibition constant by the substrate
'
determined by us KS (Table 1) proved to be even lesser
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

662
RUMSH et al.
The dose-dependent inhibition of PlmII and CatD
100
80
60
40
20
by compounds (I-1)–(I-5) and pepstatin A was studied
using the CS substrate. The obtained titration curves
correspond to the type of an inhibitor with a high affin-
ity in all the cases (Fig. 2) that is characteristic of the
inhibition of aspartate proteases by pepstatin [8]. The Ki
values are usually determined by the Henderson equa-
tion [15]:
1
2
3
4
5
6
[I]/(1 – vi/vo) = [E] + (Ki × A)v0/vi,
(2)
where v0 is the starting rate of hydrolysis of a substrate
by an enzyme with the starting concentration [E] in the
absence of an inhibitor, and vi is the same rate in the
presence of an inhibitor with the starting concentration
[I]. Noefficient A depends on the type of inhibition: A = 1 in
the case of uncompetitive inhibition, and A = ([S] + Km)/Km
for competitive inhibitors, including Pst [15].
0
1
2
3
4
5
6
3
The starting enzyme concentration is determined
from the linear dependence of the [I]/(1 – vi/vo on vo/vi
according to intersection with the ordinate axis using
the Henderson equation for strongly bound inhibitors.
The Ki value is calculated from the tangent of the angle
of the curve slope.All the studied inhibitors are the con-
current inhibitors, like pepstatin, because the tangents
of the angles of slope for the inhibitors and the enzymes
increase with the enhancement of the substrate concen-
tration (see Table 2, inhibitor (I-3)). According to the
Henderson equation [15], the value of corrective
A = ([S] + Km)/Km. We used high concentrations of the
CS substrate for enhancement of the accuracy. The
value of this increasing factor A is experimentally
established and proved to be 2.65 at [CS] = 150 or 1.33 at
[CS] = 30 µM in the case of cathepsin D (Km = 91 µM)
and 7.29 at [SC] = 60 µM in the case of plasmepsin II
(Km = 9.53 µM). The experimentally obtained values of
Ki × Ä (columns 2 and 5 for PlmII and CatD, respec-
tively) and the values of inhibition constants calculated
by the Henderson equation [15] (columns 3 and 6) are
given in Table 3. The value of inhibition constant of
CatD by pepstatin determined by us practically coin-
cides with the literature data (3.8 nM, [16]). However,
the calculated value of Ki for pepststin and plasmepsin
(31 nM, Table 3) is one order higher than that known
from the literature (3–5 nM) [8]. The effects of strong
[I]×10 , å
Fig. 2. Inhibition of plasmepsin II (30 nM) in the course of
hydrolysis of the CS substrate (60 µM) by the following
inhibitors: 1, Pst; 2, (I-1); 3, (I-2); 4, (I-3); 5, (I-4); 6, (I-5),
in 0.1 M sodium formate pH 4.4 at 37°C.
6-benzyloxyhexanoic acid (for the X fragment in com-
pound (I-2)) was prepared by deamination of Nα-Z-L-lysine
with sodium nitroprusside followed by replacement of
Z-protecting group by Boc-group and attachment of
benzyl group to free ω–OH function. The overall yield
of target product was 52%. For the synthesis of 2-tert-
butyloxycarbonylamino-7-benzyloxy-4-heptanoic acid
(for the X fragment in compounds (I-1), (I-3), an (I-5)),
N-Boc-L-serine was converted into its methyl ester by
the treatment with diazomethane. The obtained ester
was alkylated with 3-benzyloxypropylbromide, and the
free acid was prepared by alkaline hydrolysis. The
overall yield was 41%. 4-Benzyloxy-2-isopropylbu-
tanoic acid (for the Y fragment in compound (I-3) was
synthesized by alkylation of diethylester of malonic
acid with isopropylbromide, Saponification, and decar-
boxylation of the diethyl ester with 2-benzyloxy-2-iso-
propylmalonic acid. The target acid was prepared with
the overall yield of 47%.
