Selective Inhibition of an Apicoplastic Aminoacyl-tRNA Synthetase from Plasmodium falciparum

Article abstract of DOI:10.1002/cbic.201200620

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antibacterial targets and so constitute

Full text of DOI:10.1002/cbic.201200620

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DOI: 10.1002/cbic.201200620
Selective Inhibition of an Apicoplastic Aminoacyl-tRNA
Synthetase from Plasmodium falciparum
Rob Hoen,[a] Eva Maria Novoa,[b] Alba Lꢀpez,[a] Noelia Camacho,[b] Laia Cubells,[b]
Pedro Vieira,[d] Manuel Santos,[d] Patricia Marin-Garcia,[e] Jose Maria Bautista,[e]
Alfred Cortꢁs,[b] Lluꢂs Ribas de Pouplana,*[b, c] and Miriam Royo*[a]
The resistance of malaria parasites to available drugs continues
to grow, and this makes the need for new antimalarial thera-
pies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential
enzymes and well-established antibacterial targets and so con-
stitute a promising set of targets for the development of new
antimalarials. Despite their potential as drug targets, apicoplas-
tic ARSs remain unexplored. We have characterized the lysyla-
tion system of Plasmodium falciparum, and designed, synthe-
sized, and tested a set of inhibitors based on the structure of
the natural substrate intermediate: lysyl-adenylate. Here we
demonstrate that selective inhibition of apicoplastic ARSs is
feasible and describe new compounds that that specifically in-
hibit Plasmodium apicoplastic lysyl-tRNA synthetase and show
antimalarial activities in the micromolar range.
Introduction
Malaria remains one of the most important infectious diseases
in the world, causing acute illness in more than 100 million
people and leading to approximately 1 million deaths annual-
ly.[1] In addition to its human cost, malaria represents a massive
economic burden, contributing substantially to poverty in the
developing world. Effective antimalarial drugs are available,
but their efficacy is compromised by emerging resistance.[2]
There is therefore a broad consensus about the need to devel-
op new antimalarial drugs. Malaria is caused by Plasmodium,
a genus of parasitic protists. At the moment there are over
200 species known of this genus, of which at least 11 can
infect humans. Of these, Plasmodium falciparum causes the
most severe form of malaria, being responsible for 90% of the
deaths.[1]
The P. falciparum genome project revealed many new poten-
tial drug targets,[3–9] several of which are enzymes that act in
the apicoplast, a relict plastid derived from secondary endo-
symbiosis of cyanobacteria,[10] which is essential for the para-
site’s survival.[11,12] Many of its bacterial-like enzymes are sub-
stantially different from their mammalian homologues,[13–15]
and this makes them excellent drug target candidates. Several
antibacterial drugs that are clinically used for the treatment of
malaria and toxoplasmosis (e.g., doxycycline, clindamycin, and
spiramycin) act on apicoplastic targets. These drugs typically
display a “delayed death” phenotype, which is characterized by
the inhibition of parasite growth on the second erythrocytic
cycle after the drug treatment.[16–20]
[a] Dr. R. Hoen,+ A. Lꢀpez, Dr. M. Royo
Combinatorial Chemistry Unit, Barcelona Science Park
University of Barcelona
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes
and well-established antimicrobial drug targets,[21,22] and so
represent interesting new targets for antimalarial drug discov-
ery.[23] They perform a central role in the translation of the ge-
netic code by catalyzing the attachment of each amino acid to
its cognate transfer RNA (tRNA). Although these enzymes differ
widely in size, sequence, and oligomeric state, they all carry
out similar two-step reactions.[22] In a first step, the ARS catalyz-
es the activation of the amino acid, and in a second step the
aminoacyl-adenylate intermediate (AA-AMP) is transferred to
the tRNA.
