Mechanism-based inhibitors of the aspartyl protease plasmepsin II as potential antimalarial agents

Article abstract of DOI:10.1021/jm100233b

Four aspartyl proteases known as plasmepsins are involved in the degradation of hemoglobin by Plasmodium falciparum, which causes a large percentage of malaria deaths. The enzyme plasmepsin II (Plm-II) is the most extensively studied of these aspartyl proteases and catalyzes the initial step in the breakdown of hemoglobin by the parasite. Several groups have reported the design, synthesis, and evaluation of reversible peptidomimetic inhibitors of Plm II as potential antimalarial agents. We now report four peptidomimetic analogues, compounds 6-9, which are rationally designed to act as mechanism-based inhibitors of Plm II. Three of these analogues produce potent irreversible inactivation of the enzyme with IC₅₀ values in the low nanomolar range. Of these three compounds, two retain the low micromolar IC ₅₀ values of the parent compound in Plasmodium falciparum (clone 3D7) infected erythrocytes. These analogues are the first examples of fully characterized mechanism-based inactivators for an aspartyl protease and show promise as novel antimalarial agents.

Full text of DOI:10.1021/jm100233b

4234 J. Med. Chem. 2010, 53, 4234–4247
DOI: 10.1021/jm100233b
Mechanism-Based Inhibitors of the Aspartyl Protease Plasmepsin II as Potential Antimalarial Agents
Deepak Gupta,†,§ Ravikiran S. Yedidi,‡, Sheeba Varghese,†,^ Ladislau C. Kovari,‡ and Patrick M. Woster*,†
†Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 3132
Applebaum Hall, 259 Mack Avenue, Detroit, Michigan 48202, and ‡Department of Biochemistry and Molecular Biology, School of Medicine,
Wayne State University, Detroit, Michigan 48202. §Current affiliation: LECOM-School of Pharmacy, Room No. 280, 5000 Lakewood Ranch
Boulevard, Bradenton, Florida 34202. Current affiliation: ERS/HAMB-NCI/NIH, 10 Center Drive, Room No. 5A24, Bethesda, Maryland
20892. ^Current affiliation: Feik School of Pharmacy, University of the Incarnate Word, 4301 Broadway, Box 99, San Antonio, Texas 78209.
Received February 22, 2010
Four aspartyl proteases known as plasmepsins are involved in the degradation of hemoglobin by
Plasmodium falciparum, which causes a large percentage of malaria deaths. The enzyme plasmepsin II
(Plm II) is the most extensively studied of these aspartyl proteases and catalyzes the initial step in the
breakdown of hemoglobin by the parasite. Several groups have reported the design, synthesis, and
evaluation of reversible peptidomimetic inhibitors of Plm II as potential antimalarial agents. We now
report four peptidomimetic analogues, compounds 6-9, which are rationally designed to act as
mechanism-based inhibitors of Plm II. Three of these analogues produce potent irreversible inactivation
of the enzyme with IC50 values in the low nanomolar range. Of these three compounds, two retain the
low micromolar IC50 values of the parent compound in Plasmodium falciparum (clone 3D7) infected
erythrocytes. These analogues are the first examples of fully characterized mechanism-based inactiva-
tors for an aspartyl protease and show promise as novel antimalarial agents.
Introduction
Examination of differences in the regulation of gene pro-
ducts between host, vector, and parasite has resulted in
identification of target sites or alterations in biochemical
regulation, and a number of new targets for drug design have
emerged.29-32 The aspartyl proteases plasmepsin I (Plm Ia)
andplasmepsin II (Plm II) are hemoglobin-degrading aspartyl
proteases that have received considerable attention as promis-
ing targets for the design of antimalarial agents.33-35 Four
aspartic proteases, Plm I, Plm II, plasmepsin IV (PlmIV), and
histoaspartyl protease (HAP) are found in the food vacuole of
the parasite and efficiently degrade 75% of host hemoglobin
(Figure1). Plm I and Plm IImakethefirst strategic cleavageof
hemoglobin between Phe33 and Leu34 of the R-chain, result-
ing in protein unfolding and release of the heme moiety.36-39
Subsequent degradation steps are catalyzed by the cysteine
protease falcipain, the metalloprotease falcilysin, and cyto-
plasmic aminopeptidases (Figure 1).
Malaria remains one of the world’s most deadly diseases,
threatening 40% of the world’s population and infecting 300
million people worldwide. In Africa alone, more than one
million children under the age of 5 die from malaria each
year,1-3 translating to one death from malaria every 30 s.
Human infection can be caused by four distinct species of the
protozoon Plasmodium, but P. vivax and P. falciparum account
for more than 95% of malaria cases.4,5 Nearly all deaths caused
by malaria are due to infection by P. falciparum.3,4 Malaria has
become more difficult to treat because of an increase in multi-
drug resistant strains.5 In fact, the re-emergence of malaria as a
worldwide epidemic can be largely attributed to the rapid
development of parasite resistance. Some progress has been
made in identifying resistance markers in parasites that fail
conventional therapy.6-8 The disease is transmitted by the bite
of the female anopheles mosquito, at which time sporozoites of
P. falciparum are discharged into the puncture wound and are
carried to the liver, where they enter hepatic mesenchymal cells.
Lysis of the hepatocyte then releases the merozoite form of
P. falciparum, which invades host red blood cells, feeding on
hemoglobin during the erythrocytic portion of its life cycle.9
Although P. falciparum has a complex biochemistry and life
cycle, recent research has produced a number of potential new
therapies.4,10 Current drug therapies for malaria include qui-
nolines related to chloroquine,11-13 artemisinins and related
dispirotetraoxanes,14-23 and miscellaneous agents such as
febrifugine analogues24 and atovaquone and proguanil.25,26
The treatment of malaria has recently been reviewed.27,28
Plm II is the most thoroughly studied enzyme among the
aspartyl proteases of Plasmodium. It is made up of 329 amino
acids folded into two topologically similar N- and C-terminal
domains. The binding cleft contains Asp34 and Asp214,
which together represent the catalytic dyad.40 The catalytic
mechanism of plasmepsin II is shown in Figure 2.41,42 The
a Abbreviations: Plm I, plasmepsin I; Plm II, plasmepsin II; Plm IV,
plasmepsin IV; HAP, histoaspartyl protease; API, aspartyl protease
inhibitors; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBT,
1-hydroxybenzotriazole; SDS-PAGE, sodium dodecyl sulfate polyacry-
lamide gel electrophoreisis; FRET, fluorescence resonance energy transfer;
EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; DABCYL,
4-(dimethylaminoazo)benzene-4-carboxylic acid; hCatD, human cathepsin
D; CHO, Chinese hamster ovary; SOC, super optimal broth with catabolite
repression; IPTG, isopropyl β-D-1-thiogalactopyranoside; TB, terrific
broth; LB, lysogeny broth; DMEM, Dulbecco’s modified Eagle’s medium;
MES, 2-(N-morpholino)ethanesulfonic acid.
*To whom correspondence should be addressed. Phone: 313-577-
1523. Fax: 313-577-2033. E-mail: pwoster@wayne.edu.
r
pubs.acs.org/jmc
Published on Web 05/03/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4235
Figure 2. Catalytic mechanism for Plm II-mediated hydrolysis of
hemoglobin.