Table 2. Inhibition of plasmepsin II (12 nM) by compound (I-3) at different concentrations of the CS substrate (0.1 M sodium
formate buffer, pH 4.4, 37°C)
A = ([S] + Km)/Km [15]
A = (Km + [S] + [S]2/K'S)/ Km
Ki × A, nM (tangent of the angle of
[CS], µM
the slope)
A
Ki, nM
A
Ki, nM
1
2
3
4
5
6
60
30
15
12.9
6.70
1.20
7.29
4.35
2.57
1.77
1.54
0.468
82.1
22.85
7.23
0.157
0.293
0.166
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SELECTIVE INHIBITORS OF PLASMEPSIN II OF Plasmodium falciparum
663
Table 3. Inhibition of plasmepsin II (pH 4.4) and human cathepsin D (pH 3.5) in the course of hydrolysis of the CS substrate;
[S] 60 µM, 0.1 M sodium formate buffer, 37°C
PlmII
CatD
Inhibitor
1
Ki, pM
Ki × A, nM
(tangent of the angle
of the slope)
Ki × A, pM
(tangent of the angle of
the slope)
Ki, pM*
A = 7.29*
A = 82.1**
2
3
4
5
6
Pst
0.226
0.178
0.291
12.9
31.10
24.4
39.9
2.75
2.15
3.54
157
5.01***
5.32***
3.77***
(I–1)
(I–2)
(I–3)
(I–4)
(I–5)
4.00***
4.32***
5.75***
1770
3990***
623****
8000****
3000***
0.451
0.413
61.9
56.6
5.49
5.03
235****
3020****
Notes:
* By the Henderson method.
** By our method.
*** [CS] = 30 µM; A = 1.33.
**** [CS] = 150 µM; A = 2.65.
substrate inhibition of PlmII by the CS substrate dis- The general view of the Henderson equation remains
covered by us (Table 1 and Fig. 1) allowed a proposal the same, but the increasing factor A looks as:
that the Henderson equation should be modified for cal-
2
'
A = (Km + [S] + [S] /KS)/ Km.
(5)
culations of the Ki values in experiments with a high
concentration of a substrate (60 µM). Indeed, the sub-
strate inhibition was absent I the case of cathepsin D,
and the value of inhibition constant by pepstatin calcu-
lated by the Henderson equation was not distinguished
from the literature data.
Evidently, this factor dramatically increases with
the enhancement of the substrate concentration. Indeed,
tangent of the angle of slope of the Henderson graph (2)
in he case of plasmepsin II (calculated using kinetic
parameters of Table 1, and the substrate concentration
of 60 µM) is 82.1 × Ki instead of 7.29 × Ki calculated as
usual:
Dependence of the inhibition constants calculated
by the Henderson equation on the substrate concentra-
tion (Table 2, column 4) is another argument in favor of
inutility of the classic Henderson method for plasme-
psin.
We discussed the modification of Henderson equa-
tion for the special case of strong substrate inhibition.
The equation for calculation of the starting rate of
hydrolysis in the presence of competitive inhibitor
looks as follows:
A = (9.53 + 60 + 3600/5.05) ÏÍå/9.53 ÏÍå = 82.1.
It is evident that reliability of determination of the Ki
values significantly increases in the case of high con-
centrations of substrate and inhibitor. True values of the
inhibition constants of PlmII for compounds (I-1)–(I-5)
and pepstatin calculated by our modification of the
Henderson equation are presented in Table 2 (column
6) and Table 3 (column 4). The value of inhibition con-
stant of PlmII by pepstatin coincides with the literature
data [8].
Compound (I-3) is the weakest inhibitor of plasme-
psin from all the studied compounds. The Ki value for
it is two orders higher than that for pepstatin and com-
pounds (I-1), (I-2), (I-4), and (I-5) (Table 3). The Ki val-
ues for these inhibitors are of the same order than that
for pepstatin (≈pM). It should be especially noted that
the inhibition of human cathepsin D by compounds
(I-4) and (I-5) is two and three orders lower in compar-
ison with plasmepsin II, respectively. The selectivity of
the inhibitors of the target enzymes appears o be one of
vi = kcat[E][S]/{(Km + [S]) + Km[I]/Ki}.
(3)
Equation 3 results in the usual Henderson equation
(2), where A = ([S] + Km)/Km. Therefore, tangent of the
angle of slope of the Henderson graph (2) is 1.33–2.65 ×
Ki for cathepsin D and 7.29 × Ki for plasmepsin II under
our experimental conditions in the case of the strongly-
binding competitive inhibitor.