C/Baldiri Reixac 10, 08028 Barcelona, Catalonia (Spain)
E-mail: mroyo@pcb.ub.cat
[b] Dr. E. M. Novoa,+ N. Camacho, Dr. L. Cubells, Prof. A. Cortꢁs,
Prof. L. Ribas de Pouplana
Institute for Research in Biomedicine (IRB Barcelona)
C/Baldiri Reixac 10, 08028 Barcelona, Catalonia (Spain)
E-mail: lluis.ribas@irbbarcelona.org
[c] Prof. L. Ribas de Pouplana
ICREA
Passeig Lluꢂs Companys 1, 08010 Barcelona, Catalonia (Spain)
[d] P. Vieira, Prof. M. Santos
RNA Biology Laboratory, Department of Biology and
Centre for Environmental and Marine Studies (CESAM), University of Aveiro
3810-193 Aveiro (Portugal)
1) AA+ATP !AA-AMP+PPi
[e] P. Marin-Garcia, Prof. J. M. Bautista
Department of Biochemistry and Molecular Biology
Complutense University of Madrid
28040 Madrid (Spain)
2) AA-AMP+tRNA!AA-tRNA+AMP
Currently, ARS inhibition is the mechanism of action of one
commercially available antibiotic: pseudomonic acid or mupir-
ocin (GlaxoSmithKline), a natural product that inhibits bacterial
isoleucyl-tRNA synthetases with an 8000-fold selectivity over
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200620: includes Figures S1–S6, Table S1,
and supplementary methods.
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their mammalian homologues. Mupirocin has also been shown
to inhibit Plasmodium apicoplastic isoleucyl-tRNA synthetase.[24]
Other ARS inhibitors described to date include natural prod-
ucts, such as borrelidin,[25,26] granaticin,[27] indolmycin,[28] furano-
mycin,[29] ochratoxin A,[30] and cispentacin,[31] as well as several
semisynthetic products.[32–34] Most efforts in the design of new
synthetic drugs targeting ARSs have focused on mimicking the
aminoacyl adenylate intermediates (AA-AMPs).[21,35–37] Finally, it
has recently been reported that cladosporin, a fungal secon-
dary metabolite, targets P. falciparum cytosolic lysyl-tRNA syn-
thetase (PfKRS-1) with a selectivity of 100-fold with respect to
its human homologue.[38]
sults show that apicoplastic lysyl-tRNA synthetases cluster with
bacterial enzymes and are only distantly related to HsKRS.
Using a manually curated homology model of PfKRS-2 (Fig-
ure S1 in the Supporting Information), we noted that those
residues involved in the recognition of lysine in the bacterial,
human, and P. falciparum enzymes are conserved across spe-
cies. Importantly, however, other residues in the active site
cavity that are not involved in substrate recognition are not so
well conserved, and the sizes of the catalytic cavities are signif-
icantly different (Figure 1D). This suggests that the active site
in PfKRS-2 might be able to accommodate ligands that might
not be able to bind in the HsKRS cavity, due to steric restric-
tions. Altogether, our analyses suggest that specific design of
inhibitors to target the active site of PfKRS-2 is feasible.
Indeed, the main challenge in using ARSs as drug targets is
to avoid cross-reactivity with their human homologues. In this
regard, apicoplast-specific P. falciparum lysyl-tRNA synthetase
(PfKRS-2) is interesting because its cyanobacterial origin makes
it evolutionarily distant from human lysyl-tRNA synthetase
(HsKRS). In this work we present a new series of compounds
that selectively inhibit apicoplastic PfKRS-2, thus validating its
potential as an antimalarial drug target, and demonstrating
that specific inhibition of apicoplastic ARS is feasible.
Design, selection, and synthesis of a library of lysyl-
adenylate analogues
A virtual compound library was designed in order to identify
molecules that might mimic the lysyl-adenylate intermediate
(Scheme 1). To construct the library, a proline derivative was
used as a ribose mimetic, together with a heterocycle as an ad-
enylate substitute, as previously described.[36] In addition, four
more points of chemical diversity were explored: 1) both lysine
and thialysine derivatives were used as lysine analogues, 2) the
phosphate linker was replaced by other types of chemical link-
ers, 3) heterocyclic groups were used as substitutes for the ad-
enylate moiety, and 4) the stereochemistry of the proline and
the lysine derivatives was varied. With this approach a library
of 1764 compounds was designed and evaluated by docking
the molecules against the 3D structures of both PfKRS-2 and
HsKRS.
Results
Characterization of the lysylation system in P. falciparum
Malaria parasites possess two distinct lysyl-tRNA synthetases:
PfKRS-1 (PF13_0262), and PfKRS-2 (PF14_0166). Subcellular
localization prediction software[11] finds that PfKRS-1 would be
expected to be cytosolic, whereas PfKRS-2 would be expected
to be targeted to the apicoplast (Figure 1A). Immunofluores-
cence assays on PfKRS-2leader-GFP-transfected P. falciparum para-
sites (Figure 1B) indicate that, as expected, PfKRS-2 is exclu-
sively located in the apicoplast.