Figure 1. Degradation of hemoglobin by plasmepsins and asso-
ciated proteases.
enzyme-activated inactivators for any aspartyl protease have
been fully characterized (one report alludes to mechanism-based
inhibitors of cathepsin D, but these analogues were never
characterized69). It has been demonstrated that transition state
analogues containing an (S)-hydroxyl group displace the
enzyme-bound water molecule from the Plm II active site and
hydrogen-bond to the catalytic Asp-32 and Asp-214 groups.70,71
It follows that a peptidomimetic analogue containing an (S)-
hydroxyl substituent core and bearing a latent electrophile
should act as an enzyme-activated, irreversible inhibitor of
Plm II. We chose compound 2 and the close homologue
TIT1330 (5, Figure 3, Ki = 0.5 nM)72,73 as a starting point for
the design of related analogues containing latent electrophilic
groups. Putative Plm II inactivators 6-9 and their proposed
mechanism of inactivation are shown in Figure 4. We reasoned
that 6-9 would bind to Plm II and coordinate to the catalytic
dyad, as described above, followed by activation of the latent
electrophilic species.
During activation, we hypothesized that Asp-214 or an
adjacent basic amino acid could abstract an acidic proton
from the latent electrophile, resulting in the generation of an
R,β-unsaturated Michael acceptor (6), a ketenimine (7), or an
allene (8 and 9) in the active site (Figure 4). β-Chloroalanine
has been used as a mechanism-based inhibitor for pyridoxal
phosphate-containing enzymes,74 and the chloromethyl moi-
ety has been shown to be activated by elimination of HCl both
enzymatically74 and chemically.75-77 Activation by this route
is further supported by the observation that we were unable to
isolate the β-fluoro derivative analogous to 6 because of rapid
elimination to the corresponding dehydroalanine (see below).
The propargyl78-80 and cyanomethyl81,82 moieties have been
used in the design of mechanism-based inhibitors for various
enzymes, although these groups are generally directly ap-
pended to a nitrogen or sulfur. These moieties have been
shown to be activated by enzymes to the corresponding allene
or ketenimine, respectively. In the case of 9, which contains an
N-propargyl latent electrophile, this is very likely to occur.
However, because the cyanomethyl and propargyl groups of
7 and 8, respectively, are one carbon removed from an amide
nitrogen (thus raising the pKa of the methylene protons), this
issomewhat lesslikely, althoughneighboring groupassistance
from the carbonyl oxygen could facilitate this reaction.
A second less likely possibility for the activation of 7 and 8
is the intramolecular formation of a five-membered ring83
(Figure 4) that could form a covalent bond with an enzyme-
bound nucleophile via ring-opening. Following the activation
Asp34 and Asp214 residues coordinate a water molecule that,
following abstraction of a proton by Asp214, attacks the
Phe33-Leu34 peptide bond. Hydrolysis of the C-N bond
in the transition state then affords two peptides and regener-
ates the aspartates in the catalytic dyad. As products leave the
active site, Asp34 and Asp214 again coordinate a water
molecule.
There are numerous examples of aspartyl protease inhibi-
tors (APIs), such as the HIV protease inhibitors saquinavir,
ritonavir, indinavir, nelfinavir, lopinavir, and amprenavir,43-48
that have become effective therapeutic agents, and validated
aspartyl protease drug targets include renin, cathepsin D, HIV
protease, and β-secretase. A key structural feature in APIs
is the presence of a hydroxyl or hydroxyl-like moiety that
coordinates to the catalytic dyad, as well as a nonhydrolyzable
peptide bond mimic. A number of APIs, including the general
aspartyl protease inhibitor pepstatin A, contain a statin core,
which serves as a transition state mimic for pepide bond
hydrolysis.49-54 Other transition state analogue cores have
been developed for use in constructing APIs,53,55-57 the most
important of which is the hydroxyethylamine core that has
been used extensively in HIV protease inhibitors.58 Pepstatin A
coordinates with Asp-34 and Asp-214 in the active site of Plm II
0
0
(Figure 3) and contains P1, P2, P3, P1 and P2 side chain
residues that complement the corresponding binding pockets
S1, S2, etc. Thus, pepstatin A has served as lead compound in
the design of various APIs,59 and pepstatin A analogues such as
2-5 (Figure 3) represent reversible inhibitors that are selective
for the plasmepsins. The phenylalanine-statin analogue 2 has
potent inhibitory activity (Ki = 0.56 nM) with 38-fold selec-
tivity over cathepsin D.40 It also exhibits activity in cell culture
assays, inhibiting growth of cultured P. falciparum by 54% at
20 μM. The hydroxyethylene analogue 3 exhibits a Ki of
357 nM against Plm II and 10-fold selectivity over cathepsin D,
while hydroxyethylamine analogue 4 exhibits an IC50 value of
121 nM against Plm II and completely suppresses parasite
growth at 5 μM, with an ED50 of 1.6 μM, in an infected
erythrocyte assay.60,61
All of the reversible Plm II inhibitors described above under-
go noncovalent interactions with the enzyme. Mechanism-
based or enzyme-activated inhibitors have been reported for
various peptidases,62-64 including a number of serine and
cysteine proteases.65,66 However, reports of irreversible inhibi-
tors of aspartyl proteases are rare,67,68 andnomechanism-based,

4236 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Gupta et al.
Figure 3. Representation of pepstatin 1 bound in the active site of Plm II, and the structures of Plm II inhibitors 2-5.
Figure 4. Structures and proposed enzyme-mediated activation of compounds 6-9.
of 6-9 as described above, inactivation of the enzyme would
then occur when a nucleophilic amino acid residue on the
protein formed a covalent bond, forming an irreversible
enzyme-inhibitor complex. The parasite must then synthe-
size new enzyme over an extended period, preventing the
degradation of hemoglobin and effectively starving the para-
site. Such analogues would have a longer duration of action
when compared to reversible inhibitors. Ideally, these agents
will be specific for the parasitic target enzyme and thus produce
less toxicity in the host cell.84 The proposed analogues, as well

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4237
Scheme 1
as the methods used to synthesize and evaluate them as Plm II
inhibitors, appear in the following sections.
plasmid/cell ratio of 1:20 was used in transfection, according
to the standard protocol.97 Cells were grown overnight in TB
media (Terrific Broth, Qbiogene, CA) containing 0.1 mg/mL
ampicillin. Induction conditions were optimized in terms of
temperature (20 ꢀC/37 ꢀC), media (LB/TB), and IPTG (iso-
propyl 1-thio-β-D-galactopyranoside, 100-1000 μM). The
transfected cells were lysed by passing through a French press
followed by sonicating twice for 45 s. Proteins were harvested
by centrifugation at 8000g, and several rounds of suspension
and centrifugation in a range of buffers were necessary to
isolate the pure protein fraction, as outlined in the published
protocol. For each buffer, the supernatant was collected and
analyzed by SDS-PAGE electrophoriesis. Inclusion bodies
in the supernatant containing Plm II were solublized and
purified by FPLC (heparin FF and Q-Seph columns), and
the pure protein fraction was collected and concentrated. The
m-Plm II protein was allowed to refold (as verified by non-
denaturing gels) and was activated by stirring in sodium
citrate buffer, pH 4.7, at a final concentration of 0.1 M. Final
purification of m-Plm II was accomplished on a Supradex 100
column, followed by SDS-PAGE analysis. The final purified
protein was concentrated and stored at 4 ꢀC in buffer (20 mM
Tris-HCl, pH 8.5, 0.25 M NaCl).