However, equationfor the starting rate of hydrolysis
in the presence of inhibitor becomes more complex
with consideration of the substrate inhibition (1):
2
'
vi = kcat[E][S]/{(Km + [S] + [S] /KS) + Km[I]/Ki}. (4)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

664
RUMSH et al.
the important factors for creation of therapeutic agents 3.80 (H, m.s, OH), 4.48 (H, t, J 6.9, H2), 5.20 (H, m.s,
along with their high inhibiting ability. The plasmepsin
inhibitors (I-4) and, especially (I-5) studied by us are
evidently promising for pharmacology.
NH), 11.80 (H, m.s, COOH).
The solution of (2S)-2-tert-butyloxycarbonylamino-
6-hydroxyhexanoic acid (2.47 g, 10 mmol) in anhy-
drous ether (30 ml) was treated with the solution of
CH3N2 in ether (20 ml). After completion of the reac-
tion, ether was evaporated in vacuum. The residue was
dissolved in anhydrous dimethylformamide (20 ml),
cooled to 0°ë and mixed with 55% suspension (0.5 g)
of NaH in oil (11.4 mmol of NaH). After termination of
the ç2 liberation, benzyl bromide (1.71 g, 10 mmol)
was added. The reaction mixture was poured out into
water (100 ml). The pH value was brought up to 7, and
the precipitated oil was extracted with ethyl acetate
(5 × 25 ml). The extracts were washed, the solvent was
evaporated, and NaOH was added to the residue dis-
solved in methanol (60 ml). The free acid was prepared
by the treatment of the salt with the DOWEX 50 × 8
ion-exchange resin in ç+-form and purified by chroma-
tography. (2S)-6-Benzyloxy-2-tert-butyloxycarbony-
laminohexanoic acid was obtained as colorless oil
with the yield of 2.60 g (77%); 1H NMR: 1.41 (2 H, m,
H4), 1.49 (9 H, s, CH3), 1.66 (2 H, m, H5), 1.95 (2 H,
m, H3), 3.30 (2 H, t, J 7.5, H6), 4.50 (H, t, J 7.0, H2),
4.58 (2 H, s, C H2Ph), 5.18 (H, broadened s, NH), 7.10–
7.40 (5 H, m, Ph), 11.40 (H, s, COOH).
EXPERIMENTAL
Synthesis of inhibitors and substrate. TLC analy-
sis was performed on Silufol plates (Chemapol, Czech
Republic). Chromatographic purification was carried
out on columns filled with SiO2 (Merck G-60, Ger-
many) eluted with the mixture of chloroform and meth-
anol (7 : 1). 1H NMR spectra were recorded on a Unity
Inova spectrometer (400 MHz) in CDCl3. The residual
protons of solvent were used as an internal standard.
Melting points were determined on a Boetius device.
Fragment Y, 2-hydroxy-3-methylbutanoic acid used
for synthesis of compounds (I-4) and (I-5) was pur-
chased from Aldrich company (United States).
(2S)-6-Benzyloxy-2-tert-butyloxycarbonylamino-
hexanoic acid (for fragment X in (I-2)). (2S)-2-benzy-
loxycarbonyl-amino-6-hydroxyhexanoic acid was
prepared by deamination of Nα-Z-L-lysine with sodium
nitroprusside [17] with the yield of 80%. Water (10 ml)
and 10% Pd/C (0.5 g) were added to the solution of
(2S)-2-benzyloxycarbonyl-amino-6-hydroxyhexanoic
acid in methanol (100 ml), and the reaction mixture was
subjected to hydrogenolysis in the hydrogen current
until the disappearance of the starting compound. The
catalyst was filtered off and washed with water on a fil-
ter. The joined filtrate was evaporated to dryness. Water
(10 ml) and ethanol (110 ml) were added to the residue.
The precipitated crystals were separated, washed with
anhydrous ethanol, and dried in vacuum over alkali.
(2S)-2-amino-6-hydroxyhexanoic acid was obtained
Found, %: C 63.91, H 8.00, N 4.06. Calcd. for
C18H27NO5, %: C 64.07, H 8.07, N 4.15.