All 1764 compounds were docked both to PfKRS-2 and
HsKRS, and the different ligand poses obtained were ranked
by use of GlideScore.[40] Compounds to be synthesized for ex-
perimental testing were selected on the basis of their selectivi-
ty towards the PfKRS-2 enzyme. By this criterion we selected
36 compounds for further analysis (Table S1).
In general, apicoplastic-targeted enzymes tend to be of bac-
terial origin.[39] To confirm the bacterial origin of PfKRS-2 and to
evaluate its evolutionary distance from its human homologue,
we performed a structure-based phylogenetic analysis on class
II lysyl-tRNA synthetases from all kingdoms (Figure 1C). Our re-
Scheme 1. Design of the virtual library and the synthetic library. The reaction intermediate lysyl-adenylate (left) was subdivided into four parts, which have
been shaded accordingly. A virtual library of 1764 compounds (middle) based on the structure of the lysyl-adenylate complex (generic structure in (R,R) con-
figuration is depicted) was constructed. On the basis of the docking predictions, a library of 48 compounds was built (right).
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Figure 1. Characterization of the lysylation system in P. falciparum. A) Domain structures of P. falciparum KRS and KRS-2. The bipartite apicoplastic-targeting
signal, consisting of signal peptide (SP) followed by a transit peptide (TP), is only found in PF14_0166; this suggests that PF13_0262 corresponds to the cyto-
solic enzyme, whereas PF14_0166 corresponds to the apicoplastic enzyme. B) Immunofluorescence assays of infected red blood cells (iRBCs) containing
PfKRS-2leader-GFP-transfected P. falciparum parasites. The GFP-tagged sequence colocalizes with ACP and not with Mitotracker, indicating that it is being spe-
cifically targeted to the apicoplast, consistently with the bioinformatic predictions. C) Phylogenetic analysis of class II lysyl-tRNA synthetases. The plasmodial
PfKRS-1 and PfKRS-2 are boxed in red, whereas the human HsKRS is boxed in blue. PfKRS-2 clusters with bacterial sequences, whereas PfKRS-1 and HsKRS
cluster with eukaryotic sequences. D) Active site comparison between PfKRS2 and its human homologue. The active site is defined as those residues with at
least one of their atoms at less than 4 ꢄ from the ligand. Differing residues are highlighted. See also Figure S1.
Amongst the 36 compounds selected, 70% contained a hy-
droxamate group as a phosphate analogue and a proline ring
with an (S,S) configuration. A library of 50 lysyl-adenylate ana-
logues based on the (S,S)-4-aminoproline scaffold with a hy-
droxamate group as a phosphate linker mimic was thus de-
signed. Of these compounds, 25 contained lysine, whereas the
other 25 contained thialysine (Table 1; see also Scheme 1). In
addition to the predicted hits, a number of predicted nonselec-
tive and inactive compounds were also synthesized to evaluate
the performance of the docking calculations (Figure S2B).
The library of potential PfKRS-2 inhibitors was produced by
solid-phase synthesis based on an Alloc/Boc strategy
(Scheme 2).[36] Coupling of Alloc-protected hydroxyproline to
the resin was followed by the introduction of the protected
lysine or thialysine hydroxamic acid moiety under Mitsunobu
conditions. After removal of the Alloc group with Pd(PPh3)4
and PhSiH3, introduction of the different carboxylic acids was
carried out under standard peptide coupling conditions. Sub-
sequent cleavage of the products and protecting group re-
moval under strongly acidic condition produced the crude in-
hibitors. Purification by preparative HPLC yielded the desired
products with purities of ꢀ85% (Scheme 2A). A number of
thialysine-derived products were not obtained, due to degra-
dation of the products during purification. The obtained prod-
ucts were subjected to biological evaluation.
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Table 1. List of 46 synthesized compounds, ranked by their GlideScores when docked into the PfKRS-2 homology model (Pf-Gscore). The GlideScores in
the case of the human homologue (Hs-Gscore) are also shown. See also Figure S2 and Table S1.