Enzyme assays and time dependent kinetics for target
compounds 6-9 were conducted using a high-throughput
FRET-based fluorimetric assay.60,95,98-100 Analogues were
evaluated on the basis of hydrolysis kinetics.99 Each reaction
was initiated by the addition of the substrate DABCYL-Glu-
Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS, which mimics the
Hb-R Phe33-Leu34 initial cleavage site.101 The fluorophore
EDANS is quenched by DABCYL in the intact peptide and
expressed following enzymatic cleavage. Assays were per-
formed in NaOAc buffer, pH 5.0, at 37 ꢀC at a final concen-
tration of 2.5 nM enzyme and 1 μM substrate. Inhibitor,
enzyme, and substrate were incubated for 30 min at 37 ꢀC, and
fluorescence was measured on a photoluminescence fluori-
meter(Perkin-Elmer LS-55, excitationwavelength336 nm, slit
width 5 nM; emission wavelength 490 nm, slit width 10 nM).
The percentage of inhibition for each sample was calculated on
Chemistry
The synthesis of target analogues 6-9is outlined in Scheme 1.
N-Boc-(S)-phenylalanal 10 (synthesized in two steps from (S)-
phenylalanine methyl ester)85 was reacted with ethyl acetate
(LDA, dry EtOAc/THF) to form the statin precursor 11 as a
mixture of the (3S,4S)- and (3R,4S)-diastereomers, and the
N-Boc protecting group was removed (3.0 M HCl) to afford 12
as a mixture of diastereomers. The diastereomers were separated
by flash chromatography to afford the pure diastereomers,
and their absolute stereochemistry was verified by NMR and
polarimetry.86-88 The synthesis was continued with (3S,4S)-
12, which was coupled to intermediate 13 under peptide
coupling conditions (EDC, HOBT) to afford the ethyl ester
14a, followed by ester hydrolysis (1.0 N LiOH) to yield
14b.89,90 Dipeptides 15, 16, and 17 were synthesized by coupl-
ing (S)-leucinamide with the commercially available (S)-β-
chloroalanine, (S)-β-cyanoalanine, or (S)-R-propargylglycine
using the method described above. Attempts to synthesize the
(S)-β-fluoroalanine-containing dipeptide failed because of
rapid elimination to the dehydroalanine derivative during
the reaction. Subsequently, compound 14b was coupled to
synthon 15, 16, or 17 to form the desired target molecule 6, 7,
or 8, respectively. Likewise, coupling of compound 14b to
secondary amine 18 afforded target molecule 9.88,89,91,92
Enzyme Kinetic Studies
Plm II expression and purification was accomplished using
published protocols.93-95 An expression construct encoding
the pro form of Plm II was obtained (Dr. Daniel Goldberg,
Washington University, St. Louis, MO), verified by DNA
sequencing, and used to generate mature Plm II (m-Plm II).
The m-Plm II pro form contains an Arg238 codon in place of
Lys238,96 a point mutation that does not affect the kinetic
behavior of the enzyme. Escherichia coli BL21(DE3) was used
to express the pET-3d expression construct of m-Plm II, and a

4238 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Gupta et al.
Figure 5. Inhibition of Plm II by compounds 6-9 at 1.0 μM. Each
data point is the average of three determinations that differed by<5%
in each case.
the basis of the equation % inhibition = 100 ꢀ {1- [(fluorescence
at [I])/(fluorescence of control)]}. Compounds were initially
tested at 1 μM, and active compounds were subjected to a
dose-response analysis to find exact IC50 values. Compounds
6, 8, and 9 completely inhibited Plm II at 1.0 μM, while
compound 7 produced 88.2% inhibition (Figure 5). All four
analogues produced inhibition comparable to pepstatin at
the same concentration (82.8% inhibition). Dose-response
curves were then generated for 6-9 (Figure 6) by assuming
competitive inhibition, and IC50 values were determined using
Kaleidagraph 3.6 (Synergy Software) (compound 6, 28 nM;
compound 7, 141 nM; compound 8, 333 nM; compound 9,
22 nM). Compounds 6 and 8 were selected for further analysis
and were subjected to time-dependence studies.102,103 The
reduction of enzyme activity was measured over a 30 min
period at 0, 25, 50, 75, and 100 nM inhibitor (Figure 7). A kobs
value was derived from each line, and the data were replotted
according to the method of Kitz and Wilson,104 revealing that
compound 6 has a Ki value of 35.7 nM and a kinact of 0.025 per
min for Plm II. A partition ratio of 84 and a turnover number
of 85 were calculated from the corresponding plot of %
activity remaining vs [I]/[E]. A similar analysis for compound
8 (Figure 7) revealed the following kinetic constants: Ki =
333.3 nM; kinact = 0.191 per min; partition ratio 98; turnover
number 99. Interestingly, although compounds 7 and 9 were
both potent inhibitors of Plm II, and both featured potentially
activatable electrophiles, neither exhibited time-dependent,
pseudo-first-order inhibition kinetics (data not shown).
Dialysis experiments were performed to determine
whether compounds 6-9 were irreversibly bound to the
enzyme or whether dialysis resulted in regeneration of enzyme
activity.102,103,105 For each assay, a fixed amount of enzyme
and inhibitor in 500 μL of buffer was incubated at 37 ꢀC for
1-2 h, and residual activity was measured using a 50 μL
aliquot. The remaining portion was dialyzed overnight at
room temperature in sodium acetate buffer at pH 5.0 contain-
ing 10% glycerol and 0.01% Tween-20. Compounds 6-9
(1 μM) reduced enzyme activity by 76.9-93.2% under the
assay conditions described above, while pepstatin produced a
97.8% inhibition at the same concentration. Following dia-
lysis, enzyme activity was restored to 85.2% and 88.0% for
pepstatin and 7, respectively, and to 36.1% for compound 9,
compared to control However, there was no significant
Figure 6. Dose-response curves for the inhibition of PlmII by
6-9. Each data point is the result of three determinations that in
each case differed by 5% or less.
restoration of activity in the cases of 6 and 8 (Figure 8),
suggesting that 6 and 8 were covalently bound to the enzyme.
These data are consistent with the time-dependent loss of
activity observed following treatment with 6 and 8 but not
with 7 and 9. A substrate protection experiment was then
employed to demonstrate that 6 and 8 are active-site directed
inactivators, as shown in Figure 9. As the ratio of inhibitor
to substrate concentration increased, the inactivation of Plm
II also increased in a roughly linear fashion, suggesting that
the observed time-dependent inactivation is mediated at the
active site.
Selectivity and in Vitro Antimalarial Activity
The selectivity of compounds 6-9 for Plm II was deter-
mined by evaluating these analogues as inhibitors of human
cathepsin D (hCatD).60,95,98 The results of these studies are
shown in Figure 10. The IC50 values for 6-9 against hCatD
were determined by assessing percent activity remaining over
a range of concentrations (50-2000 nM) using the assay
described above (data not shown). The hCatD IC50 values
for compounds6-9 were then comparedtothe corresponding
values for Plm II as determined above. Compound 8 did not
exhibit any selectivity for hCatD of Plm II. However, com-
pounds 6, 7, and 9 were found to possess 3.3-, 1.4-, and 10.6-
fold selectivity for Plm II. The cytotoxicity of the target
molecules in mammalian cells was measured in cultured
Chinese hamster ovary (CHO) cells at an inhibitor concentra-
tion of 10 μM.106 As seen in Figure 11, none of the target
analogues 6-9 produced significant toxicity in the CHO cell
line compared to control or DMSO-treated cells. Compound
6 was further tested at 100 and 250 μg/mL and was found to be
nontoxic even at these elevated concentrations. These data
suggest that compounds 6-9 are nontoxic to uninfected
mammalian cells.