2-tert-Butyloxycarbonylamino-7-benzyloxy-4-
oxaheptanoic acid (for fragment X in compounds
(I-1), (I-3), and (I-5). The solution of N-Boc-L-Ser-OH
(4.1 g, 20 mmol) in methanol (20 ml) was treated with
CH3N2 in ether. After completion of the reaction, acetic
acid (0.5 ml) was added to the reaction mixture. The
solvents were evaporated, and the residue was purified
by flesh-chromatography (SiO2, Merck G-60) in the
mixture of chloroform and ethyl acetate (1 : 1). N-Boc-
L-Ser-OMe was prepared as colorless oil with the yield
of 3.94 g (90%). It was dissolved in anhydrous dimeth-
ylformamide (30 ml) and cooled to 0°C. The 55% sus-
pension (0.87 g) of NaH (20 mmol) in oil was added,
and the reaction mixture was stirred for 1 h.
PhCH2O(CH2)3Br (4.85 g) was added dropwise, and
the reaction mixture was stirred for 2 h at 0°C and kept
for a night at room temperature. The free acid was iso-
lated as described above. Chromatographic purification
yielded 3.18 g (45%) of 2-tert-butyloxycarbonyl-7-
with the yield of 2.63 g (95%); mp 251°ë; [α]2D0 + 23°
[18]; 1H NMR spectrum (D2O): 1.45 (2 H, m, H4), 1.62
(2 H, m, H5), 1.91 (2 H, m, H3), 3.64 (2 H, t, J 7.5, H6),
3.78 (H, t, J 6.8, H2).
Triethylamine (2.5 ml, 18 mmol) and di(tert-
butyl)pyrocarbonate (4.44 g, 20.5 mmol) were added to
the solution of (2S)-2-amino-6-hydroxyhexanoic acid
(2.50 g, 17 mmol) in the mixture of THF and water
(1 : 1, 30 ml). The reaction mixture was stirred for 6 h,
diluted with water (50 ml), and THF was evaporated in
vacuum. The aqueous solution was washed with hexane
(2 × 10 ml) and acidified to pH 3. The target product
was extracted with ethyl acetate (5 × 20 ml) and dried
over Na2SO4. Ethyl acetate was evaporated. The residue
was purified by chromatography (ethyl acetate was
used as an eluent). (2S)-2-tert-butyloxycarbony-
lamino-6-hydroxyhexanoic acid was prepared with
1
benzyloxy-4-oxaheptanoic acid as colorless oil; H
NMR: 1.49 (9 H, s, CH3), 1.83 (2 H, m, H6), 3.45 (2 H,
m, H3), 3.65 (4 H, m, H5 and H7), 4.38 (H, m, H2),
4.50 (2 H, s, C H2Ph), 5.80 (H, broadened s, NH), 7.18–
7.23 (5 H, m, Ph), 11.20 (H, s, COOH).
the yield of 3.70 g (88%); mp 112°ë, [α]2D0 – 6.36° [19];
1H NMR: 1.43 (2 H, m, H4), 1.50 (9 H, s, CH3), 1.68
Found, %: C 61.00, H 7.74, N 4.01. Calcd. for
C18H27NO6, %: C 61.17, H 7.70, N 3.96.
(2 H, m, H5),1.95 (2 H, m, H3),3,66 (2 H, t, J 7.5, H6),
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SELECTIVE INHIBITORS OF PLASMEPSIN II OF Plasmodium falciparum
665
4-Benzyloxy-2-isopropylbutanoic acid for frag- (10/1) at 0°C for 1 h. When reaction was finished, HF
was removed in vacuum. The peptides were precipi-
tated from reaction mixtures by the addition of diethyl
ether (15 ml). The resin and a deprotected peptide were
filtered off, washed with ether (5 × 10 ml), and dried on
a filter. The peptides were extracted with 50–95% solu-
tion of acetic acid depending on their hydrophobicity,
frozen, and lyophilized.
