Peptidomimetic plasmepsin inhibitors with potent anti-malarial activity and selectivity against cathepsin D

Article abstract of DOI:10.1016/j.ejmech.2018.11.068

Following up the open initiative of anti-malarial drug discovery, a GlaxoSmithKline (GSK) phenotypic screening hit was developed to generate hydroxyethylamine based plasmepsin (Plm) inhibitors exhibiting growth inhibition of the malaria parasite Plasmodium falciparum at nanomolar concentrations. Lead optimization studies were performed with the aim of improving Plm inhibition selectivity versus the related human aspartic protease cathepsin D (Cat D). Optimization studies were performed using Plm IV as a readily accessible model protein, the inhibition of which correlates with anti-malarial activity. Guided by sequence alignment of Plms and Cat D, selectivity-inducing structural motifs were modified in the S3 and S4 sub-pocket occupying substituents of the hydroxyethylamine inhibitors. This resulted in potent anti-malarials with an up to 50-fold Plm IV/Cat D selectivity factor. More detailed investigation of the mechanism of action of the selected compounds revealed that they inhibit maturation of the P. falciparum subtilisin-like protease SUB1, and also inhibit parasite egress from erythrocytes. Our results indicate that the anti-malarial activity of the compounds is linked to inhibition of the SUB1 maturase plasmepsin subtype Plm X.

Full text of DOI:10.1016/j.ejmech.2018.11.068

Accepted Manuscript
Peptidomimetic plasmepsin inhibitors with potent anti-malarial activity and selectivity
against cathepsin D
Rimants Zogota, Linda Kinena, Chrislaine Withers-Martinez, Michael J. Blackman,
Raitis Bobrovs, Teodors Pantelejevs, Iveta Kanepe-Lapsa, Vita Ozola, Kristaps
Jaudzems, Edgars Suna, Aigars Jirgensons
PII:
S0223-5234(18)31027-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.068
EJMECH 10926
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2018
Revised Date: 26 November 2018
Accepted Date: 28 November 2018
Please cite this article as: R. Zogota, L. Kinena, C. Withers-Martinez, M.J. Blackman, R. Bobrovs,
T. Pantelejevs, I. Kanepe-Lapsa, V. Ozola, K. Jaudzems, E. Suna, A. Jirgensons, Peptidomimetic
plasmepsin inhibitors with potent anti-malarial activity and selectivity against cathepsin D, European
Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.068.
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ACCEPTED MANUSCRIPT
TOC graphic
R and R1 are selectivity inducing substituents
Ph
S3
O
O
Me
Me
V
V
Plm IV/ Cat D selectivity up to 50-fold
R
OMe
N
N
N
H
H
H
EC50(Pf) = 6.0-0.3 nM
OH
evidence of Plm X sub-type inhibition
R1
S4

ACCEPTED MANUSCRIPT
Peptidomimetic Plasmepsin Inhibitors with Potent anti-Malarial Activity and
Selectivity Against Cathepsin D
Rimants Zogotaa, Linda Kinenaa, Chrislaine Withers-Martinezb, Michael J
Blackmanb,c, Raitis Bobrovsa, Teodors Pantelejevsa, Iveta Kanepe-Lapsaa, Vita Ozolaa,
Kristaps Jaudzemsa, Edgars Sunaa*, Aigars Jirgensonsa*
aLatvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
bMalaria Biochemistry Laboratory, The Francis Crick Institute, 1 Midland Road,
London NW1 1AT, UK
cFaculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical
Medicine, London WC1E 7HT, UK
Abstract
Following up the open initiative of anti-malarial drug discovery, a GlaxoSmithKline
(GSK) phenotypic screening hit was developed to generate hydroxyethylamine based
plasmepsin (Plm) inhibitors exhibiting growth inhibition of the malaria parasite
Plasmodium falciparum at nanomolar concentrations. Lead optimization studies were
performed with the aim of improving Plm inhibition selectivity versus the related
human aspartic protease cathepsin D (Cat D). Optimization studies were performed
using Plm IV as a readily accessible model protein, the inhibition of which correlates
with anti-malarial activity. Guided by sequence alignment of Plms and Cat D,
selectivity-inducing structural motifs were modified in the S3 and S4 sub-pocket
occupying substituents of the hydroxyethylamine inhibitors. This resulted in potent
anti-malarials with an up to 50-fold Plm IV/Cat D selectivity factor. More detailed
investigation of the mechanism of action of the selected compounds revealed that they
inhibit maturation of the P. falciparum subtilisin-like protease protease SUB1, and
also inhibit parasite egress from erythrocytes. Our results indicate that the anti-
malarial activity of the compounds is linked to inhibition of the SUB1 maturase
plasmepsin subtype Plm X.
Key words: Plasmepsins; Malaria; Plasmodium falciparum; cathepsin D; Inhibitors;
Hydroxyethylamine
1. Introduction
Malaria is a life-threatening disease caused by Plasmodium parasites which are
transmitted by mosquitoes.1 More than half of the earth’s population lives in malaria

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endemic areas, rendering the disease a global health problem. Extensive eradication
campaigns have been implemented, leading to considerably reduced malaria
morbidity.2 A key future goal, according to the Global Technical Strategy for Malaria
2016–2030, is a 90% reduction in clinical cases and deaths by 2030 as compared with
2015.3 However, these efforts are impeded by widespread resistance of the parasite to
all currently used drugs, including artemisinins, the current front line drug class.4-6
Consequently, new antimalarial drugs with new modes of action are urgently needed.
Their development faces notable hurdles, one of which is a low expected profit after
market approval. This has prompted several open innovation initiatives by private and
academic organizations, including the disclosure of preclinical research data to the
scientific community.7-10 To support one such initiative, GlaxoSmithKline (GSK)
recently published the results of a large-scale cell-based (phenotypic) HTS screening
campaign that provided a number of starting points for anti-malarial drug discovery.7
From the pool of parasite growth inhibitory compounds we selected
hydroxyethylamine derivative 1a for further development (Figure 1).11 In our
previous studies we showed that compound 1a is an inhibitor of the Plasmodium
falciparum aspartic proteases - plasmepsin subtypes Plm I, Plm II and Plm IV with
particularly high potency against Plm IV. Structurally simplified potent Plm IV
inhibitors 1b,c were developed as compound 1a analogues, retaining high potency in
P. falciparum growth assays.
Table 1. Representative Plm inhibitors 1a-c from previous studies11
IC50
Plm IV,
IC50
Cat D, µΜ
EC50
Pf growth, µΜ
Comp.
R
µΜ
(S,R)-1a
(S,R)-1b
(S,R)-1c
0.029
0.024
0.006
0.043
0.042
0.054
0.002
0.006
0.002
Ph
It is important to note that despite more than two decades of research on plasmepsin
inhibitor discovery, only a few compounds (including inhibitors 1a-c) exhibiting