Lysine derivatives
Thialysine derivatives
R group
Code[a]
M-01
Pf-Gscore
Hs-Gscore
Yield [mg]
Purity [%]
84.4
Code
Pf-Gscore
Hs-Gscore
Yield [mg]
Purity [%]
96.0
ꢁ12.94
ꢁ9.63
3.5
M-26
ꢁ11.99
ꢁ10.85
3.2
M-02
M-03
M-04
ꢁ12.23
ꢁ10.76
ꢁ11.46
ꢁ8.85
ꢁ10.32
ꢁ9.66
9.3
1.9
1.6
95.0
53.0
90.2
M-27
M-28
M-29
ꢁ11.06
ꢁ9.28
ꢁ7.94
ꢁ8.40
8.1
–
90.9
–
ꢁ10.88
ꢁ10.60
–
–
M-06
M-07
ꢁ11.22
ꢁ11.74
ꢁ9.91
ꢁ9.40
3.9
4.0
98.7
93.9
M-31
M-32
ꢁ9.68
ꢁ9.73
ꢁ8.40
–
–
ꢁ10.43
11.2
98.5
M-08
ꢁ12.70
ꢁ8.86
1.1
90.2
M-33
ꢁ12.00
ꢁ9.89
6.5
92.0
M-09
M-11
M-12
ꢁ9.38
ꢁ10.50
ꢁ10.97
ꢁ8.63
ꢁ8.68
ꢁ9.12
3.4
12.0
6.3
91.4
85.8
89.4
M-34
M-36
M-37
ꢁ9.25
ꢁ9.75
ꢁ10.89
ꢁ9.50
ꢁ8.23
5.1
10.1
8.3
>99.9
96.9
ꢁ10.54
88.2
M-13
M-14
ꢁ11.00
ꢁ11.76
ꢁ8.88
ꢁ8.23
10.4
4.2
92.7
86.5
M-38
M-39
ꢁ10.54
ꢁ9.84
ꢁ8.23
ꢁ9.37
20.1
12.6
86.8
84.9
M-15
ꢁ9.78
ꢁ10.68
1.4
99.0
M-40
ꢁ10.42
ꢁ10.66
12.7
90.0
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Table 1. (Continued)
Lysine derivatives
Thialysine derivatives
R group
Code[a]
Pf-Gscore
Hs-Gscore
Yield [mg]
Purity [%]
93.9
Code
M-41
Pf-Gscore
Hs-Gscore
Yield [mg]
Purity [%]
88.1
M-16
M-17
ꢁ10.43
ꢁ11.62
0.9
1.0
ꢁ10.11
ꢁ8.38
11.4
ꢁ9.89
ꢁ9.59
ꢁ9.42
95.3
86.7
M-42
M-43
ꢁ9.89
ꢁ9.66
ꢁ10.21
ꢁ9.83
4.9
8.6
92.8
92.6
M-18
M-19
ꢁ10.55
4.2
2.0
ꢁ10.26
ꢁ8.12
86.5
M-44
ꢁ11.17
ꢁ9.54
3.9
96.5
M-20
M-21
ꢁ10.22
ꢁ9.75
ꢁ8.86
1.3
1.7
95.3
76.5
M-45
M-46
ꢁ10.49
ꢁ9.99
ꢁ9.10
ꢁ8.54
–
–
ꢁ11.09
6.5
91.3
M-22
M-23
ꢁ10.62
ꢁ11.19
ꢁ10.69
ꢁ10.45
2.0
3.0
89.5
95.9
M-47
M-48
ꢁ12.49
ꢁ9.94
ꢁ9.85
9.0
3.5
92.7
93.1
ꢁ11.59
M-24
ꢁ9.52
ꢁ9.59
ꢁ11.16
ꢁ10.77
0.5
4.7
99.0
92.0
M-49
M-50
ꢁ9.79
ꢁ10.53
ꢁ8.90
9.0
95.8
M-25
ꢁ10.08
–
–
[a] The subset of selected compounds that were resynthesized for further in vitro analyses is shown in bold.
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Scheme 2. Synthesis of the library of lysyl-adenylate analogues. A) Solid-phase synthesis of the library. a) 20% piperidine in DMF; b) HOBt, DiPCDI, DMF;
c) DIAD, CH2Cl2, PPh3; d) Pd(PPh3)4, PhSiH3, CH2Cl2, 2ꢅ20’; e) RCOOH, HOBt, DIPCDI, DMF, 2 h; f) TFA/CH2Cl2/TIS (95:2.5:2.5). B) Synthesis of the active com-
pounds in solution. g) 2m NaOH(aq), CH2Cl2 (57%); h) WSC·HCl and HOBt·H2O; i) 32% NH4OH(aq) (66%); j) PPh3, DEAD, THF; k) 10% Pd/C, H2, MeOH;
l) WSC·HCl, HOBt·H2O, DMF/DMF (9:1), RCOOH; m) 40% TFA in CH2Cl2. See also Figure S4.