Gel filtration was performed on a column (800 ×
25 mm) with Sephadex LH-20 (Pharmacia, Sweden) in
methanol at a flow rate of 0.8 ml/min at 226 nm (Mul-
tirac 2158, LKB, Sweden). The preparative HPLC was
ment Y in compound (I-3). Isopropylmalonic ester
(5.05 g, 25 mmol) [20] was added to the 55% suspen-
sion of NaH (1.1 g) in anhydrous dimetgylformamide
(30 ml) at room temperature on stirring. The reaction
mixture was stirred for 1 h, and 2-benzyloxyethyl bro-
mide (5.38 g, 25 mmol) [21] was added. The reaction
mixture was stirred for 6 h at 75°C and 12 h at 50°C,
cooled, and poured out into water (150 ml). The pH
value was brought out to 7, and the precipitate was
extracted with ethyl acetate (4 × 40 ml). The joined
extracts was washed and dried over Na2SO4 and evapo-
rated. The yield of diethyl ester of 2-benzyloxy- carried out on a System Gold chromatograph (Beck-
man, Sweden) equipped with an Ultrasphere ODS col-
ethyl(isopropyl)malonic acid was 6.30 g (75%);
1H NMR: 0.95 (6 H, d, J 6.8, CHCH3), 1.18 (6 H, t, J
6.5, CH2CH3), 2.19 (2 H, t, J 6.5, OCH2CH2), 2.30 (H,
m, CH), 3.50 (2 H, t, J 6.0, OCH2CH2), 4.10 (4 H, q, J
6.5, CH2CH3), 4.41 (2 H, s, CH2Ph), 7.18–7.25 (5 H,
m, Ph).
umn (7 µm, 25 × 2.0 cm) in a gradient of acetonitrile
(from 10 to 90%) in 0.23 M (NH ) NaPO (pH 6.1) at
4 2
4
a flow rate of 6 ml/min at 220 nm. A fraction containing
a target product was evaporated, and another gel filtra-
tion on the column with Sephadex LH-20 in 80% meth-
anol was performed.
2 M NaOH (20 ml) was added to the solution of
diethyl ester of 2-benzyloxyethyl(isopropyl)malonic
acid (5.38, 16 mmol) in ethanol (100 ml). The reaction
mixture was boiled for 8 h, cooled, acidified to pH 3,
and evaporated to dryness. 2-Benzyloxyethyl(isoporo-
pyl)malonic acid was extracted from the residue with
ether, ether was evaporated, and the acid was decarbox-
ylated by heating for 1 h at 180°C. The product was dis-
tilled in a vacuum. 4-Benzyloxy-2-isopropylbutanoic
acid was obtained as colorless oil in yield of 2.64 g
(70%); mp 116–120°ë/0.1 mm of Hg column; 1H NMR:
0.92 (6 H, d, J 6.8, ëç3), 1.90 (2 H, m, ëçëç2), 2.16
(H, m, CHCOOH), 2.32 (H, m, ëç(ëç3)2), 3.43 (2 H,
m, éëç2ëç2), 4.44 (2 H, s, CH2Ph), 7.18–7.25 (5 H,
m, Ph), 10.0 (H, s, COOH).
Analytical HPLC was carried out on a System Gold
chromatograph (Beckman, Sweden) equipped with an
Ultrasphere ODS column (5 µm, 250 × 2.0mm) in the
same gradient regime at a flow rate of 0.25 ml/min at
220 nm.
Purity of the synthesized peptides was 95% accord-
ing to the analytical HPLC. The peptides were charac-
terized by mass spectrometry on a MALDI-TOF Vision
2000 mass spectrometer (United States). The deter-
mined molecular masses were: 760 Da for (I-1), 744 Da
for (I-2), 804 for (I-3), 702 for (I-4), 776 for (I-5), and
1103 for the substrate.
Expression and isolation of PlmII. Gene for the
target protein encoding the short form of pro-PlmII (the
76-453 fragment according to the numeration of the
full-size zymogen) were earlier cloned in the pET-
23a(+) vector (Invitrogen, United States). Cells of the
BL21 (DE3) strain were transformed, grown at 37°C
for a night in the presence of 100 mg/ml of ampicillin,
and reseeded into preliminary agarized medium to
achieve the 100-fold dilution of the night culture. The
culture was induced at the suspension density of 0.5 OU600
by the addition of 1 mM of isopropyl-β-D-thiogalactopy-
ranoside and incubated in a regime of intensive stirring
for 3 h under the same temperature. The cell biomass
(10 g from 5 l of the culture) was separated by centrif-
ugation and used for protein isolation.