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parasite growth inhibition at low nanomolar concentration have been identified.11,12
This is likely attributable to the fact that most of the efforts so far have been devoted
to inhibiting plasmepsin subtypes involved in hemoglobin digestion (Plms I-IV).13-22
Gene disruption studies have revealed that none of these hemoglobinase Plms are
essential in the parasite asexual blood stage lifecycle, indicating a high degree of
redundancy in the hemoglobin catabolic pathway.23-25 In contrast, the three other
plasmepsin subtypes expressed in the P. falciparum blood stages, Plms V,26-28 IX,
and X,29-32 all appear to be essential for parasite viability. The hemoglobinase
plasmepsins (Plm I, II, IV) share high sequence homology with Plms IX and X, but
not Plm V. It might therefore be expected that inhibitors developed to target the
hemoglobinase Plms would exhibit activity in cell-based assays only if they
additionally target Plms IX and/or Plm X. Recombinant expression of both Plms IX
and X has been recently reported,29,30 but this could be achieved only in higher
eukaryotic protein expression systems, such as insect or mammalian cells. For our
further work to develop the hydroxyethylamine based inhibitors (S,R)-1a-c as anti-
malarials we therefore used Plm IV as a readily accessible model plasmepsin, an
approach also supported by the previously observed good correlation between
inhibition of this enzyme and potency in parasite growth assays in erythrocytes.11,20
The successful development of protease inhibitors as drugs requires optimization of
on-target potency and minimization of undesirable off-target activity, particularly
against related host proteases. The human lysosomal aspartic protease cathepsin D
(Cat D) plays critical roles in protein catabolism and retinal function.33 Recent work
focused on development of inhibitors of the human aspartic protease β-secretase
(BACE1) revealed the importance of ensuring selectivity against Cat D in order to
avoid off-target ocular toxicity.34,35 In view of this, we decided that the next step for
optimization of the hydroxyethylamine based Plm inhibitors 1 should aim to improve
their selectivity for their malarial target(s) over human Cat D.
2. Results and discussion
2.1. Structural factors determining the selectivity of inhibitor binding to Plms vs
Cat D
Our previous SAR investigations revealed that the substituents of the inhibitor (S,R)-1
occupying the prime sub-pockets (part S’, Table 1) are optimal for Plm inhibition.11
Therefore, we focused our efforts on optimisation of selectivity inducing motifs in the

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substituents occupying the non-prime sub-pockets (Table 1). To assess the differences
in inhibitor recognition between Plms IV, IX, X and Cat D, we generated a structure-
based sequence alignment of these proteins and compared their interactions with
inhibitor 1b in docking models. Since Plm IX and X lack experimentally determined
structures and in order to avoid possible inaccuracies associated with the use of
homology models, the docking studies were performed on the crystal structure of Plm
IV (PDB ID 2ANL), which is the closest homologue with an available crystal
structure 36
As can be seen from Fig. 1A, the S3 sub-pocket shows the largest
.
differences in amino acid composition between the Plms and Cat D. Additionally, this
revealed that the S3 sub-pocket of Plm IV is wider, more shallow and more
hydrophobic than that of Cat D (Fig. 1B). For these reasons, selectivity improvement
was first attempted by modifying the N,N-dipropylamide moiety in inhibitors (S,R)-1
which occupies the S3 sub-pocket.
Fig. 1. (A) Structure-based sequence alignment of the amino acid residues making up
the S1’ and S1-S4 pockets of Plm IV, Plm IX, Plm X and Cat D. (B) Surface
representation of the S3 and S4 pockets in Plm IV and Cat D docking models with the
hydroxyethylamine based inhibitor (S,R)-1b. The figure was prepared in PyMol.37
Surface oxygen atoms are colored in red, nitrogens in blue, sulfurs in yellow and
hydrogens and carbons in grey.

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Several N,N-disubstituted amide analogues (S,R)-2a,b,d-f were synthesized (see
Section 2.3) bearing hydrophobic groups that would prefer the more hydrophobic S3
sub-pocket of Plm IV. Unexpectedly the N,N-diethyl and N,N-dimethyl substituted
analogues (S,R)-2a and (S,R)-2b showed the highest selectivity factor for Plm IV
inhibition over Cat D, even though our docking studies indicated that the wider S3
sub-pocket of Plm IV could accommodate bulkier groups. Analogue (S,R)-2c bearing
N-hydroxyethyl groups showed 3-fold weaker Plm IV inhibition potency than
compound (S,R)-2a. Introduction of larger linear substituents such as N,N-dipropyl
(compound (S,R)-1b), N,N-di(methoxyethyl) (compound (S,R)-2d) and N,N-di(3,3,3-
trifluoropropyl) (compound (S,R)-2e) resulted in improved Cat D inhibition and
correspondingly lower selectivity factors, suggesting that these groups fit well in the
deep S3 sub-pocket of Cat D. Analogue (S,R)-2f bearing a N,N-diisobutyl amide
showed poor inhibition of both enzymes, indicating that this group is too large to fit
into the S3 sub-pocket of both Plm IV and Cat D.
Table 2. SAR of N,N-disubstituted amide analogues (S,R)-1b, 2a-f
Comp.
R
IC50
IC50
Sa
Plm IV, µΜ
Cat D, µΜ
(S,R)-1b
(S,R)-2a
(S,R)-2b
n-Pr
Et
0.024b
0.014
0.087
0.068
0.037
0.21
0.042b
0.25
0.5
1.8
17.9
5.7
Me
(S,R)-2c HOCH2CH2
(S,R)-2d MeOCH2CH2
(S,R)-2e CF3CH2CH2
(S,R)-2f (CH3)2CHCH2
0.27
0.10
0.12
1.3
4.0
2.7
0.57
2.6
0.5
b
a Selectivity factor of Plm IV/Cat D inhibition; Data from literature11