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Table 2. In vitro inhibition of the five resynthesized compounds.[a] See also Figure S4.
Screening (purity >85%, 150 mm)
Resynthesized hits (purity >95%, 50 mm)
Smears [% inhib.] IC50 [mm]
48 h
Purity
[%]
LDH [% inhib.]
Smears [% inhib.]
Purity
[%]
Selectivity
[fold]
48 h
96 h
48 h
96 h
48 h
96 h
96 h
M-12
M-24
M-26
M-33
M-37
93
92
96
85
88
0
4.42
0
24.7
15.7
45.9
58.7
35.5
100
65.5
23.2
0
0.18
76.6
49.7
58.0
58.3
66.0
98.9
99.5
99.5
100
98.6
100
31.7
32.6
3.90
43.5
6.20
70.4
42.4
41.6
77.9
72.7
172
518
551
48.1
151
83.2
427.1
84.7
29.5
38.4
2.2
1.2
6.5
1.4
3.9
100
[a] Inhibition of P. falciparum cultures measured by: 1) LDH (lactate dehydrogenase) assay, and 2) visual inspection of Giemsa-stained smears.
In vitro testing of the compounds
the reaction intermediate. In agreement with this observation,
analogues showing a LAD-like binding mode (Figure 3; see
also Figure S3A) tend to have higher docking scores. Interest-
ingly, both M-26 and M-37 display a LAD-like binding mode in
PfKRS-2, whereas in HsKRS they have either an adenine-like or
a lysine-like binding mode, respectively (Figure S3A). These di-
verse binding modes are due to differences in the sizes of the
active site cavities of the two enzymes. Whereas the PfKRS-2
cavity can accommodate the inhibitors and maintain recogni-
tion of both the lysine and adenine moieties, the catalytic
cavity of HsKRS cannot accommodate both moieties of these
compounds at the same time.
All of the synthesized compounds were initially tested by the
pLDH assay for their ability to kill P. falciparum parasites.[41]
Inhibitors of apicoplastic protein synthesis kill the parasite in a
retarded manner,[16–18] so we used the “delayed death” pheno-
type as an initial indication that a compound in our library
might be preferentially targeting apicoplastic lysyl-tRNA syn-
thetase. Our initial screening allowed us to select five com-
pounds that showed clear delayed inhibitory effects (Table 2).
The activities of these compounds were further confirmed by
visual inspection of smears.
The five most active compounds from the library (M-12, M-
24, M-26, M-33, and M-37) were resynthesized. Solution syn-
thesis was used to improve purities and yields and to minimize
possible side-reactions (Scheme 2B). All products were ob-
tained in purities of >98.5%. The antimalarial activities of the
resynthesized compounds were evaluated by visual analysis of
P. falciparum smears. The highest inhibition rates were ob-
served for compounds M-12, M-33, and M-37 (Table 2). In
order to select specific inhibitors of the apicoplastic translation
machinery it was decided to focus on those compounds that
produced clear delayed effect phenomena. Compounds M-26
and M-37 were thus chosen as drug candidates for further in
vitro and in vivo analyses, given that these compounds show
the greatest differences in their inhibitory rates at 48 and 96 h.
In order to investigate the selectivities and specificities of M-
26 and M-37, we first verified their abilities to inhibit HsKRS,
PfKRS-1, and PfKRS-2. In vitro aminoacylation assays were per-
formed with radiolabeled lysine and in-vitro-transcribed
tRNALys, and the effects of the compounds upon these amino-
acylation reactions were quantified. Both M-26 and M-37 were
found to inhibit PfKRS-2, but were not active against HsKRS or
PfKRS-1 (Figure 2), in accordance with our docking predictions.
We also verified that the compounds did not inhibit other plas-
modial ARSs (Figure S2). We can therefore conclude that M-26
and M-37 are selective inhibitors of apicoplastic PfKRS-2.
Discussion and Conclusions
In recent years cell-based screening has been presented as an
attractive way to find new leads for malaria drug development.
However, although these approaches are capable of identifying
large numbers of hits, they also have serious limitations. If an
initial hit is not suitable for chemical modification, for instance,
or its target is not known, there might be no opportunity to
proceed to hit-to-lead optimization. In this regard, the initial
validation of targets based on chemoinformatic predictions
can be a useful approach.