The antiparasitic activity of 6, 8, and 9 was determined in an
infected erythrocyte model conducted as previously
described.107,108 A culture of Plasmodium falciparum (clone

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4239
Figure 7. Time-dependent inactivation of Plm II by 6 and 8 and the corresponding Kitz-Wilson replots. Each data point in the time-
dependence assay is the average of three determinations that differed by 5% or less in all cases.
Figure 9. Substrate protection of Plm II from inactivation by 6 and 8.
Plm II was incubated with increasing [I]/[S] at constant [E] for 2 h
before measuring activity. Each data point is the average of three
determinations that differed by 5% or less in all cases.
Figure 8. Plm II activity following treatment with 1 μM pepstatin
or 6-9 before and after dialysis. Each data point is the average of
three determinations that differed by 5% or less in all cases.
Figure 12. Compound 6 had unremarkable activity, while
compounds 8 and 9 exhibited IC50 values of 7.7 and 9.2 μM,
respectively.
3D7) at 2% parasitemia in red blood cells was incubated with
various concentrations of inhibitor. This treatment was fol-
lowed by addition of 1 μCi (17.2 Ci/mmol) of [3H]hypoxan-
thine, a nucleic acid precursor required for parasite growth.
Parasites were harvested, and the cells were isolated by
filtration. The amount of radioactive hypoxanthine taken
up by the cells was determined by scintillation counting of
the dry filters. Parasitemia in the cultures is directly propor-
tional to hypoxanthine incorporation. The IC50 value for each
of the three inhibitors was calculated from the plot shown in
In preliminary molecular modeling studies, the binding of
compound 6 Plm II was simulated using the MOE-Dock
2004.03 software docking program. The X-ray coordinates
for compound 5 bound to PlmII were obtained from the
Protein Data Bank (1W6H.pdb).73 The structure of the ligand
was altered to produce a model of ligand 6, and the enzyme-
inhibitor complex was reminimized. Conformational search-
ing was conducted using simulated annealing (a global opti-
mization technique based on the Monte Carlo method) and

4240 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Gupta et al.
Figure 10. Selectivity between hCatD and Plm II based on IC50
values for 6-9.
Figure 12. Dose-response curves for compounds 6, 8, and 9
against erythrocytes infected with 3D7 P. falciparum. Each data
point is the average of three determinations that differed by 5% or
less in all cases.
Compounds 6 and 8 (Ki of 35.7 and 333.3 nM, respectively)
were significantly less potent inhibitors of Plm II than the
parent compounds 2 (Ki = 0.56 nM) and 5 (Ki = 0.5 nM), but
their Ki values were still found to be in the nanomolar range.
Compounds 6 and 8 also possessed reasonable partition ratio
values (84 and 98, respectively), indicating they were modestly
efficient inactivators. Unfortunately, addition of the latent
electrophilic group to 2 and 5 resulted in a significant loss of
hCadD/Plm II specificity. Of the analogues where the electro-
phile was placed adjacent to the statin core, only compound 6
showed a modest preference for Plm II (3.3-fold preference),
compared to a 38-fold selectivity for compound 2.40 Interest-
ingly, the compound with the greatest selectivity for Plm II, 9,
exhibited a 10.6-fold selectivity for Plm II, despite the fact that
the propargyl latent electrophile was located in a terminal posi-
Figure 11. Cytotoxicity of compounds 6-9 in cultured CHO cells.
Each data point is the average of three determinations that differed
by 5% or less in all cases.
the molecular mechanics force field MMFF94. A total of
25 runs were completed, with 6 cycles per run and an 8000
iteration limit, and a 1000 K temperature was maintained
during the first cycle. During energy minimization, the chir-
ality of 6 was constrained, and a proximity radius of 7.5 was
set. As shown in Figure 13, the (S)-hydroxyl group is
complexed to both Asp34 and Asp214 at distances of 2.4
˚
and 2.6 A, respectively. In this orientation, there are a number
of nucleophilic residues in proximity to the latent electrophilic
chloromethyl moiety present in 6 that could react with an
˚
enzyme-generated Michael acceptor, including Tyr77 (3.7 A),
0
tion at P2 rather than adjacent to the transition state core. This
˚
˚
˚
Ser37 (3.5 A), Thr217 (3.7 A), and Tyr192 (3.4 A). The specific
amino acid residues involved in the covalent binding of 6 to
Plm II will be determined by X-ray crystallographic studies
and reported in a subsequent publication.
value is comparable to the 10-fold selectivity observed for 3.60
As was mentioned above, compound 6 was a poor inhibitor
of parasitic growth in vitro, while compounds 8 and 9
exhibited IC50 values of 7.7 and 9.2 μM, respectively, in the
infected erythrocyte assay. These values are in the same range
as the IC50 values of 5-20 μM for parent compounds 2-5.
These dataimply thatpeptidomimetic inhibitors of Plm IIthat
contain activatable groups retain their in vitro activity against
Plasmodium, and this observation encourages the synthesis
of additional analogues in an effort to improve selectivity
between hCatD and Plm II. Recent studies involving a Plm
knockout mutant Plasmodium strain demonstrate that the
parasite produces redundant proteolytic enzymes to ensure
an adequate supply of amino acids, and this suggests that
additional targets may be involved in the antimalarial activity
of peptidomimetics designed to target Plm II.109 Studies in-
volving previously reported reversible Plm II inhibitors with
antimalarial activity suggest that these inhibitors do appear to
act on multiple targets.110 Because Plm II inhibitors 8 and 9
retain the in vitro antimalarial activity observed with rever-
sible Plm II inhibitors, it is reasonable to assume that 8 and 9
Discussion
Taken together, the results of the time dependence, dialysis,
and substrate protection studies strongly suggest that com-
pounds 6 and 8 represent the first examples of mechanism-
based, enzyme-activated inactivators for any aspartyl pro-
tease. A similar case could be made for compound 9, since
extensive dialysis resulted in only a 30% increase in Plm II
activity following inhibition. However, kinetic analysis did
not reveal why compound 7 did not appear to produce
inactivation (as evidenced by a nearly complete return of
enzyme activity following dialysis). This behavior could poten-
tially be attributed to significantly slower activation of the
cyanomethylene latent electrophile by Plm II. Until a crystal
structure of the inhibitor/Plm II adducts can be solved, we will
not be sure of the mechanism of activation of 6, 8, and 9, and
these experiments are now being attempted.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4241
Figure 13. Representation of the transition state mimic hydroxyl of compound 6 coordinated to Asp34 (left) and Asp214 (right) in the active
site of Plm II.4.
may also interact with multiple targets. This possibility will be
explored as part of our ongoing antimalarial research efforts.