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Fig. 2. Docking models of compounds (S,R)-2e and (S,R)-3d in crystal structures of
Plm IV and Cat D
We further explored N-monosubstituted amide analogues (S,R)-3a-m (see Section 2.3
for synthesis). The best compounds in this series showed similar Plm IV inhibition
potency compared to the most potent N,N-disubstituted amides (S,R)-2. Gratifyingly,
these appeared to be less potent Cat D inhibitors, leading to improved selectivity
factors. The removal of one N-substituent was more beneficial for compounds with
larger or branched substituents (e.g. (S,R)-2e and (S,R)-2d compared to (S,R)-3d and
(S,R)-3f) while for compounds bearing smaller substituents a slight decrease in Plm
IV inhibitory activity was observed e.g. (S,R)-1b (Table 2) compared to (S,R)-1d
(Table 3). The docking studies suggested that the introduction of a hydrogen bond
donor (inhibitors (S,R)-3c,e,i,j,) or hydrogen bond acceptor ((S,R)-3a,l,f) group in the
S3 sub-pocket substituents could potentially enable additional electrostatic
interactions with electron-rich functional groups in this pocket. Hydrogen bond donor
groups could interact with Asn13 and Leu14 backbone carbonyl in Plm IV; and
Asp323, Tyr15, Gln14 and Ala13 in CatD, whereas for the hydrogen bond acceptors
the most likely interactions are with Asn13 in Plm IV and Gln14 in Cat D (Fig. S1,
see supporting information). Although the introduction of hydrogen bond donor or
acceptor groups in the S3 sub-pocket occupying substituent reduced Plm IV inhibition
activity (up to 2 times if comparing 1b and 3a), it also produced the most selective

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ligand in this series - (S,R)-3a, as the activity decrease for Cat D is even higher (up to
19 times if comparing (S,R)-1b and (S,R)-3a). The docking studies suggest that the
relatively higher drop in activity against Cat D for the compounds bearing the
hydrogen bond donor or acceptor groups is due to an unfilled hydrophobic sub-pocket
(resulting in an entropic penalty of solvating the non-polar sub-pocket). That is, the
position of the amide substituent remains the same in both enzymes due to additional
interactions with aforementioned residues (Fig.3), but by doing so it creates a
situation where the Cat D hydrophobic sub-pocket is filled with water resulting in an
entropic penalty and reduced Cat D inhibition activity.
Fig. 3. Docking models of compound 3a in complex with Plm IV and Cat D.
Despite varying hydrogen bond donor and acceptor groups in the S3 sub-pocket
filling substituents, docking studies also suggest that the main factor affecting Plm IV
and Cat D inhibition potency is the size and shape of the substituent; branched and
long substituents cannot fit into the narrow S3 recess of Cat D whereas the open S3
sub-pocket of Plm IV can accommodate such substituents. The weakest inhibitor of
the N-mono-substituted amide analogues was compound (S,R)-3g. This could be
explained by the protonated amine group as the amide substituent which, according to
the docking studies, tends to form hydrogen bonds and ionic interactions with
residues outside of the S3 sub-pocket. Altogether these results indicate that the
selectivity against Cat D can be improved by targeting the S3 sub-pocket with mono-
substituted amide moieties containing linear or branched hydrophobic groups.

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Table 3. SAR of N-mono-substituted amide analogues (S,R)-1d, 3a-m
Comp.
R
IC50
IC50
Sa
Plm IV, µΜ Cat D, µΜ
(S,R)-1d
n-Pr
0.038b
0.048
0.030
0.093
0.024
0.10
0.11b
2.1
2.9
43.8
25.3
24.2
24.2
16.6
15.0
13.3
14.8
12.2
6.8
(S,R)-3a MeOC(CH3)2CH2
(S,R)-3b c-PrCH2
(S,R)-3c HOCH2CH2CH2
0.76
2.25
0.58
1.66
0.75
4.8
(S,R)-3d
(S,R)-3e
(S,R)-3f
(S,R)-3g
(S,R)-3h
(S,R)-3i
(S,R)-3j
(S,R)-3k
(S,R)-3l
(S,R)-3m
CF3CH2CH2
HOC(CH3)2CH2
MeOCH2CH2
Me2NCH2CH2
t-BuCH2
0.05
0.36
0.027
0.12
0.40
1.46
1.42
0.22
0.15
0.15
HOCH2C(CH3)2
HOCH2CH2
PhCH2
0.21
0.038
0.031
0.09
5.8
t-BuOCH2CH2
c-HexCH2
4.8
1.7
a Selectivity factor of Plm IV/Cat D inhibition; b Data from literature11
The S4 sub-pocket is another inhibitor binding region which is notably distinct
between Plm IV and Cat D (Fig. 1A). Although predominantly hydrophobic in both
enzymes, the S4 sub-pocket of Plm IV is flatter and more solvent exposed. Therefore,
we investigated SAR for substituents occupying the S4 sub-pocket in the series of
compounds (S,R)-4a-g (see Section 2.3. for synthesis). Installation of an N,N-
dipropylamide group (compound (S,R)-4g) resulted in practically unchanged Plm IV
inhibitory activity compared to parent compound 1b, however this was paralleled by a

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more than 15-fold drop in activity against Cat D resulting in improvement of the
selectivity factor. Installation of fluorine or removal of the S4 filling substituent
provided compounds 4b,c with slightly decreased Plm IV inhibitory potency, whereas
inhibition of Cat D was considerably lower which again improved the selectivity
factor. Installation of chlorine in the benzene ring (compound 4d) improved the
activity for both Plm IV and Cat D. Cyano, methyl and trifluoromethyl groups in the
S4 sub-pocket (compounds 4a,e-f) made less difference for inhibitor binding to Plm
IV and Cat D. Collectively, these effects of S4 occupying substituents are difficult to
explain from our molecular modelling data, and presumably arise from an interplay of
hydrophobic and polar non-covalent interactions.
Table 4. SAR of phenylgroup substitution in analogues 1b,c, 4a-g
Comp.
R
IC50
IC50
Sa
Plm IV, µΜ Cat D, µΜ
(S,R)-1b
(S,R)-1c
(S,R)-4a
(S,R)-4b
(S,R)-4c
(S,R)-4d
(S,R)-4e
(S,R)-4f
(S,R)-4g
1-piperidinyl
0.024b
0.006b
0.023
0.058
0.050
0.008
0.015
0.059
0.018
0.042b
0.054b
0.21
1.8
9.0
Ph
Me
9.1
H
1.15
19.8
20
F
1.0
Cl
CF3
0.096
0.067
0.56
12.0
4.5
CN
9.5
(n-Pr)2NC(=O)
0.7
38.9
a Selectivity factor of Plm IV/Cat D inhibition; b Data from literature11
Considering that the lack of an S4 occupying substituent considerably improves
selectivity against Cat D, analogues (S,R)-5a,b of the most potent N-mono-
substituted amide inhibitors (S,R)-3d,h were prepared and tested (Table 5, see

