Plasmepsin inhibitory activity and structure-guided optimization of a potent hydroxyethylamine-based antimalarial hit

Article abstract of DOI:10.1021/ml4004952

Antimalarial hit 1SR (TCMDC-134674) identified in a GlaxoSmithKline cell based screening campaign was evaluated for inhibitory activity against the digestive vacuole plasmepsins (Plm I, II, and IV). It was found to be a potent Plm IV inhibitor with no selectivity over Cathepsin D. A cocrystal structure of 1SR bound to Plm II was solved, providing structural insight for the design of more potent and selective analogues. Structure-guided optimization led to the identification of structurally simplified analogues 17 and 18 as low nanomolar inhibitors of both, plasmepsin Plm IV activity and P. falciparum growth in erythrocytes.

Full text of DOI:10.1021/ml4004952

Letter
pubs.acs.org/acsmedchemlett
Plasmepsin Inhibitory Activity and Structure-Guided Optimization of
a Potent Hydroxyethylamine-Based Antimalarial Hit
Kristaps Jaudzems,† Kaspars Tars,‡ Gundars Maurops,† Natalija Ivdra,† Martins Otikovs,† Janis Leitans,‡
Iveta Kanepe-Lapsa,† Ilona Domraceva,† Ilze Mutule,† Peteris Trapencieris,† Michael J. Blackman,§
and Aigars Jirgensons*,†
†Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
‡Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia
§Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill, Hill, London NW7 1AA, U.K.
S
* Supporting Information
ABSTRACT: Antimalarial hit 1SR (TCMDC-134674) iden-
tified in a GlaxoSmithKline cell based screening campaign was
evaluated for inhibitory activity against the digestive vacuole
plasmepsins (Plm I, II, and IV). It was found to be a potent
Plm IV inhibitor with no selectivity over Cathepsin D. A
cocrystal structure of 1SR bound to Plm II was solved,
providing structural insight for the design of more potent and
selective analogues. Structure-guided optimization led to the
identification of structurally simplified analogues 17 and 18 as
low nanomolar inhibitors of both, plasmepsin Plm IV activity
and P. falciparum growth in erythrocytes.
KEYWORDS: Malaria, Plasmodium falciparum, plasmepsins, Cathepsin D, inhibition, structure-guided optimization,
hydroxyethylamine
Chart 1. Structure of GSK Cell Based HTS Antimalarial Hit
espite extensive eradication campaigns, malaria caused by
DPlasmodium parasites remains a devastating disease with
an estimated 219 million cases and 660 thousand lethal
outcomes in 2010.1 Widespread resistance to practically all
currently used drugs has activated the search for antimalarials
with novel mechanisms of action.2−4 Low profit potential of
antimalarial drugs has promoted collaboration between
academic, private, and charitable organizations to establish
novel drug discovery programs. As a part of an antimalarial
initiative, pharmaceutical companies contribute with their
unique resources to the development of antimalarials, making
their data publicly available.5 Recently, researchers at
GlaxoSmithKline (GSK) published the structures of 13 533
hits from the screening of nearly 2 million compounds that
inhibited malaria parasite growth by at least 80% at 2 μM
concentration.6 These hits were further analyzed using
cheminformatics to identify 47 series of high-quality starting
points for lead optimization.7 The series 3 included 74
compounds based on a hydroxyethylamine scaffold that is
characteristic for plasmepsin inhibitors.8−11 Given the interest
in the digestive vacuole plasmepsins (Plm I, II, and IV) as
targets for antimalarial drug discovery, we selected the most
active compound from this series, 1SR (TCMDC-134674), for
investigation of its Plm I, II, and IV inhibitory activity (Chart
1).12
that it is a potent Plm IV inhibitor (IC50 = 29 nM) while being
a less effective inhibitor of Plm II (IC50 = 0.15 μM) and Plm I
(IC50 = 0.70 μM) (Table 1). Selectivity studies of compound
1SR showed that it was not a selective inhibitor of plasmesins
over the human aspartic protease Cathepsin D (CatD, IC50 of
43 nM, Table 1).