Aminoacyl-tRNA synthetases (ARSs) have been recognized
for decades as useful targets for drug design.[42,43] Indeed, ARSs
continue to be used as targets in antibacterial and antiparasitic
drug discovery programs[34,44,45] and have recently received sig-
nificant attention as promising targets for the development of
next-generation antimalarials.[24,38,46,47] Targeting the ARS of a
microorganism without inhibiting the human counterpart,
however, remains a major challenge. The use of methods for
phylogenetic inference helps in recognition of targets with
evolutionary histories that might favor the identification of se-
lective compounds. Remarkably, apicomplexan protozoa have
incorporated a second form of KRS during the endosymbiotic
event that gave rise to apicoplasts. This is evident from the
phylogenetic positions assigned in our trees to the apicoplast
KRSs of P. falciparum and P. yoelii (Figure 1C). Whether the api-
coplast ancestor was a green or a red alga is still a matter of
debate,[11,15] but our data indicate that some of the genes
acquired from the apicoplast genome by Plasmodium could be
direct descendants of bacterial symbionts.
Structural basis for selectivity
Through analysis of the binding mode of the natural lysyl-ade-
nylate (LAD) ligand, it was observed that both the adenine and
the lysine moieties of LAD are recognized at the binding site
and are major contributors to the free energy of binding of
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these molecules inhibit PfKRS-2, whereas the activity
of its human homologue remains unaffected by
these compounds. Finally, through in silico predic-
tions, we have identified PfKRS-2-specific features of
the active site that can explain the selectivities of
these compounds. The two compounds described
here strongly and specifically inhibit the aminoacyla-
tion reaction catalyzed by the PfKRS-2, demonstrating
the potential of apicoplastic lysyl-tRNA synthetases as
a druggable enzyme targets that can be selectively
inhibited and should be further explored for the de-
velopment of new antimalarial chemotherapies.
Thanks to their cyanobacterial origin, apicoplastic
proteins are interesting targets with potential to aid
the development of selective drugs. Multiple com-
monly used antimalarials (e.g., doxycycline, clindamy-
cin, and azithromycin) exert their effects by interfer-
ing with apicoplastic proteins. Although apicoplastic
proteins might not be the most promising targets for
acute malaria treatment, they are nevertheless useful
for the development of prophylactic compounds,
which in turn are critical for the eradication of malar-
ia.[15,16]
Experimental Section
Homology modeling of PfKRS-2: The sequence of the
apicoplastic lysyl-tRNA synthetase of P. falciparum was
retrieved from the PlasmoDB database (http://Plasmo
DB.org). A PSI-BLAST[48] search was performed against
the Uniprot database (http://www.uniprot.org) in order
to obtain a Position-Specific Scoring Matrix, which was
used as input to perform a new BLAST search against
the PDB database (http://www.rscb.org), to afford a list
of candidate templates to build the model (1lyl, 1bbu,
1e1o). The templates were structurally aligned with the
aid of STAMP[49] to create a profile by use of HMMER,[50]
which was introduced as meta-template for alignment
with the target sequence. Finally, the 9v5 version of
MODELLER was employed to create structural models
with use of default options.[51] The models generated
were manually refined by several methods, including
corrections of the alignment with use of the PSI-PRED[52]
secondary structure predictions as guideline, and then
followed by a new rebuilding of the model. The final
model was analyzed with ProSA[53] and validated by
using PROCHECK.[54]
Figure 2. In vitro inhibition of the aminoacylation reactions catalyzed by PfKRS-2, PfKRS-
1, and HsKRS. The IC50 values for the two most promising inhibitors—M-26 (A) and M-37
&
*
(E)—were computed both at 48 ( ) and at 96 h ( ). Both inhibitors show the clear de-
layed-inhibition effects typical of apicoplastic inhibitors. To verify the target of these in-
hibitors, we checked their effects on the aminoacylation reactions catalyzed not only by
PfKRS-2 (B and F), but also with PfKRS-1 (C and G) and HsKRS (D and H). As can be seen,
both compounds inhibit PfKRS-2, whereas the aminoacylation activities of HsKRS and
PfKRS-1 remain practically unaffected.
Virtual screening and docking: Ligand screening and
docking was performed with Glide 5.0.[40] Ligands were
prepared such that several conformations were generat-
Both our structural and evolutionary data strongly point to
ed for each input ligand, with use of the LigPrep[55] facility of
MAESTRO,[56] whereas the setting-up of the proteins (PfKRS-2 and
HsKRS) was carried out with the Protein Preparation Wizard facility.