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Section 2.3. for synthesis). As expected, a slight drop in Plm IV inhibitory potency
was observed; however the modifications appeared to be additive for a considerable
improvement in the selectivity factor.
Table 5. Combining selectivity inducing structural motives in analogues (S,R)-5a,b
IC50
IC50
Comp.
R
Sa
Plm IV, µΜ Cat D, µΜ
(S,R)-5a
(S,R)-5b
t-BuCH2
0.076
0.15
3.8
4.9
50.0
32.6
CF3CH2CH2
a Selectivity factor of Plm IV/Cat D inhibition
2.2. Relation of Plm subtype inhibition with P. falciparum growth inhibition.
The capacity to inhibit growth in vitro of asexual blood stage P. falciparum was
determined for selected compounds (S,R)-2a,3a,b,d,h,4b,c,g,5a,b (Table 6). In all
cases, the compounds showed strong growth inhibitory potency with EC50 values in
the low nanomolar range. Inhibitory activity against Plm I, Plm II and Plm IV was
determined for the same compounds. This revealed no correlation between inhibitory
potency in the parasite growth assay and inhibition of Plm I and II enzyme activity. In
contrast, there was a much better correlation between parasite growth inhibition
potency and Plm IV inhibition potency, although even here there were some notable
exceptions; for instance, the most potent growth inhibitory compound, (S,R)-4b, was a
3-fold weaker inhibitor of Plm IV than compound (S,R)-4g, yet showed a 20-fold
better inhibition of parasite growth than (S,R)-4g.

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Table 6. Plm I, II, IV inhibition and P. falciparum growth inhibition activity of
selected compounds
b
IC50
IC50
IC50
Sa
EC50
Comp.
(S,R)-2a
(S,R)-3a
(S,R)-3b
(S,R)-3d
(S,R)-3h
(S,R)-4b
(S,R)-4c
(S,R)-4g
(S,R)-5a
(S,R)-5b
Plm I, µΜ Plm II, µΜ Plm IV, µΜ
Pf Growth, nΜ
0.8
7.4
0.16
5.4
0.014
0.048
0.030
0.024
0.027
0.058
0.050
0.018
0.076
0.15
17.9
43.8
25.3
24.2
14.8
19.8
20
1.5
2.0
1.8
2.0
6.0
0.3
1.5
6.0
2.0
6.0
1.8
0.5
2.5
2.2
2.0
0.85
1.7
3.1
1.1
1.1
0.78
5.6
0.27
7.1
38.9
50.0
32.6
10.3
10.4
b
a Selectivity factor of Plm IV/Cat D inhibition; The EC50 values for P. falciparum
growth were determined using a SYBR Green-based assay with an incubation time of
96 h (2 erythrocytic cycles). Samples were each measured in triplicate, in 2 separate
biological assays. Compound TCMDC-13467411,20 was used as a positive control (see
Supporting Information).
These results implied that the important parasite target(s) engaged by the growth
inhibitory compounds are not the hemoglobinase plasmepsins. Recent reports have
shown that inhibitors of the non-hemoglobinase plasmepsins Plm IX and Plm X
(which are structurally similar to Plm I, II and IV) can potently block parasite
replication.29,30 A key biological function of Plm X is the proteolytic maturation of
SUB1, a parasite subtilisin-like serine protease that plays an essential role in
regulating parasite release (egress) from the infected host erythrocyte.38 SUB1
maturation comprises 2 steps in which the initial ~82 kDa pre-proenzyme is cleaved
to form first a 54 kDa protein (p54) then a 47 kDa terminal product (p47) which
accumulates during the latter ~12 h of intra-erythrocytic parasite development. Whilst
the first SUB1 processing step is autocatalytic, the second p54-to-p47 step is believed
to be mediated by Plm X.29, 30 We used a Western blot-based assay to examine the
effects of selected compounds (S,R)-2a, (S,R)-4b and (S,R)-4c on both SUB1
maturation and parasite egress (Fig. 4).

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Fig.4. Inhibition of P. falciparum SUB1 maturation and egress by selected
compounds indicates that they target Plm X. (A) Synchronous cultures of immature
intracellular parasites were treated for ~8 h with compounds (S,R)-2a or (S,R)-4b,c
(10 nM), or vehicle only (DMSO, 1% v/v), or the cGMP-dependent protein kinase
inhibitor (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3-
yl]pyrimidin-2-amine (compound 2; C2, 2 µM) which inhibits egress but not SUB1
maturation. Extracts of the parasites were then analyzed by Western blot, probing
with an antibody to SUB1. The positions of migration of the SUB1 p54 and p47
forms (green arrow) are indicated. The schematic below indicates the mode by which
Plm X converts SUB1 p54 to the terminal p47 form. (B) Parasites treated for ~ 24 h as
in (A) were transferred to fresh medium containing the various compounds and
allowed to undergo egress for 4 h before the culture supernatants were analysed by
Western blot, probing with antibodies to the parasite protein SERA5. The positions of
migration of the SERA5 precursor, a processing intermediate and the terminal P50
form (green arrow) are indicated. The schematic below indicates the mode by which
SUB1 converts the SERA5 precursor to the P50 form.
Egress was quantified by measuring the release of a soluble parasite protein called
SERA5 into parasite culture supernatants. SERA5 is also a SUB1 substrate, and is
generally released in a P50 form that results from SUB1-mediated cleavage of a larger
precursor. Release of correctly processed SERA5 P50 is therefore an indicator of both
SUB1 activity and efficiency of egress. As shown in Fig. 4A, treatment of developing
intracellular parasites with the three Plm inhibitors (S,R)-2a, (S,R)-4b and (S,R)-4c
resulted in a relative enrichment of the p54 form of SUB1, indicating inhibition of
conversion of p54 to the terminal p47 form. Analysis of culture supernatants from
treated parasites allowed to proceed to egress (Fig. 4B) showed that compounds
(S,R)-4b and (S,R)-4c both produced a clear reduction in SERA5 P50 release, whilst
all three compounds produced an increase in the release of SERA5 precursor or