While a number of digestive vacuole plasmepsin inhibitors
have been previously identified, to the best of our knowledge
none of them displayed low nanomolar activity in cell based
models. Therefore, it was an exciting finding that compound
1SR, which was reported as highly active against intra-
erythrocytic P. falciparum cell growth (IC50 of 30 nM),6 is a
Received: December 3, 2013
Accepted: January 13, 2014
Compound 1SR was resynthesized (see Supporting
Information) and tested in enzymatic assays, which showed
© XXXX American Chemical Society
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Table 1. SAR of R1 Group
Chart 2. Binding of 1 (TCMDC-134674)6 within the Active
Site of Plm II
a
a
(a) X-ray crystal structure showing the ligand and residues located
within 4 Å from it as stick models (colored in yellow and cyan,
respectively). (b) 2Fo − Fc electron density of the bound ligand
contoured at 1σ. The figure was generated using PyMOL.22 (c) Ligand
interaction diagram. Hydrogen bonds are shown as dashed lines, and
distances between heavy atoms are indicated. Potentially repulsive
interactions between the ligand and protein atoms are shown as red
curves, and distances between the closest heavy atoms are indicated in
Å.
potent Plm IV inhibitor. The critical role of Plm IV is still
unclear, ; however, gene knockout studies have revealed that
out of all individual digestive vacuole plasmepsin knockouts
only Δpfpm4 produced a statistically significant reduction in
hemozoin accumulation indicating impaired hemoglobin
digestion.13 In addition, the quadruple-plasmepsin knockout
mutant (Δpfpm1−4) showed a significantly slower rate of
growth in contrast to a triple plasmepsin knockout mutant
(Δpfpm1−3).14 On the basis of the available data, it was
intriguing to speculate that inhibition of Plm IV could be
responsible for the activity of compound 1SR in the cell based
assay. Nevertheless, it cannot be excluded that any other of the
nondigestive plasmepsins structurally similar to PlmIV could be
the target (or an additional target) for compound 1SR.15,16
To provide a structural basis for the design of structurally
simplified, potent and selective analogues of compound 1SR,
the X-ray crystal structure of Plm II in complex with compound
1SR was solved (Chart 2). Plm II was chosen for the
crystallization studies because it was easily obtainable in
milligram quantities, and its crystallization conditions have
been described previously.17 Plm II mutant M205S was used,
which displays enhanced resistance to self-cleavage compared
to the wild type enzyme.18 The obtained crystals diffracted to
1.85 Å resolution and belonged to space group C2 with six
molecules in the asymmetric unit. The electronic density of the
inhibitor was very similar in all six subunits and did not improve
significantly upon averaging. As shown in Chart 2a, electron
density was good for the central part of the inhibitor but was
weak or lacking for aliphatic propyl groups, the methoxy moiety
and part of the 1,2-thiazinane-1,1-dioxide ring. As expected, the
structure shows an interaction between the hydroxyethylamine
core of 1SR and the Plm II catalytic dyad Asp34−Asp214
(Chart 2b). However, in contrast to other structures of
transition state analogue−Plm II complexes,17,19,20 the hydroxyl
group is not within the characteristic hydrogen-bond distances
to both carboxylic acids, but only Asp34 (O−O distance of 2.69
Å) and Asp214 is hydrogen-bonded with the secondary amino
group (N−O distance of 2.47 Å, Chart 2c). The secondary
amine is linked to the isopropyl-2-(3-methoxyphenyl) moiety
that occupies the S1′ and part of the S2 pockets. The other side
of the hydroxyethylamine core is linked via a methine bridge to
the benzyl group positioned in the S1 pocket and the 3,5-
disubstituted benzamide moiety, whose N,N-dipropylamide and
2-(1,2-thiazinane-1,1-dioxide)-substituents bind in the S3 and
S4 pockets, respectively. The interactions in the S1−S4 and S1′
pockets are predominantly hydrophobic, while a few H-bond
interactions are formed with the more central groups of the
inhibitor, i.e., between the benzamide and Gly216 and the N,N-
dipropylamide and Ser218 (Chart 2c). Additionally, the
secondary amino group seems to interact simultaneously with
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the carboxylate of Asp214 and the carbonyl of Gly36. Detailed
inspection of the structure highlighted three sites of potentially
repulsive interactions between the inhibitor and Plm II active-
site residues (Chart 2c). First, the sulfonyl group of the 2-(1,2-
thiazinane-1,1-dioxide) moiety is only 3.0 Å from the
hydrophobic Val78 side chain. This repulsion seems to interfere
with flap closing, making the binding cavity more open than in
other Plm II−inhibitor complexes.21 Second, the 6-C atom of
the 3-methoxyphenyl group lies 3.3 Å from the hydrophilic side
chains of Thr217 and Asp214, and third, one methyl group of
the isopropyl-2-(3-methoxyphenyl) moiety is 3.6 Å from the
carbonyl of Gly36. Importantly, these interactions may also be
present in complexes with Plm I and IV as the interacting
residues are conserved among the food vacuole plasmepsins,
except Val78, which is replaced by a Gly in Plm IV. It was also
apparent that the aliphatic chains of the N,N-dipropylamide
moiety make only weak contacts with the protein as their
electron density was indiscernible (Chart 1a).