The receptor grid defining the docking universe was defined by
means of a cubic box centered on the lysyl-adenylate. Schrçding-
er’s GlideScore scoring function was used to score the poses.
PfKRS-2 as a promising target for the development of inhibi-
tors of apicoplast metabolism in apicomplexan organisms,
given that they are only distantly related to its human counter-
parts. Here we demonstrate that this enzyme can indeed be
specifically inhibited. Using a combination of computational
and experimental approaches, we have built a series of lysyl-
adenylate analogue inhibitors designed to be effective against
apicoplastic lysyl-tRNA synthetases. We show that some of
Solid-phase synthesis: Each solid-phase synthesis was carried out
manually in a polypropylene syringe fitted with a polyethylene
porous disk. Solvents and soluble reagents were removed by suc-
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mum likelihood methods with use of the PHYLIP 3.68 package,[63]
with 1000 bootstrap replicates in the distance calculations and
100 bootstrap replicates for the maximum likelihood trees.
Cloning and expression of P. falciparum PfKRS-2: Soluble and
active PfKRS-2 was obtained by the following procedure: the nu-
cleotide sequence of the gene coding for PfKRS-2 without the pre-
dicted bipartite signal sequence (DPfKRS-2) was codon-optimized
for E. coli, synthesized (MrGene), and inserted into the plasmid
pQE70. Expression screening was carried out with the In-Fusion
based Vector Suite at the IRB Protein Expression Core Facility. Dif-
ferent tagged DPfKRS-2 constructions were built and inserted into
both E. coli Rosetta and B834 strains. Three soluble proteins were
finally selected for activity assays: these were DPfKRS-2-Sumo,
DPfKRS-2-His, and DPfKRS-2-Z, which contained a C-terminal Sumo
tag,[64] a N-terminal His tag, and a C-terminal Z tag,[65] respectively.
The final concentrations of the His tag, Sumo-His tag, and Z tag en-
zymes were 3.2, 2.7, and 3.4 mm, respectively. Final aminoacylation
assays were performed with DPfKRS-2-His.
Figure 3. Structural analysis of M-26 and M-37 binding modes. In the top
panel the binding modes of M-26 (left) and M-37 (right) docked into PfKRS-
2 are shown. The natural ligand (lysyl-adenylate; LAD) is shown in pale gray.
Both compounds have a lysyl-adenylate-like binding mode in the P. falcipa-
rum binding site (GlideScores of ꢁ11.99 and ꢁ10.54 for M-26 and M-37, re-
spectively). In the lower panel the binding modes of M-26 (left) and M-37
(right) docked into HsKRS are shown. M-26 has an adenine-like binding
mode in H. sapiens (GlideScore=ꢁ10.85), whereas M-37 has a lysine-like
binding mode (GlideScore=ꢁ8.23).
Pyrophosphate exchange assay: The pyrophosphate (PPi) ex-
change assay was used to measure the amino acid activation activ-
ities of the three soluble PfKRS-2 recombinant proteins (Figure S6).
This assay measures the rate of isotopic exchange of PPi into ATP,
and is therefore focused on the first step of the aminoacylation
reaction.[66] Enzyme (5 mL, 2 mm) was added to the reaction mix (2-
(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES)/NaOH
(pH 7.2, 50 mm), MgCl2 (20 mm), dithiothreitol (DTT, 1 mm), ATP
(2 mm), lysine (4 mm), NaPPi (2 mm), [32P]NaPPi (2 MBqmmolꢁ1)),
and aliquots (22 mL) were withdrawn at 2, 10, and 30 min time
points into quenching solution [250 mL, perchloric acid (3.5%, w/v),
activated charcoal (3%, w/v), NaPPi (100 mm)]. The quenched ali-
quots were filtered (Whatmann 25 mm filters), and washed once
with washing solution [perchloric acid (1.4%, w/v), NaPPi (40 mm)],
once with Milli-Q water, and finally with ethanol (100%). The filters
were placed into scintillation liquid and counted by the manufac-
turer’s protocol for 32P detection with QuantaSmart Operating Soft-
ware.
tion. Peptide synthesis for this work employed a combined Boc/
Alloc solid-phase strategy on an Fmoc-Rink-Amide-MBHA resin.