Found, %: C 71.00, H 8.50. Calcd. for C14H20O3, %:
C 71.16,H 8.53.
Synthesis of peptide inhibitors (I-1)–(I-5) and the
H-Leu-Glu-Arg-Ile-Phe-Phe(NO2)-Ser-Phe-OH sub-
strate (CS) was performed by the solid phase method
according to the Boc-strategy on a modernized Beck-
man 990 synthesizer (United States). Reagents and
amino acid derivatives were purchased from Reanal
(Hungary), PRF (Japan), Advanced Chem. Tech., Ald-
rich (United States), and Fluka (Switzerland). The start-
ing polymeric carrier of the peptide synthesis was pre-
pared by attachment of Boc-Sta-OH (Neosystem,
France) to the chlormethylated polymer (Advanced
The cell paste (5 g) was resuspended in 20 ml of
Chem. Tech., United States) according to the standard buffer A (50 mM Tris-HCl, pH 8.0, o.1 M NaCl, 2 mM
procedure via cesium salt of Boc-Sta-OH [22]. The EDTA) treated with ultrasound, diluted with the cooled
amino acid content on the polymer was 0.25 mmol/g. buffer A in 6 times, and centrifuged for 1 h at 100000 g.
Condensations were carried out by DCC in the pres- Insoluble fraction of lysate that contained the target
ence of HOBu (1 : 1) with 10-min preactivation at 0°C. protein was resuspended in 60 ml of buffer B (50 mM
The reagents were taken in 3.5-fold excess. Temporary Tris-HCl, pH 8.0, 1% Triton X-100). The suspension
Boc-group was removed by the treatment with 30% was stirred for 30 min and centrifuged at 100000 g for
solution of trifluoroacetic acid in chloroform. The pep- 20 min. The supernatant was removed, and the residue
tides were cleaved from the resin and deprotected by was washed by centrifugation with buffer B (120 ml)
the treatment with liquid HF in the presence of p-cresol without the detergent two times. Incorporation corpus-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

666
RUMSH et al.
cles were dissolved in 20 ml of the denaturing buffer
(6 M urea, 0.5 M Tris-HCl, pH 8.0, 1 mM EDTA,
50 mM β-mercaptoethanol), stirred for a night, and
centrifuged at 100000 g for 30 min. The supernatant
was diluted with buffer C (50 mM Tris-HCl, pH 8.5) in
50 times by dropping through a peristaltic pump at a
flow rate of 0.5 ml/min. The protein refolding was per-
formed for 16 h (25°ë) at slow stirring of the solution.
The solution of the enzyme after the refolding (1 l) was
filtered through a membrane (pore size of 0.45 µm),
concentrated in 20 times on a cellulose membrane that
retained proteins with molecular masses higher than
10 kDa, and used for anion-exchange chromatography.
The protein solution was applied onto Q-Sepharose
(10 ml) equilibrated with buffer C (pH 8.0) at a flow
rate of 1 ml/min. The column was washed with ten vol-
umes of the same buffer and eluted with a linear gradi-
ent of NaCl (from 0 to 1 M) in buffer C (pH 8.0) (ten
volumes of the column) at the same flow rate. Fraction
of 2 ml were collected and analyzed by electrophoresis.
Fractions containing the target protein were joined and
concentrated to the volume of 2 ml usingAmicon Ultra-
15 centrifuge concentrators.
'
(KS) were calculated by equation (1). The inhibition
constants were determined form the Henderson equa-
tion (2) with the corrective of the competitive character
of the inhibition in the case of CatD and from the Hend-
erson equation modified by us with the corrective of the
substrate inhibition (5). The standard deviation was not
higher than 20% in all the cases.
ACKNOWLEDGMENTS
We thank Dr. Richard Weller, Richard Ornstein,
James Morris, and Evguenia Rainina from the Pacific
Northwest National Laboratory for cooperation and
active support of this project and Prof. D. Goldberg. We
are grateful to the Howard Hughes Medical Institute for
the expression plasmid for preparation of plasmepsin
II. This study was supported by the U.S. Department of
Energy, contract no. 312665-A-G2 and by the State
Contract of Rosnauka, project no. 02.512.11.2007
(February 19, 2007).
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