activity. The phenyl-substituted analogue (compound 2) of 1
was 2- to 3-fold less active, while the corresponding analogue
(compound 5) of 3 was of similar potency, indicating some
cooperativity between the R1 and R4 substituents in the case of
5. In order to explore possible H-bonding interactions with the
hydroxyl group of Thr217, we prepared compounds 6−8 by
replacing the 3-methoxyphenyl group with 2-, 3-, and 4-pyridyl,
respectively, hoping that the nitrogen atom might act as the H-
bond acceptor. However, this did not result in improved
activity, possibly, due to protonation of the pyridine nitrogen
atom at the pH of the assay (pH 4.6). The 3-pyridyl-
substitution (compound 7) was tolerated best from this series
and in combination with the 5-methoxy group yielded
compound 9 with similar activity (IC50 of 23 nM) against
Plm IV as 3 (IC50 of 24 nM).
Next, we investigated SARs associated with R2 (Table 2).
Although the crystal structure showed that one methyl group is
Guided by these insights from the crystal structure, we
started our SAR study of 1SR and defined four positions, where
appropriate chemical modifications were likely to improve
intermolecular interactions (Tables 1−4, R1 to R4). Mod-
ifications of R1, R2, and R4 were also expected to contribute to
the selectivity versus CatD as these moieties are located in the
S1′ and S2 and S4 pockets that are most different between the
two enzymes.21 Keeping R4 substituent (2-(1,2-thiazinane-1,1-
dioxide)) constant while optimizing substituents R1−R3 proved
to be impractical due to the very lengthy synthesis. Therefore,
we preferred to use the more accessible piperidinyl group as the
R4 substituent for optimization around R1−R3. To confirm that
this modification does not significantly alter the bound
conformation observed in the crystal structure, a few analogues
were synthesized with both piperidinyl and 1,2-thiazinane-1,1-
dioxide groups. Comparison of their activities (compound pairs
2SR and 5, 10, and 12) led to similar conclusions about
changes in inhibition activity suggesting that the piperidinyl
analogue binds similarly to compound 1SR.
Previous SAR studies of statine- and hydroxyethylamine-
based Plm II inhibitors have indicated a preference for the S-
configuration at the central hydroxyl group;21,23 therefore, we
were surprised that compound 1 has the R-configuration at this
stereogenic center. We prepared both diastereoisomers of two
of the studied compounds (1 and 2) and tested their inhibitory
activity against Plm I, II, and IV. The stereoisomers 1SS and
2SS showed 15−90 -fold lower activity than the isomers 1SR
and 2SR indicating that the R-configuration of the central
hydroxyl-bearing stereo center is critical for plasmepsin
inhibition (see Table 1) opposed to statine based inhibitors.21
This may be explained by the different binding mode of the
hydroxyethylamine core of 1SR in the Plm II catalytic center as
was observed in the crystal structure of the complex and
discussed above.
Table 2. SAR of R2 Group
IC50 (μM)
compd
R2
CH3
Plm I
Plm II
Plm IV
CatD
1SR
10
3
0.70
18
0.15
2.7
0.029
0.20
0.043
0.36
H
CH3
−CH2−
H
0.30
0.33
6.1
0.070
0.15
0.6
0.024
0.025
0.18
0.042
0.10
11
12
0.20
located in a polar environment while the other one lies in a
hydrophobic environment, we synthesized symmetrically
substituted analogues to avoid the complication associated
with an additional stereogenic center. Removal of both methyl
groups (compounds 10 and 12) resulted in a significant
decrease in activity, whereas a cyclopropyl functionality
(compound 11) was well tolerated at this position and at the
same time slightly enhanced selectivity for Plm IV over Cat D.
Further we examined the importance of the propyl groups in
the N,N-dipropylamide moiety (R3) that showed poor electron
density in the crystal structure. Removal of one propyl group
(compound 13) resulted in 2- to 7-fold decreased activity. A
decrease in activity was also observed upon changing the N,N-
dipropyl for a pyrrolidinyl group (compound 14) (Table 3).
Removal of both propyl groups (compound 15) resulted in
substantial activity loss suggesting that the propyl chains may
make transient contacts with the protein that are important for
binding affinity.