Washing between deprotection, coupling, and subsequent depro-
tection steps was carried out with DMF (5ꢅ1 min) and CH2Cl2 (5ꢅ
1 min) with 10 mL of solvent per g of resin each time. All the cou-
plings and Fmoc removal were monitored by the Kaiser test. See
also Figures S4 and S5 and the supplementary methods.
Synthesis in solution: Unlike in the solid-phase synthesis, the
amine protecting group used was now the UV-active p-nitrobenzyl
carbamate (PNZ), which is more stable under acidic conditions and
more readily cleaved by hydrogenolysis than the related benzyl
carbamate (Z). Straightforward introduction of the PNZ protecting
group followed by conversion of the carboxylic acid into the corre-
sponding primary amide, under standard peptide coupling condi-
tions, gave 60 in good yield. Subsequently, lysine or thialysine
were introduced by means of a Mitsonubu reaction. Separation of
the formed triphenylphosphine oxide from the corresponding
products was laborious. In spite of intensive attempts, complete
removal of the triphenylphosphine oxide from the desired product
could not be achieved. It was decided to continue the synthesis
with the mixture, because the triphenylphosphine oxide should
not interfere with the outcomes of the subsequent reactions. De-
protection of the proline derivative by hydrogenolysis (Pd/C under
H2), followed by coupling of the corresponding carboxylic acids
under standard peptide coupling conditions (WSC·HCl/HOBt·H2O),
gave products 65–69 (Scheme 2) in good yields. Full deprotection
of 65–69 with trifluoroacetic acid (40%) in CH2Cl2 and immediate
purification of the crude products by preparative HPLC gave the
final products (M-12, M-24, M-26, M-33, and M-37) in good yields.
The final products were characterized by standard techniques such
In vitro aminoacylation assays: To characterize the activities of
our compounds, their effects on the lysylation of tRNALys by
DPfKRS-2-His were tested. In-vitro-transcribed P. falciparum tRNALys
was prepared as described.[67] Aminoacylation was performed at
378C in Hepes/KOH (pH 7.2, 100 mm), aqueous KCl (20 mm), aque-
ous MgCl2 (30 mm), aqueous DTT (0.5 mm), ATP (5 mm), bovine
serum albumin (BSA, 0.1 mgmLꢁ1), [3H]lysine (20 mm, 500 Cimolꢁ1
,
PerkinElmer), and in-vitro-transcribed tRNA (5 mm). Reaction ali-
quots were spotted on 3 mm filter disks and washed in aqueous
trichloroacetic acid (5%) with aqueous lysine (100 mm). Radioactivi-
ty was determined by liquid scintillation counting. Aminoacylation
rates of human lysyl-tRNA synthetase (HsKRS) were also deter-
mined to check for possible cross-reactivity of PfKRS-2 inhibitors
towards the human homologue. The reactions were performed by
addition of in-vitro-transcribed H. sapiens tRNALys to human HEK
293T cell extracts (5 mL) under the same conditions as for PfKRS-2.
Similarly, aminoacylation rates of P. falciparum cytosolic lysyl-tRNA
synthetase (PfKRS-1) were determined by addition of in-vitro-tran-
scribed nuclear-encoded tRNALys of P. falciparum to plasmodial ex-
tracts (5 mL). Additional controls to test the specificities of the com-
pounds were performed against the tryptophanyl-tRNA synthetase
of P. falciparum. We used recombinant P. falciparum tryptophanyl-
tRNA synthetase (a kind gift from Amit Sharma) and in-vitro-tran-
scribed P. falciparum tRNATrp for this purpose. Aminoacylation re-
actions were performed under the same conditions as described
above.
1
as H and 13C NMR (Figure S5) and exact mass and HPLC-MS.
Phylogenetic analysis of lysyl-tRNA synthetases: The sequences
of lysyl-tRNA synthetases reported here are available in Uniprot.[57]
We applied the method of structure-based alignment of the active
sites of the enzymes, as described elsewhere.[58] Archaeal LysRS-II
sequences were initially included in our analysis, but were later
dropped for two reasons: 1) they are generally believed to have
emerged through lateral gene transfer events,[59–62] and 2) in our in-
itial analyses they clustered consistently within the bacterial clade.
Phylogenetic distributions were calculated by distance and maxi-
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ChemBioChem 2013, 14, 499 – 509 507

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the IC50 values of the most active compounds on 3D7 P. falciparum
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Keywords: aminoacyl-tRNA synthetases
·
combinatorial
chemistry · drug design · enzyme inhibitors · malaria ·
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Received: September 26, 2012
Published online on February 21, 2013
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