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processing intermediates, indicating defects in egress and SERA5 processing. These
results strongly suggest that the mechanism of parasite growth inhibition by
compounds (S,R)-4b and (S,R)-4c (as well as possibly (S,R)-2a) involves Plm X
inhibition, since the effects on egress were linked to inhibition of SUB1 maturation
and its enzymatic activity against an endogenous substrate. Given their structural
similarity, it is likely that the other compounds 3a,b,d,h, 4g and 5a,b exerting potency
in the parasite growth assay (Table 6) also target Plm X.
2.3. Synthesis of inhibitors (S,R)–2-5
Plm inhibitors (S,R)–2-5 were synthesized from substituted benzoic acids 15, 17 and
19 and either enantiomerically pure amino alcohol (R,S)–11 or the corresponding
racemate rac-11 (Schemes 1-5). Inhibitors (S,R)–2b-e, (S,R)–3a,c,e-g,i,j,l,m, (S,R)–
4c,f,g and (S,R)–5a,b were obtained from enantiomerically pure amino alcohol (R,S)–
11, whereas targets 2a,f, 3b,d,h,k and 4a,b,d,e were obtained as mixtures of
stereoisomers from racemic alcohol rac-11. Enantiomerically pure inhibitors (S,R)–
2a,f, (S,R)–3b,d,h,k and (S,R)–4a,b,d,e were obtained by separation of diastereomers
using chromatography on silica gel, followed by separation of enantiomers using the
chromatography on a chiral stationary phase.
Synthesis of aminoalcohol 11 commenced with N-Boc protection of rac-6 (Scheme
1). Dihydroxylation of the resulting carbamate rac–7 with AD-mix-α afforded diol 8
as a 2:3 mixture of syn:anti diastereomers and a 2:1 mixture of enantiomers.
Enantiomerically pure diol (S,S)–8 was obtained by an initial separation of syn/anti
diastereomers using chromatography on silica gel, followed by separation of syn–8
enantiomers by chromatography on a chiral stationary phase. The major enantiomer
turned out to be the desired diol (S,S)–8 as evidenced by comparison of optical
rotation data with that from the literature.39 Conversion of (S,S)–8 into epoxide (S,S)–
9 under Mitsunobu conditions was followed by aminolysis with 2-(3-
methoxyphenyl)propan-2-amine11 to afford N-Boc protected amino alcohol (S,R)–10.
Finally, the cleavage of N-Boc protecting group yielded amino alcohol (R,S)–11.
Benzoic acids 15a-s were prepared from dimethyl 5-bromoisophthalate (Scheme 2).
Pd-catalyzed amination with piperidine afforded 12a, which was hydrolysed to
isophthalic monoester 13a under basic conditions. Subsequent HBTU-mediated

ACCEPTED MANUSCRIPT
condensation with amines afforded amides 14a-s, which were hydrolysed to benzoic
acids 15a-s.
Scheme 1. Synthesis of amino alcohol intermediate (R,S)–11. Reagents and
conditions: a) Boc2O (1.25 equiv), NEt3 (2 equiv), DCM, rt, 2 h, 87%. b) AD-mix-α
(1 equiv), 1:1 (v/v) t-BuOH:water, rt, 20 h. c) preparative HPLC on chiral stationary
phase (Chiralpack-ID), 25% in two steps. d) Ph3P (1.1 equiv), DEAD (1.1 equiv),
o
CHCl3, 85 C, 48 h, 64%. e) 2-(3-Methoxyphenyl)propan-2-amine11 (1.05 equiv), i-
PrOH, 70 oC, 40 h, 73%. f) 4 M HCl in dioxane, rt, 6h, 99%.
Scheme 2. Synthesis of Plm inhibitors (S,R)–2a-f and (S,R)–3a-m. Reagents and
conditions: a) piperidine (1 equiv), Pd(OAc)2 (5 mol%), rac-BINAP (5 mol%),
o
Cs2CO3 (1.5 equiv), toluene, 100 C, 18 h, 88%. b) General procedure A: aqueous 1
M NaOH (1 equiv), MeOH, rt, 16 h. c) General procedure B: R1R2NH (1.2 equiv),
HBTU (1 equiv), NEt3 (2 equiv), DMF, rt, 2 h. d) General procedure C: aqueous 1 M
NaOH (1.5 equiv), MeOH, 50 oC, 18 h. e) General procedure D: amino alcohol (R,S)–
11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv), DMF, rt, 16 h. f) amino alcohol

ACCEPTED MANUSCRIPT
rac–11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv), DMF, rt, 16 h; then separation
of enantiomers by HPLC on chiral stationary phase (Chiralpack-ID).
Table 7. Substitution pattern of compounds in Scheme 2.
R1
R2
No.
14a
14b
14c
14d
14e
14f
14g
14h
14i
No.
15a
15b
15c
15d
15e
15f
15g
15h
15i
No.
Comments
Et
Et
(S,R)-2a from rac–11 (step f)
Me
Me
(S,R)-2b
(S,R)-2c
(S,R)-2d
(S,R)-2e
HOCH2CH2
HOCH2CH2
MeOCH2CH2 MeOCH2CH2
CF3CH2CH2 CF3CH2CH2
(CH3)2CHCH2 (CH3)2CHCH2
(S,R)-2f
(S,R)-3a
from rac–11 (step f)
H
H
H
H
H
H
H
H
H
H
H
H
H
MeOC(CH3)2CH2
c-PrCH2
(S,R)-3b from rac–11 (step f)
HOCH2CH2CH2
CF3CH2CH2
HOC(CH3)2CH2
MeOCH2CH2
Me2NCH2CH2
t-BuCH2
(S,R)-3c
(S,R)-3d from rac–11 (step f)
14j
14k
14l
15j
15k
15l
(S,R)-3e
(S,R)-3f
(S,R)-3g
14m 15m
(S,R)-3h from rac–11 (step f)
14n
14o
14p
14q
14r
14s
15n
15o
15p
15q
15r
15s
HOCH2C(CH3)2
HOCH2CH2
PhCH2
(S,R)-3i
(S,R)-3j
(S,R)-3k from rac–11 (step f)
t-BuOCH2CH2
c-HexCH2
(S,R)-3l
(S,R)-3m
Similar synthetic approach was also used for the preparation of acids 17b-i (Scheme
3). Accordingly, monoesters 13c-f were obtained from commercially available

ACCEPTED MANUSCRIPT
dimethyl isophthates. The synthesis of methyl ester 13b required an initial Pd-
catalyzed alkylation of dimethyl 5-bromoisophthalate with methylboronic acid,
followed by hydrolysis of one of the two ester moieties. Benzoic acids 13b-f were
converted into amides 16b-f and ester moieties were hydrolysed to afford acids 17b-f.
Benzoic acid 17h was obtained directly from trifluoromethyl isophthalic acid and n-
Pr2NH in the presence of HBTU, whereas the synthesis of 17i was accomplished by
Cu(I)-catalyzed substitution of bromide in benzoic acid 17c for cyano group (Scheme
3).
O
O
MeO
OMe
R
O
O
O
O
R=H, F, Cl, Br
HO
OMe
c
(n-Pr)2N
OMe
b
a (for R=Br)
R
R
O
O
13b-f
16b-f
MeO
OMe
d
O
O
O
O
Me
12b
c
(n-Pr)2N
OH
HO
OH
R
CF3
O
O
f or g
17b-f,h
Me
Me
OMe
(n-Pr)2N
N
N
e (for R=Br)
H
H
OH
O
O
R
4a-f
(S,R)-
(n-Pr)2N
OH
CN
17i
Scheme 3. Synthesis of Plm inhibitors (S,R)–4a-f. Reagents and conditions: a)
MeB(OH)2 (1.2 equiv), Pd(dppf)Cl2xCH2Cl2 (5 mol%), K3PO4 (3 equiv), toluene, 90
°C, 18 h. b) General procedure A: aqueous 1 M NaOH (1 equiv), MeOH, rt, 16 h. c)
General procedure B: (n-Pr)2NH (1.2 equiv), HBTU (1 equiv), NEt3 (2 equiv), DMF,
rt, 2 h. d) General procedure C: aqueous 1 M NaOH (1.5 equiv), MeOH, 50 oC, 18 h.
e) CuCN (2 equiv), NMP, 160 oC, 6 h. f) General procedure D: amino alcohol (R,S)–
11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv), DMF, rt, 16 h. g) amino alcohol
rac–11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv), DMF, rt, 16 h; then separation
of enantiomers by HPLC on chiral stationary phase (Chiralpack-ID).