Initial optimization of R4 was achieved by replacing the 2-
(1,2-thiazinane-1,1-dioxide) moiety with the piperidinyl group
to ease the synthesis of new analogues. This resulted in a 2-fold
higher activity against Plm I and II, but only a small
improvement against Plm IV (Table 4). A similar, but more
pronounced trend was observed also by changing R4 to a
cyclohexyl moiety (compound 16). In contrast, changing R4 to
pyrrolidinyl (compound 17) and phenyl (compound 18)
resulted in more than 4-fold higher activity against Plm IV,
while the improvement against Plm I and II was up to 2-fold. It
SAR studies on R1 (Table 1) were performed to evaluate the
importance of the 3-methoxy group as well as to explore the
possibility of introducing an H-bond acceptor for the adjacent
hydroxyl group of Thr217. After preparing compound 2, the
unsubstituted phenyl analogue of 1, we decided to perform
further studies with piperidinyl group as the R4 substituent for
the reasons described above and synthesized compounds 3−5.
It should be noted that this R4 substitution yielded 2-fold
higher activity against Plm I and II, while the activity increase
against Plm IV was negligible. Replacing the methoxy group
with a hydroxyl functionality (compound 4) did not change its
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Compounds 17 and 18, which were high nanomolar inhibitors
of Plm I and Plm II, were still very active in the cell based assay.
In addition, diastereomer 1SS, which was a micromolar
inhibitor of PlmI and II but a high nanomolar inhibitor of
Plm IV, exhibited considerable activity in the cell based assay
(Table 4).
Table 3. SAR of R3 Group
In conclusion, we have identified Plm IV as the putative
target of 1SR (TCMDC-134674) in the malaria parasite P.
falciparum. A cocrystal structure of 1SR bound to Plm II was
solved and provided structural insight for the design of more
potent and selective analogues. Structure-guided optimization
then led to the identification of simplified analogues 17 and 18
as a low nanomoalr inhibitors of Plm IV activity and P.
falciparum growth in erythrocytes. Selectivity of PlmIV versus
CatD inhibition for these compounds was improved by
increasing their PlmIV inhibitory activity. Structure guided
optimization studies provide a base to further optimize drug-
like properties of this series of compounds.
IC50 (μM)
compd
R3
Plm I
Plm II
Plm IV
CatD
3
N(Pr)2
NHPr
0.30
0.75
1.6
0.070
0.50
0.50
4.4
0.024
0.038
0.11
0.042
0.11
13
14
15
N(CH2)4
NH2
0.038
0.50
7.6
0.17
Table 4. SAR of R4 Group
ASSOCIATED CONTENT
* Supporting Information
■
S
Description of the synthesis of compounds 1−18, description
of crystallographic studies, description of PlmI, II, IV, and CatD
enzymatic assays, and parasite growth inhibition assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(A.J.) E-mail: aigars@osi.lv.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Financial support from ERAF 2010/2DP/2.1.1.1.0./10/APIA/
VIAA/074 is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
a
3D7 strain of P. falciparum
ACKNOWLEDGMENTS
■
We would like to thank Prof. B. Dunn for providing Plm II-
transformed BL21(DE3)pLysS cells. We also thank the
personnel at MAX-Lab for their help during our stay at the
synchrotron.
implies that nonplanar hydrophobic groups such as piperidinyl
and cyclohexyl at this position are better accommodated by
Plm I and II because of the favorable interaction with Val78,
while planar and more compact groups are preferred by Plm IV,
possibly, due to favorable flap-closing interactions. Importantly,
inhibitory activity of CatD for compounds 17 and 18 remained
at the level of starting hit 1, which improved selectivity for Plm
IV versus CatD inhibition (ratios of 4.6 and 9, respectively) as
compared to all other analogues. This indicates that
interactions with flap residues are important for Plm IV
selectivity.
Selected compounds exhibiting high Plm IV inhibitory
activity were tested for inhibition of P. falciparum parasite
growth in erythrocytes using a SYBR green assay (Table 4).
Compound 1SR showed somewhat higher activity to inhibit the
growth 3D7 parasite strain in erythrocytes compared to GSK
LDH assay.6 This deviation can be due to the different assays
used to monitor parasite growth. The results of parasite growth
inhibition of compounds 1SS, 1SS, 3, and 16−18 again showed
a clear correlation between parasite growth suppression and
Plm IV inhibition, and not with Plm I or Plm II inhibition.
ABBREVIATIONS
GSK, GlaxoSmithKline; Plm I, plasmepsin I; Plm II, plasmepsin
I; Plm IV, plasmepsin IV; Cat D, cathepsin D
■
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