In addition, the synthesis of additional compounds in this
seriesandcrystallography studies todeterminethe mechanism
of activation of 6 and 8 and the specific amino acid residues
involved in covalent bond formation are ongoing concerns in
our laboratory.
determined to be 95% pure or greater by HPLC chromatography
using an Agilent series 1100 high-performance liquid chromato-
graph fitted with a C18 reversed-phase column.
(3S,4S)-Ethyl 4-(tert-Butoxycarbonylamino)-3-hydroxy-5-
phenylpentanoate ((3S,4S)-11). A solution of 2.46 g of lithium
diisopropylamide (0.023 mol, 11.5 mL of a 2.0 M solution in
THF/hexane/ethylbenzene) was cooled to -78 ꢀC, and 2.02 g of
anhydrous ethyl acetate (0.023 mol, 2.25 mL) in 10 mL of
anhydrous THF was added via syringe. The mixture was allowed
to stir for 15 min, after which a 2.26 g portion of 10 (0.009 mol) in
20mLofTHF was slowlyadded. The mixture was thenstirred for
10 min, warmed to 0 ꢀC, and acidified to pH 3 with 1 N HCl. The
resulting two-phase mixture was washed with three 50 mL
portions of ethyl acetate, and the organic layer was washed with
brine and dried over anhydrous magnesium sulfate. The mixture
was filtered and the solvent removed in vacuo to yield crude 11 as
a mixture of the (3S,4S)- and (3R,4S)-diastereomers. The dia-
stereomers were then separated by chromatography on silica gel,
eluting with 9:1 benzene/ethyl acetate. The desired (3S,4S)-
diastereomer of 11 was isolated as a light-yellow solid (0.576 g,
19.0%). 1H NMR (400 MHz, CDCl3) δ 1.24 (t, J = 6.8 Hz, 3H),
1.35 (s, 9H), 2.34-2.39 (m, 1H), 2.55-2.62 (m, 1H), 2.91 (d, J =
7.6 Hz, 2H), 3.47 (d, J = 2.4 Hz, 1H), 3.73 (q, J = 8.4 Hz, 1H),
3.97-3.99 (m, 1H), 4.13 (q, J = 6.8 Hz, 2H), 4.93 (d, J = 9.6 Hz,
1H), 7.19-7.36 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 14.4,
25.6, 28.5, 36.0, 38.4, 45.0, 50.9, 52.5, 61.0, 61.7, 70.4, 74.4, 86.3,
126.7, 128.7, 161.8, 167.1, 172.3, 173.4. IR (KBr, cm-1) 3352.5,
2981.9, 2936.2, 1733.2, 1681.9, 1525.1, 1368.1, 1314.6, 1168.1,
1031.1. (M þ H)þ 283.2. Mp 87 ꢀC. [R]2D0 -36.3 (c 1.0, MeOH). A
1.19 g portion of the corresponding (3R,4S)-diastereomer(39.2%)
Experimental Section
General Methods. All reagents were purchased from Sigma/
Aldrich Chemical (Milwaukee, WI), Acros Chemical (Chicago, IL),
or Bachem (Torrence, CA) and were used without further
purification except as noted below. Triethylamine was distilled
from potassium hydroxide and stored in a nitrogen atmosphere.
Methanol was distilled from magnesium and iodine under a
nitrogen atmosphere and stored over molecular sieves. Methy-
lene chloride was distilled from phosphorus pentoxide, and
chloroform was distilled from calcium sulfate. Tetrahydrofuran
was purified by distillation from sodium and benzophenone.
Dimethylformamide was dried by distillation from anhydrous
calcium sulfate and was stored under nitrogen. Preparative scale
chromatographic procedures were carried out using E. Merck
silica gel 60, 230-440 mesh. Thin-layer chromatography was
conducted on Merck precoated silica gel 60 F-254. Ion exchange
chromatography was conducted on a Dowex 1X8-200 anion-
exchange resin. Compound 10 was synthesized from (S)-pheny-
lalanine methyl ester as previously described,85 and compound
13 was synthesized from picolinic acid and (S)-valine tert-butyl
ester as previously reported.72 Compound (S)-18 was synthe-
sized from (S)-phenylalanine and N-propargylamine as pre-
viously described.111
1
was also isolated as a light-yellow solid. H NMR (400 MHz,
CDCl3) δ 1.24 (t, J = 6.8 Hz, 3H), 1.41 (br s, 9H), 2.37 (dd, J =
2.4, 21.2 Hz, 1H), 2.58 (dd, J = 10.4, 15.2 Hz, 1H), 2.91 (d, J =
7.6 Hz, 2H), 3.47 (d, J = 2.4 Hz, 1H), 3.72 (q, J = 8.4 Hz, 1H),
3.96-3.99 (m, 1H), 4.13 (q, J = 6.8 Hz, 2H), 4.93 (d, J = 9.6 Hz,
1H, NH), 7.19-7.36 (m, 5H). 13C NMR (100 MHz, CDCl3) δ
14.3, 27.6, 28.5, 38.4, 45.0, 52.5, 70.4, 126.7, 128.7, 167.1, 172.3.
Mp 130 ꢀC. [R]2D0 þ31.4 (c 1.0, MeOH).
1
All H, 13C and 19F and NMR spectra were recorded on a
Varian Mercury 400 MHz or Bruker 300 MHz spectrometer,
and all chemical shifts are reported as δ values referenced to
TMS or DSS. Infrared spectra were recorded on a Nicolet
5DXB FT-IR spectrophotometer and are referenced to poly-
styrene. In all cases, 1H NMR, 13C NMR, and IR spectra were
consistent with assigned structures. Melting points were recorded
on a Thomas-Hoover capillary melting point apparatus and are
uncorrected. Mass spectra were recorded on a Kratos MS
80 RFA (EI and CI) or Kratos MS 50 TC (FAB) mass spectro-
meters. Prior to biological testing, target molecules 6-9 were
(3S,4S)-Ethyl 3-Hydroxy-4-amino-5-phenylpentanoate
((3S,4S)-12). To a stirring solution of 0.11 g (0.0003 mol) of
(3S,4S)-11 in 15 mL of ethyl acetate was added 3.0 mL of 3.0 M
HCl, and the resulting mixture was allowed to stir at room
temperature for 30-40 min. After completion of reaction was

4242 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Gupta et al.
verified by thin-layer chromatography (ethyl acetate/benzene,
1:4), the solvents were removed in vacuo and the resulting solid
was triturated with three 20 mL portions of ethyl ether. The
combined ether layers were dried (anhydrous MgSO4) to afford
(3S,4S)-12 (0.066 g, 91.7%). This solid was used without further
purification in the subsequent reaction.
added and the mixture was stirred for another 15 min at 0 ꢀC. A
2.6 g (0.020 mol) portion of (S)-leucinamide dissolved in 5 mL of
DMF was then added by syringe, and the mixture was warmed to
room temperature and allowed to stir for 8 h at room tempera-
ture. The solvent was then removed under high vacuum to yield a
dark-yellow semisolid. The semisolid was dissolved in 50 mL of
water and extracted with three 50 mL portions of chloroform.