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Table 8. Substitution pattern of compounds in Scheme 3.
R
No.
No.
13b
13c
13d
13e
13f
-
No.
16b
16c
16d
16e
16f
-
No.
17b
17c
17d
17e
17f
17h
17i
No.
Comments
Me
Br
H
(S,R)–4a
-
from rac–11 (step g)
12b
-
-
-
-
-
-
(S,R)–4b
(S,R)–4c
(S,R)–4d
(S,R)–4e
(S,R)–4f
from rac–11 (step g)
F
Cl
CF3
CN
from rac–11 (step g)
from rac–11 (step g)
-
-
Synthesis of benzoic acid 17g (Scheme 4) commenced with hydrolysis of
commercially available dimethyl iodo-isophthalate to the corresponding isophthalic
acid 18, followed by Pd-catalyzed methoxycarbonylation40 to afford ester 13g.
Subsequent conversion to diamide 16g in the presence of HBTU was followed by
ester hydrolysis to form benzoic acid 17g (Scheme 4). The amide bond formation-
hydrolysis sequence was also used for the preparation of benzoic acids 19a,b (Scheme
5).
Scheme 4. Synthesis of Plm inhibitor (S,R)–4g. Reagents and conditions: a) aqueous
o
1 N NaOH (3 equiv), MeOH, 40 C, 18 h. b) Pd(dppf)Cl2xCH2Cl2 (10 mol%), NEt3
o
(2.2 equiv), CO (70 psi), MeOH, 100 C, 18 h. c) General procedure B: (n-Pr)2NH
(2.2 equiv), HBTU (2 equiv), NEt3 (4 equiv), DMF, rt, 2 h. d) General procedure C:
aqueous 1 M NaOH (1.5 equiv), MeOH, 50 oC, 18 h. e) General procedure D: amino
alcohol (R,S)–11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv), DMF, rt, 16 h.

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Scheme 5. Synthesis of Plm inhibitors 5a,b. Reagents and conditions: a) General
procedure B: (n-Pr)2NH (1.2 equiv), HBTU (1 equiv), NEt3 (2 equiv), DMF, rt, 2 h. b)
General procedure C: aqueous 1 M NaOH (1.5 equiv), MeOH, 50 oC, 18 h. c) General
procedure D: amino alcohol (R,S)–11 (1.0 equiv), HBTU (1.0 equiv), NEt3 (4 equiv),
DMF, rt, 16 h.
Summary
The optimization of hydroxyethylamine based Plm inhibitors was performed with the
aim of improving selectivity against the related human aspartic protease Cat D. The
studies were performed using Plm IV as a readily accessible model protein, the
inhibition of which was previously found to correlate with Plasmodium falciparum
growth inhibition. Based on sequence alignment of Plm IV and Cat D, putative
selectivity inducing structural motifs were sought in S3 and S4 sub-pocket-occupying
substituents of the inhibitors. Installation of an S3 sub-pocket targeting mono-
substituted amide moiety in compounds (S,R)-3 containing linear or branched
hydrophobic groups resulted in up to 40-fold selectivity (compound (S,R)-3a) against
Cat D. Plm IV inhibitors (S,R)-4b,c with no substituents or fluorine targeting the S4
sub-pocket led to 20-fold selectivity against Cat D, though with some loss of Plm IV
inhibition potency. Surprisingly, installation of amide as the S4 sub-pocket filling
group in compound (S,R)-4g resulted in potent Plm IV inhibition with almost 40-fold
selectivity against Cat D. Selectivity-inducing factors in S3 and S4 positions were
additive as evidenced by compound (S,R)-5a (50-fold selectivity). Determination of
P. falciparum growth inhibition potency for ten of the new Plm inhibitors showed
them to display activities in the low nanomolar range. The potent anti-malarial
activity did not correlate with the relatively weak inhibition of Plm I and II, whilst in
contrast there was a much better correlation with Plm IV inhibition. More detailed
investigation of the mechanism of action of the selected compounds showed that they
interfered with parasite egress and maturation of the parasite serine protease SUB1,
indicating a strong link between anti-malarial activity and inhibition of the non-
hemoglobinase plasmepsin and SUB1 maturase Plm X. Future studies should clarify

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whether cooperative Plm IV and Plm X inhibition or only Plm X inhibition is
necessary to achieve optimal anti-malarial activity.
Acknowledgement
This work was supported by the European Regional Development Fund (Agreement
No. 1.1.1.1/16/A/290).
References
(1) WHO, World Malar. Rep. 2016 World Health Organ. Geneva 2016
http://www.who.int/malaria/publications/world-malaria-report-2016/report/en/
(2) Wells, T. N. C.; van Huijsduijnen, R. H.; Van Voorhis, W. C. Malaria
Medicines: A Glass Half Full? Nat. Rev. Drug. Discov. 2015, 14 (6), 424–442.
(3) WHO, http://www.Who.Int/Malaria/Publications/Atoz/9789241564991/En/.
(4) Wells, T. N. C.; Alonso, P. L.; Gutteridge, W. E. New Medicines to Improve
Control and Contribute to the Eradication of Malaria. Nat. Rev. Drug. Discov.
2009, 8 (11), 879–891.
(5) Hyde, J. E. Drug-Resistant Malaria − an Insight. FEBS J. 2007, 274 (18),
4688–4698.
(6) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K.
M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; et al. Artemisinin Resistance in
Plasmodium Falciparum Malaria. N. Engl. J. Med. 2009, 361 (5), 455–467.
(7) Gamo, F.-J.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alvarez, E.; Lavandera, J.-L.;
Vanderwall, D. E.; Green, D. V. S.; Kumar, V.; Hasan, S.; et al. Thousands of
Chemical Starting Points for Antimalarial Lead Identification. Nature 2010,
465 (7296), 305–310.
(8) Calderón, F.; Barros, D.; Bueno, J. M.; Coterón, J. M.; Fernández, E.; Gamo, F.
J.; Lavandera, J. L.; León, M. L.; Macdonald, S. J. F.; Mallo, A.; et al. An
Invitation to Open Innovation in Malaria Drug Discovery: 47 Quality Starting
Points from the TCAMS. ACS Med. Chem. Lett. 2011, 2 (10), 741–746.
(9) Wells, T. N. C.; Willis, P.; Burrows, J. N.; van Huijsduijnen, R. H. Open Data
in Drug Discovery and Development: Lessons from Malaria. Nat. Rev. Drug
Discov. 2016, 15, 661.
(10) Pieroni, M.; Azzali, E.; Basilico, N.; Parapini, S.; Zolkiewski, M.; Beato, C.;
Annunziato, G.; Bruno, A.; Vacondio, F.; Costantino, G. Accepting the