The combined organic layers were dried over anhydrous magne-
sium sulfate and filtered, and the solvent was removed to afford
crude 15, which was purified by column chromatography to
Ethyl (3S,4S)-3-Hydroxy-4-[(R)-3-methyl-2-(picolinamido)-
butanamido]-5-phenylpentanoic Acid (14a). A 3.6 g (0.016 mol)
portion of 13 was dissolved in 25 mL of anhydrous DMF at 0 ꢀC,
and 2.48 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) (0.016 mol) and 2.16 g of hydroxybenzotriazole (HOBT)
(0.016 mol) were added. The mixture was allowed to stir for 15
min at 0 ꢀC. Then 1.62 g (1.78 mL, 0.0160 mol) of N-methyl-
morpholine was added, and the resulting mixture was stirred for
another 15 min at 0 ꢀC. A 4.38 g (0.016 mol) portion of (3S,4S)-
12 dissolved in 15 mL of anhydrous DMF was then added to the
reaction mixture, which was stirred for 8 h at room temperature.
Removal of the solvent under reduced pressure yielded a dark-
yellow semisolid. The semisolid was dissolved in water and
extracted with three 50 mL portions of chloroform, the com-
bined organic layers were dried over anhydrous magnesium
sulfate and filtered, and the solvent was removed to afford the
crude ethyl ester of 14. This intermediate was purified on silica
gel to yield 5.39 g of the pure ethyl ester of 14 (76.3%). 1H NMR
(400 MHz, CDCl3) δ 0.87-1.05 (m, 6H), 1.24 (t, J = 7.2 Hz,
3H), 2.17-2.35 (m, 1H), 2.38, 2.43 (dd, J = 2.8 Hz, 2.8 Hz, 1H),
2.52, 2.56 (dd, J = 10.0 Hz, 10.0 Hz, 1H), 2.88-2.96 (m, 2H),
3.78 (m, 1H), 4.1 (q, J = 6.4 Hz, 2H), 4.40, 4.42 (dd, J = 6.4 Hz,
2.8 Hz, 1H), 6.23 (s, 1H, OH), 7.04-7.24 (m, 4H), 7.41-7.48 (m,
1H), 7.57 (dt, J = 8 Hz, 0.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H),
7.87 (dt, J = 7.6 Hz, 1.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H, NH),
8.18 (d, J = 8.0 Hz 1H), 8.43 (d, J = 9.2 Hz, 1H, NH), 8.59 (d,
J = 4.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 14.3, 17.98,
19.84, 30.61, 38.42, 39.07, 54.25, 59.32, 61.08, 67.47, 85.30,
109.62, 120.41, 122.59, 125.27, 126.56, 126.67, 128.61, 128.74,
128.88, 129.0, 129.54, 137.58, 138.03, 148.52, 149.48, 164.87,
171.09, 173.42. IR (KBr, cm-1) 3333.8, 2962.3, 1732.2, 1651.4,
1521.3, 1434.5, 1260.7, 1153.0, 1087.7.
(3S,4S)-3-Hydroxy-4-[(R)-3-methyl-2-(picolinamido)butanamido]-
5-phenylpentanoic Acid (14b). A 5.39 g (0.012 mol) portion of the
ethyl ester of 14a was dissolved in 25 mL of dioxane/water (4:2)
and cooled to 0 ꢀC, and 20 mL of 2.0 N NaOH was added to the
mixture by dropwise addition. The solution was warmed to room
temperature and allowed to stir for 16 h, during which time the
reaction was monitored by TLC. The mixture was again cooled to
0 ꢀC and neutralized by the dropwise addition of 1.0 N HCl. The
resultant solution was washed with two 25 mL portions of diethyl
ether (2ꢀ), and the aqueous layer was extracted with three 50 mL
portions of ethyl acetate. The ethyl acetate layers were combined,
washed with brine, and dried over anhydrous magnesium sulfate.
Removal of the solvent in vacuo yielded compound (3S,4S)-14b
as a light-brown hygroscopic solid (4.70 g, 94.7%) of sufficient
purity to be used in the next reaction. 1H NMR (400 MHz,
CDCl3) δ 0.71-0.91 (m, 6H), 2.29 (d, J = 7.2 Hz, 1H), 2.55-2.57
(m, 1H), 2.59-2.61 (m, 1H), 2.74-2.85 (m, 1H), 2.98-3.06 (m,
1H), 3.71 (m, 1H, OH), 4.05-4.15 (m, 1H), 4.20-4.36 (m, 1H),
7.06-7.22 (m, 3H), 7.43-7.52 (m, 2H), 7.76-7.92 (m, 2H),
8.55-8.62 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 14.41,
19.34, 21.28, 38.47, 59.43, 60.66, 126.47, 126.72, 128.51, 128.71,
129.47, 137.93, 148.95, 171.33, 175.63. IR (KBr, cm-1) 3316.7,
3059.2, 2964.9, 2927.5, 1715.2, 1651.8, 1525.3, 1434.7, 1259.2,
1096.2, 914.3.
1
afford 2.92 g of pure 15 as a pale-yellow oil (77.5% yield). H
NMR (400 MHz, CDCl3) δ 0.81-0.86 (m, 6H), 1.47-1.57 (m,
3H), 3.66 (dd, J = 5.2 Hz, 1H), 3.79 (d, J = 4.8 Hz, 1H),
4.33-4.36 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 25.72, 26.96,
44.99, 52.41, 53.26, 53.68, 59.59, 172.50, 213.21.
(S)-2-[(S)-2-Amino-3-cyanopropanamido]-4-methylpentanamide
(16). Compound 16 was synthesized from 2-aminopent-4-ynoic
acid and (S)-leucinamide exactly as described for the synthesis of
15 in 83.7% yield. 1H NMR (400 MHz, CDCl3) δ 0.93-0.99 (m,
6H), 1.60-1.74 (m, 3H), 2.76 (dd, J = 8.8 and 9.2 Hz, 1H), 2.96
(dd, J = 4.0 and 4.4 Hz, 1H), 4.18-4.24 (m, 1H), 4.19-4.48 (m,
1H). 13C NMR (100 MHz, CDCl3) δ 25.55, 28.68, 52.45, 52.88,
53.09, 53.52, 59.37, 137.68, 170.99, 175.47.
(S)-2-Amino-N-[(S)-1-amino-4-methyl-1-oxopentan-2-yl]pent-
4-ynamide (17). Compound 17 was synthesized from 2-amino-3-
cyanopropanoic acid and (S)-leucinamide exactly as described
1
for the synthesis of 15 in 74.2% yield. H NMR (400 MHz,
CDCl3) δ 0.88-0.94 (m, 6H), 1.56-1.58 (m, 1H), 1.65-1.75 (m,
2H), 2.61-2.96 (m, 2H), 2.88 (s, 1H), 4.31 (br s, 1H), 4.50-4.56
(m, 1H), 5.48 (d, J = 6.4 Hz, 1H), 6.00 (br s, 1H), 6.70 (br s, 1H),
7.08 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 20.71,
22.03, 23.68, 27.24, 39.67, 50.48, 52.22, 70.96, 78.15, 169.54,
173.52.