ACCEPTED MANUSCRIPT
Invitation to Open Innovation in Malaria Drug Discovery: Synthesis, Biological
Evaluation, and Investigation on the Structure–Activity Relationships of
Benzo[b]Thiophene-2-Carboxamides as Antimalarial Agents. J. Med. Chem.
2017, 60 (5), 1959–1970.
(11) Jaudzems, K.; Tars, K.; Maurops, G.; Ivdra, N.; Otikovs, M.; Leitans, J.;
Kanepe-Lapsa, I.; Domraceva, I.; Mutule, I.; Trapencieris, P.; et al. Plasmepsin
Inhibitory Activity and Structure-Guided Optimization of a Potent
Hydroxyethylamine-Based Antimalarial Hit. ACS Med. Chem. Lett. 2014, 5
(4), 373–377.
(12) Ciana, C.-L.; Siegrist, R.; Aissaoui, H.; Marx, L.; Racine, S.; Meyer, S.;
Binkert, C.; de Kanter, R.; Fischli, C.; Wittlin, S.; et al. Novel in Vivo Active
Anti-Malarials Based on a Hydroxy-Ethyl-Amine Scaffold. Bioorg. Med.
Chem. Lett. 2013, 23 (3), 658–662.
(13) Haque, T. S.; Skillman, A. G.; Lee, C. E.; Habashita, H.; Gluzman, I. Y.;
Ewing, T. J. A.; Goldberg, D. E.; Kuntz, I. D.; Ellman, J. A. Potent, Low-
Molecular-Weight Non-Peptide Inhibitors of Malarial Aspartyl Protease
Plasmepsin II. J. Med. Chem. 1999, 42 (8), 1428–1440.
(14) Nöteberg, D.; Hamelink, E.; Hultén, J.; Wahlgren, M.; Vrang, L.; Samuelsson,
B.; Hallberg, A. Design and Synthesis of Plasmepsin I and Plasmepsin II
Inhibitors with Activity in Plasmodium Falciparum-Infected Cultured Human
Erythrocytes. J. Med. Chem. 2003, 46 (5), 734–746.
(15) Ersmark, K.; Samuelsson, B.; Hallberg, A. Plasmepsins as Potential Targets for
New Antimalarial Therapy. Med. Res. Rev. 2006, 26 (5), 626–666.
(16) Ersmark, K.; Feierberg, I.; Bjelic, S.; Hamelink, E.; Hackett, F.; Blackman, M.
J.; Hultén, J.; Samuelsson, B.; Åqvist, J.; Hallberg, A. Potent Inhibitors of the
Plasmodium Falciparum Enzymes Plasmepsin I and II Devoid of Cathepsin D
Inhibitory Activity. J. Med. Chem. 2004, 47 (1), 110–122.
(17) Boss, C.; Corminboeuf, O.; Grisostomi, C.; Meyer, S.; Jones, A. F.; Prade, L.;
Binkert, C.; Fischli, W.; Weller, T.; Bur, D. Achiral, Cheap, and Potent
Inhibitors of Plasmepsins I, II, and IV. ChemMedChem 2006, 1 (12), 1341–
1345.
(18) Meyer, M. J.; Goldberg, D. E. Recent Advances in Plasmepsin Medicinal
Chemistry and Implications for Future Antimalarial Drug Discovery Efforts -
Curr. Top. Med. Chem. 2012, 12 (5), 445–455.

ACCEPTED MANUSCRIPT
(19) Kumar Singh, A.; Rajendran, V.; Singh, S.; Kumar, P.; Kumar, Y.; Singh, A.;
Miller, W.; Potemkin, V.; Poonam; Grishina, M.; et al. Antiplasmodial Activity
of Hydroxyethylamine Analogs: Synthesis, Biological Activity and Structure
Activity Relationship of Plasmepsin Inhibitors. Bioorg. Med. Chem. 2018, 26
(13), 3837–3844.
(20) Rasina, D.; Otikovs, M.; Leitans, J.; Recacha, R.; Borysov, O. V.; Kanepe-
Lapsa, I.; Domraceva, I.; Pantelejevs, T.; Tars, K.; Blackman, M. J.; et al.
Fragment-Based Discovery of 2-Aminoquinazolin-4(3H)-Ones As Novel Class
Nonpeptidomimetic Inhibitors of the Plasmepsins I, II, and IV. J. Med. Chem.
2016, 59 (1), 374–387.
(21) Rasina, D.; Stakanovs, G.; Borysov, O. V.; Pantelejevs, T.; Bobrovs, R.;
Kanepe-Lapsa, I.; Tars, K.; Jaudzems, K.; Jirgensons, A. 2-Aminoquinazolin-
4(3H)-One Based Plasmepsin Inhibitors with Improved Hydrophilicity and
Selectivity. Bioorg. Med. Chem. 2018, 26 (9), 2488–2500.
(22) Kinena, L.; Leitis, G.; Kanepe-Lapsa, I.; Bobrovs, R.; Jaudzems, K.; Ozola, V.;
Suna, E.; Jirgensons, A. Azole-Based Non-Peptidomimetic Plasmepsin
Inhibitors. Arch. Pharm. 2018, 351 (9), 1800151.
(23) Bonilla, J. A.; Bonilla, T. D.; Yowell, C. A.; Fujioka, H.; Dame, J. B. Critical
Roles for the Digestive Vacuole Plasmepsins of Plasmodium Falciparum in
Vacuolar Function. Mol. Microbiol. 2007, 65 (1), 64–75.
(24) Bonilla, J. A.; Moura, P. A.; Bonilla, T. D.; Yowell, C. A.; Fidock, D. A.;
Dame, J. B. Effects on Growth, Hemoglobin Metabolism and Paralogous Gene
Expression Resulting from Disruption of Genes Encoding the Digestive
Vacuole Plasmepsins of Plasmodium Falciparum. Int. J. Parasitol. 2007, 37 (3–
4), 317–327.
(25) Moura, P. A.; Dame, J. B.; Fidock, D. A. Role of Plasmodium Falciparum
Digestive Vacuole Plasmepsins in the Specificity and Antimalarial Mode of
Action of Cysteine and Aspartic Protease Inhibitors. Antimicrob. Agents
Chemother. 2009, 53 (12), 4968–4978.
(26) Hodder, A. N.; Sleebs, B. E.; Czabotar, P. E.; Gazdik, M.; Xu, Y.; O’Neill, M.
T.; Lopaticki, S.; Nebl, T.; Triglia, T.; Smith, B. J.; et al. Structural Basis for
Plasmepsin V Inhibition That Blocks Export of Malaria Proteins to Human
Erythrocytes. Nat. Struct. Mol. Biol. 2015, 22 (8), 590–596.

ACCEPTED MANUSCRIPT
(27) Sleebs, B. E.; Gazdik, M.; O’Neill, M. T.; Rajasekaran, P.; Lopaticki, S.;
Lackovic, K.; Lowes, K.; Smith, B. J.; Cowman, A. F.; Boddey, J. A.
Transition State Mimetics of the Plasmodium Export Element Are Potent
Inhibitors of Plasmepsin V from P. Falciparum and P. Vivax. J. Med. Chem.
2014, 57 (18), 7644–7662.
(28) Nguyen, W.; Hodder, A. N.; de Lezongard, R. B.; Czabotar, P. E.; Jarman, K.
E.; O’Neill, M. T.; Thompson, J. K.; Jousset Sabroux, H.; Cowman, A. F.;
Boddey, J. A.; et al. Enhanced Antimalarial Activity of Plasmepsin V Inhibitors
by Modification of the P2 Position of PEXEL Peptidomimetics. Eur. J. Med.
Chem. 2018, 154, 182–198.
(29) Pino, P.; Caldelari, R.; Mukherjee, B.; Vahokoski, J.; Klages, N.; Maco, B.;
Collins, C. R.; Blackman, M. J.; Kursula, I.; Heussler, V.; et al. A Multistage
Antimalarial Targets the Plasmepsins IX and X Essential for Invasion and
Egress. Science 2017, 358 (6362), 522.
(30) Nasamu, A. S.; Glushakova, S.; Russo, I.; Vaupel, B.; Oksman, A.; Kim, A. S.;
Fremont, D. H.; Tolia, N.; Beck, J. R.; Meyers, M. J.; et al. Plasmepsins IX and
X Are Essential and Druggable Mediators of Malaria Parasite Egress and
Invasion. Science 2017, 358 (6362), 518.
(31) Boddey, J. A. Plasmepsins on the Antimalarial Hit List. Science 2017, 358
(6362), 445.
(32) Crunkhorn, S. Blocking Malaria Parasite Invasion and Egress. Nat. Rev. Drug
Discov. 2017, 17, 17.
(33) Steinfeld, R.; Reinhardt, K.; Schreiber, K.; Hillebrand, M.; Kraetzner, R.;
Brück, W.; Saftig, P.; Gärtner, J. Cathepsin D Deficiency Is Associated with a
Human Neurodegenerative Disorder. Am. J. Hum. Genet. 2006, 78 (6), 988–
998.
(34) Zuhl, A. M.; Nolan, C. E.; Brodney, M. A.; Niessen, S.; Atchison, K.; Houle,
C.; Karanian, D. A.; Ambroise, C.; Brulet, J. W.; Beck, E. M.; et al.
Chemoproteomic Profiling Reveals That Cathepsin D Off-Target Activity
Drives Ocular Toxicity of β-Secretase Inhibitors. Nat. Commun. 2016, 7,
13042.
(35) Ghosh, A. K.; Reddy, B. S.; Yen, Y.-C.; Cárdenas, E. L.; Rao, K. V.; Downs,
D.; Huang, X.; Tang, J.; Mesecar, A. D. Design of Potent and Highly Selective

ACCEPTED MANUSCRIPT
Inhibitors for Human β-Secretase 2 (Memapsin 1), a Target for Type 2
Diabetes. Chem. Sci. 2016, 7 (5), 3117–3122.
(36) Clemente, J.C.; Govindasamy, L.; Madabushi, A.; Fisher, S.Z.; Moose, R.E.;
Yowell, C.A.; Hidaka, K.; Kimura, T.; Hayashi, Y.; Kiso, Y.; Agbandje-
McKenna, M.; Dame, J.B.; Dunn, B.M.; McKenna, R. Structure of the aspartic
protease plasmepsin 4 from the malarial parasite Plasmodium malariae bound to
anallophenylnorstatine-based inhibitor. Acta Crystallogr. 2006, D 62, 246–252.
(37) The PyMOL Molecular Graphics System, Version 1.7.4; Schrödinger, LLC.
(38) Thomas, J. A; Tan, M. S. Y; Bisson, C.; Borg, A.; Umrekar, T. R. ; Hackett, F.;
Hale, V.L.; Vizcay-Barrena, G.; Fleck, R. A.; Snijders, A. P.; Saibil, H. R.;
Blackman, M. J. A protease cascade regulates release of the human malaria
parasite Plasmodium falciparum from host red blood cells. Nat Microbiol. 2018
3 (4), 447-455.
(39) Ghosh, A. K.; Fidanze, S. Transition-State Mimetics for HIV Protease
Inhibitors:ꢀ
Stereocontrolled
Synthesis
of
Hydroxyethylene
and
Hydroxyethylamine Isosteres by Ester-Derived Titanium Enolate Syn and Anti-
Aldol Reactions. J. Org. Chem. 1998, 63 (18), 6146–6152.
(40) Kaganovsky, L.; Gelman, D.; Rueck-Braun, K. Trans-Chelating Ligands in
Palladium-Catalyzed Carbonylative Coupling and Methoxycarbonylation of
Aryl Halides. J. Organomet. Chem. 2010, 695 (2), 260–266.

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Highlights:
•
•
•
•
Peptidomimimetic plasmepsin inhibitors are developed using Plm IV as a model enzyme
Up to 50-fold selectivity against Cathepsin D is reached
Compounds show growth inhibition of P. falciparum at nanomolar concentrations
Inhibition of SUB1 maturation and parasite egress imply (co)inhibition of Plm X