N-((S)-1-(((2S,3S)-5-(((R)-1-(((S)-1-Amino-4-methyl-1-oxo-
pentan-2-yl)amino)-3-chloro-1-oxopropan-2-yl)amino)-3-hydro-
xy-5-oxo-1-phenylpentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-
picolinamide (6). Compound 6 was prepared from 14b and 15
using the peptide coupling method described for the synthesis
of 15 and was isolated as a light-yellow solid in 25.5% yield. 1H
NMR (100 MHz, CDCl3) δ 0.81-0.99 (m, 12H), 1.60-1.76 (m,
3H), 2.24-2.43 (m, 2H), 2.58 (d, J = 8.0 Hz, 1H), 2.85-3.06 (m,
2H), 3.49 (s, 1H, OH), 3.65-3.93 (m, 2H), 4.04-4.68 (m, 5H),
7.13-7.25 (m, 3H), 7.39-7.52 (m, 2H), 7.83-7.90 (m, 1H),
8.08-8.17 (m, 1H), 8.42-8.51 (m, 1H), 8.58-8.62 (m, 1H). 13
C
NMR (100 MHz, CDCl3) δ 9.95, 13.04, 21.96, 22.72, 27.90, 28.68,
29.33, 30.27, 37.7, 39.9, 44.8, 50.34, 67.14, 84.07, 108.40, 119.17,
124.05, 127.78, 129.88, 131.43, 166.77. MSESIþ 631.5 (M þ Hþ).
N-((S)-1-(((2S,3S)-5-(((S)-1-(((S)-1-Amino-4-methyl-1-oxo-
pentan-2-yl)amino)-1-oxopent-4-yn-2-yl)amino)-3-hydroxy-5-
oxo-1-phenylpentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-
picolinamide (7). Compound 7 was prepared from 14b and 16
using the peptide coupling method described for the synth-
esis of 15 and was isolated as a white solid in 24.1% yield. 1H
NMR (400 MHz, CDCl3) δ 0.77-1.01 (m, 12H), 1.25-1.34
(m, 3H), 2.16-2.48 (m, 3H), 2.85-3.03 (m, 4H), 4.03-4.17
(m, 4H), 4.32-4.44 (m, 2H), 7.12-7.26 (m, 3H), 7.40-7.52
(m, 2H), 7.88 (m, 2H, 1H = NH), 8.11-8.19 (m, 2H, 1H =
NH), 8.38-8.42 (m, 1H), 8.42-8.60 (m, 2H, 1H = NH). 13
C
NMR (100 MHz, CDCl3) δ 16.36, 28.68, 63.76, 65.71, 84.06,
93.63, 108.41, 110.29, 119.20, 124.04, 127.77, 136.01, 147.38,
151.49, 166.38, 180.68, 202.12, 208.63. MS (m/e) ESIþ 660.4
(Mþ þ K).
(S)-2-[(R)-2-Amino-3-chloropropanamido]-4-methylpentanamide
(15). A 2.0 g (0.016 mol) portion of 2-amino-3-chloropropanoic
acid was dissolved in 30 mL of anhydrous DMF, and the solution
was cooled to 0 ꢀC in an ice bath. To this mixture was added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 3.72 g,
0.020 mol) and 1-hydroxybenzotriazole (HOBt, 2.6 g, 0.020 mol),
and the mixture was allowed to stir for 15 min at 0 ꢀC. After 15
min 2.02 g (0.020 mol, 2.2 mL) of N-methylmorpholine was
N-((S)-1-(((2S,3S)-5-(((S)-1-(((S)-1-Amino-4-methyl-1-oxo-
pentan-2-yl)amino)-3-cyano-1-oxopropan-2-yl)amino)-3-hydroxy-
5-oxo-1-phenylpentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pico-
linamide (8). Compound 8 was prepared from 14b and 17 using the
peptide coupling method described for the synthesis of 15 and was
1
isolated as a white solid in 18.6% yield. H NMR (400 MHz,
CDCl3) δ 0.67-0.90 (m, 12H), 1.50-1.17 (m, 3H), 2.33-2.42 (m,
2H), 2.53-2.97 (m, 6H), 4.08-4.56 (m, 6H), 7.06-7.24 (m, 4H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4243
7.41-7.52 (m, 1H), 7.84-7.91 (m, 1H), 8.11-8.21 (m, 1H), 8.26
(d, J=8.0 Hz, 1H, NH), 8.43 (dd, J= 6.8 Hz, 6.4 Hz, 1H), 8.56 (d,
J = 4.4 Hz, 1H), 8.61 (d, J = 4.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 18.6, 21.4, 24.9, 29.9, 31.7, 46.7, 54.4, 66.1, 70.6, 92.5,
97.7, 112.7, 122.3, 123.0, 128.8, 129.4, 152.6, 162.9, 173.7, 174.9,
181.9, 191.4. MS ESIþ 643.3 (M þ Naþ).
collected in the flow through and again loaded onto a mono-
Q-Sepharose column equilibrated in buffer D. Proteins were
eluted from the mono-Q-Sepharose column with a linear gradient
of 0-1.0 M NaCl. The eluent was dialyzed in buffer E (20 mM
Tris-HCl, pH 8.5, 1 mM EDTA, 10% glycerol) at room tem-
perature while stirring overnight at room temperature. Refolding
of the protein was ensured using native gels. The major portion of
protein was collected from the heparin FF column by running a
0-1.0 MNaClgradient.Thiswasagainloadedontoa heparinFF
column, and a 0-1.0 M NaCl gradient was run. This eluent was
again loaded onto a Q-Sephrose column equilibrated with buffer
D followed by refolding in buffer E. Autoactivation was then
achieved by stirring in 0.1 M sodium citrate solution at pH 4.7.
Small molecular fragments were removed by loading them on
Q-Sepharose column equilibrated in buffer E, and the column
was eluted with a salt gradient (0-1.0 M NaCl). In the final
purification step, the resultant eluant was loaded onto a Superdex
100 column using buffer F (20 mM Tris-HCl, pH 8.5, 1 mM
EDTA, 10% glycerol, 0.25 M NaCl) and the protein was then
stored at 4 ꢀC in buffer G (20 mM 2-(N-morpholino)ethanesulfo-
nic acid (MES), pH 6.0, 0.25 M NaCl) and concentrated to give
the desired concentration.
Plasmepsin II Inhibition Assay. Enzyme activity was deter-
mined on the basis of FRET (fluorescence resonance energy
transfer) assays using internally quenched fluorescent peptide
substrates. The substrate used was EDANS-CO-CH2-CH2-CO-
ALERMFLSFP-diaminopimelate-(DABCYL)OH. A Perkin-
Elmer LS 55 luminescence spectrophotometer was used for initial
assays, followed by quantitation using a SpectraMax M5 micro-
plate reader (Molecular Devices) using an excitation wavelength
of 336 nm (slit width 5) and an emission wavelength of 490 nm
(slit width 10). Assays were performed in 0.5 M sodium acetate
buffer at pH 5.0 containing 10% glycerol and 0.01% Tween-20.
Stock solutions in DMSO were diluted to give a final DMSO
concentration of 1%. Typically, 2.5 nM enzyme was incubated
with different concentrations of each inhibitor to make a final
1 μM concentration of the substrate. Enzyme activity was
measured as an increase in fluorescence intensity after a 30 min
incubation period. Inhibition was reported as percent inhibition
compared to control, and active compounds were further titrated
toget initial IC50 and Ki values by assuming competitive inhibition.
Logarithmic fitting of the data was achieved using the Kaleida-
Graph graphing software. For time-dependent inactivators, Ki
values were derived using the method of Kitz and Wilson.104
Time-Dependent Kinetics. Depending upon the IC50 value of
the analogues, different concentrations of analogues were incu-
bated with a 2.5 nM enzyme concentration so that in the final
reaction mixture 6 was present at 0, 25, 50, 75, and 100 nM, while 8
was present at 0, 40, 80, 160, 240, 320 nM. Incubations were
conducted for 0, 2.5, 5, 10, 20, 30, 40 min at 37 ꢀC. After each
incubation, 10 μL from each reaction mixture was added to a
96-well plate containing 90 μL of substrate (1 μM final concen-
tration). Fluorescence was then measured after 2 min of incuba-
tion at 37 ꢀC, and a control sample was used to calculate the
percent enzyme activityremaining. These values were converted to
ln(percent activity) and plotted against time to give a pseudo-first-
order rate constant (kobs) for each concentration of inhibitor. This
rate constant was then replotted againstthe inverse of the inhibitor
N-((S)-1-(((2S,3S)-3-Hydroxy-5-oxo-5-(((S)-1-oxo-3-phenyl-
1-(prop-2-yn-1-ylamino)propan-2-yl)amino)-1-phenylpentan-2-yl)-
amino)-3-methyl-1-oxobutan-2-yl)picolinamide (9). Compound 9
was prepared from 14b and 18 using the peptide coupling method
described for the synthesis of 15 and was isolated as a white solid
1
in 22.1% yield. H NMR (400 MHz, CDCl3) δ 0.73-1.11 (m,
6H), 1.98-2.70 (m, 4H), 2.78-2.82 (m, 1H), 2.94-3.48 (m, 3H),
3.72-4.31 (m, 5H), 4.72-5.30 (m, 1H), 7.14-7.31 (m, 8H),
7.41-7.53 (m, 1H), 7.84-7.89 (m, 2H), 8.07-8.31 (m, 1H),
8.46-8.61 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 19.44,
19.65, 28.68, 53.52, 89.64, 90.08, 93.76, 106.61, 111.67, 116.13,
121.35, 127.42, 128.28, 145.18, 147.39, 158.65, 159.01, 165.19,
181.17, 183.67, 190.91, 198.29. MS ESIþ 598.3 (M þ Hþ).
Plasmepsin II Expression and Purification. Competent BL21-
DE3 cells were thawed on ice and gently mixed, and an amount
of 100 μL of these cells was added to 15 mL of prechilled Falcon
2059 culture tubes and kept on ice. Then 2.5 μL of a 1.0 M stock
2-mercaptoethanol solution was added to each culture tube and
kept on ice for 10 min, with gentle swirling every 2 min. This step
was followed by the addition of 1, 2, and 3 μL of plasmid to
corresponding tubes. For the positive control transformation
reaction, 1 μL of the pUC18 control plasmid (empty vector) was
added to a separate 100 μL aliquot of the competent cells and
swirled gently. A negative control of BL-21CE-3 cells without
any transformation was included. Cells were incubated on ice
for 30 min, after which super optimal broth with catabolite
repression (SOC) medium was preheated to 42 ꢀC, and the cells
were heat-shocked for 45 s in water bath and returned to ice for
2 min. A 900 μL portion of SOC medium (42 ꢀC) was added to
each culture, and cells were incubated at 37 ꢀC for 1 h with
shaking at 225-270 rpm. After 1 h, 100 μL of the cell culture was
plated on LB agar plates containing 100 mg/mL ampicillin and
incubated overnight at 37 ꢀC. An isolated colony on agar plate
was chosen and put into 2 mL of LB media containing 1%
ampicillin, and this sample plus an identical duplicate was
agitated in a cell shaker at 220 rpm (37 ꢀC) overnight. The next
morning, 50 μL of each culture was pipetted into fresh 1 mL
aliquots of LB broth containing no ampicillin and reincubated
at 37 ꢀC for 2 h with shaking at 220 rpm. The culture was split
into two 2 mL samples, one of which was used for induction and
the second as control. Induction was started by adding isopropyl
β-D-1-thiogalactopyranoside (IPTG) to a final concentration of
1 mM when the optical density was g4. These samples were
incubated at 220 rpm in a cell shaker at 37 ꢀC until the optical
density reached 1.0. Protein expression was then analyzed by
SDS-PAGE. Induction conditions were varied in terms of
media (LB/TB), temperature (20 ꢀC/37 ꢀC) and IPTG concen-
tration (0.1-1 mM) for optimum expression of the desired
protein. Cell cultures were diluted to 1.0 L and centrifuged,
and the resulting cell pellet was resuspended in 20 mL of buffer A
(20 mM Tris-HCl, pH 8.5, 1 mM EDTA, 150 mM NaCl). The
cells were lysed with two passes through a French press and then
sonicated two times for 15 s. The resulting mixture was then
centrifuged at 15 000 rpm for 20 min, and the supernatant was
saved for SDS-PAGE. Cells were again resuspended in buffer
A and repelleted. Inclusion bodies were then washed two times
each with buffer B (20 mM Tris-HCl, pH 8.5, 1 mM EDTA, 150 mM
NaCl, 1% Triton X-100) and then buffer C (20 mM Tris-HCl, pH
8.5, 1 mM EDTA). Inclusion bodies were then solublized in
buffer D (6 M urea, 20 mM Tris-HCl, pH 8.5, 1 mM EDTA). The
supernatant was dialyzed overnight in buffer D, and the protein
solution was filtered through a 45 μm and then a 20 μm filter.
The resulting protein solution was then loaded onto a heparin-
Sepharose FF column equilibrated in buffer D. Plm II was
concentration to derive the kinetic parameters Ki and kinact
.
Substrate Protection Experiments. To determine whether
increasing substrate concentration protected the enzyme from
inactivation by 6 or 8, inhibitor and substrate were mixed in
ratios varying from 1:1 to 1:5 and incubated at a 2.5 nM final
concentration of enzyme. Following a 2 min incubation period
at 37 ꢀC, fluorescence values were measured and compared to
control values.
Dialysis and Spun Column Chromatography. A 300 μL aliquot
of Plm II solution (3 nM final concentration) was incubated with
300 μL of inhibitors 6-9 (1 μM final concentration) for 1 h to
achieve complete inactivation. Enzymes without any inhibitor

4244 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Gupta et al.
or with 1 μM pepstatin-A were used as controls. After 1 h, a
50 μL aliquot was removed and assayed against 50 μL of
substrate (final concentration 1 μM) to assess the remaining
enzyme activity. The reaction mixture was then transferred to
dialysis cassettes (molecular weight cutoff of 10 000 Da) and
dialyzed overnight against 0.1 M sodium acetate buffer, pH 5.0,
at room temperature. The buffer was changed three times during
this period. After overnight dialysis, activity was again mea-
sured as described above. Spun chromatography was also
utilized to validate the data from the dialysis experiments. In
this method, each incubated reaction mixture was loaded onto
spin column cartridges according to the manufacturer protocol
and centrifuged for 5 min at 1800 rpm at 4 ꢀC. The eluent was
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aliquot of trypsin was added, and the mixture was incubated at
37 ꢀC and 5% CO2 until cells became detached. Cells were then
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medium. The MTS reagent was prepared by mixing 20 μL of
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then incubated for 2 h at 37 ꢀC and 5.0% CO2. The plate was then
read with the plate reader set at OD 490. Absorbance values were
compared with control to measure cytotoxicity.
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Article
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