Inhibition of Plasmodium falciparum Lysyl-tRNA Synthetase via a Piperidine-Ring Scaffold Inspired Cladosporin Analogues

Article abstract of DOI:10.1002/cbic.202100212

Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising therapeutic anti-malarial target. Cladosporin was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with X-ray crystallography reveal compounds that are moderate in potency. Co-crystals of Cla?B and Cla?C with PfKRS reveal key atomic configurations that allow drug binding to and inhibition of the enzyme. Collectively these piperidine ring scaffold inhibitors lay a framework for further structural editing and functional modifications of the cladosporin scaffold to obtain a potent lead.

Full text of DOI:10.1002/cbic.202100212

Combining Chemistry and Biology
Accepted Article
Title: Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via a
piperidine-ring scaffold inspired Cladosporin analogues
Authors: Palak Babbar, Mizuki Sato, Yogavel Manickam, Siddhartha
Mishra, Karl Harlos, Swati Gupta, Suhel Parvez, Haruhisa
Kikuchi, and Amit Sharma
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To be cited as: ChemBioChem 10.1002/cbic.202100212
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1/2020

ChemBioChem
10.1002/cbic.202100212
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Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via a piperidine-
ring scaffold inspired Cladosporin analogues
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Palak Babbar §, ±, Mizuki Sato Yogavel Manickam , Siddhartha Mishra , Karl Harlos ≠,
‡,
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§, €
§
±
‡, #, *
, Amit Sharma, §, €, *
Swati Gupta , Suhel Parvez , Haruhisa Kikuchi
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Molecular Medicine – Structural Parasitology Group, International Centre for Genetic Engineering and
Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India
‡
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aza-Aoba, Aramaki, Aoba-ku, Sendai
980-8578, Japan
≠
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Division of Structural Biology, Wellcome Trust Centre for Human Genetics, The Nuffield Department of
Medicine, University of Oxford, Oxford OX3 7BN, U.K.
±
Department of Medical Elementology and Toxicology, School of Chemical and Life Sciences, Jamia
Hamdard, New Delhi 110062, India
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€ ICMR-National Institute of Malaria Research (NIMR), Sector 8, Dwarka, New Delhi 110077, India
# Present affiliation: Division of Natural Medicines, Faculty of Pharmacy, Keio University
*
Corresponding Authors: Haruhisa Kikuchi and Amit Sharma
H.K.: e-mail, hal@mail.pharm.tohoku.ac.jp
A.S.: e-mail, directornimr@gmail.com
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Abstract
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Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising
therapeutic anti-malarial target. Cladosporin, a tool compound was identified as a
selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report
chemical synthesis, biological evaluation and structural characterization of analogues
where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine
ring bearing functional group variations. Thermal binding, enzymatic, kinetic and
parasitic assays complemented with x-ray crystallography reveals compounds to be of
moderate potency. Co-crystals of Cla-B and Cla-C with PfKRS reveals key atomic
configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these
piperidine ring scaffold inhibitors lay a framework for further structural editing and
functional modifications of cladosporin scaffold to obtain a potent lead.
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Introduction
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Malaria, a mosquito-borne infectious disease caused by Plasmodium species, affects
millions worldwide each year. [1] Concerns about rising drug resistance call for new drugs
to be developed.[2] In recent times, amino-acyl tRNA synthetases (AARSs) have been
validated as a potential drug target against various diseases. [2] AARSs are enzymes that
catalyze the aminoacylation reaction by covalently linking an amino acid to its cognate
tRNA in the first step of protein translation.[2] Since AARSs are essential for protein
biosynthesis, direct inhibition of these enzymes affects protein metabolism. Mupirocin,
indolmycin, ochratoxin A, borrelidin and halofuginone are some known potent inhibitors
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of AARSs. [2-7] Halofuginone (HF), a halogenated derivative of naturally occurring
febrifugine (FF) acts on prolyl-tRNA synthetase (PRS). [8,9] The drug is FDA approved to
treat coccidiosis in poultry and cryptosporidiosis, but due to its mammalian toxicity it
could not be utilized as an antimalarial drug.[10] In this effort, our group had developed
quinazolinone based inhibitors that are active against PRS in diseases such as malaria,
leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis (Figure 1).[9]
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Figure 1- The chemical structure and biological activity reported for febrifugine,
halofuginone and the most potent quinazoline derivative (ln-5) of them.[9]
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Malaria parasite genome encodes for two different lysyl-tRNA synthetases (KRSs) that
play a role in translation in either the cytoplasm or in the apicoplast. [2,12,40] Cladosporin,
a fungal secondary metabolite isolated from Cladosporium cladosporioides [14,15], was
discovered to be an inhibitor of cytoplasmic PfKRS with activity in nanomolar range and
>100-fold selectivity as compared to the human counterpart. [11,12,13] It is a chiral
molecule with the core scaffold consisting of a 6,8-dihydroxylisocoumarin ring joined to
a trans-2,6-disubstituted tetrahydropyran moiety (THP) bearing a chiral methyl
functionality. [14,15] These together mimic an adenosine moiety and thereby inhibit
enzyme activity by competing with ATP. [11,12,13] The species-specific selectivity of
cladosporin has been accounted to Val329 and Ser346 which seem to be sterically crucial
for accommodating the methyl moiety of THP ring in case of Plasmodium. [11,12,13] Previous
studies have also shown that cladosporin effectively targets KRSs from other species like
Cryptosporidium parvum, Loa loa and Schistosoma mansoni. [16,17] Unfortunately, because
of its low bioavailability or high metabolic instability, cladosporin is embarked as a tool
compound and not a marketed drug. [17,22] Due to its potent activity we and others have
previously reported the discovery and optimization of cladosporin inspired inhibitors.
[17,18,19,29]
Cladosporin binds to PfKRS with a ~300-fold tighter affinity than the natural ligand
ATP.[13] Previous studies have shown that cladosporin possesses a high selectivity index
against Plasmodium parasites compared to mammalian cells (IC50/CC50> 111) [11] but
still binds to both human and Plasmodium KRSs in a nearly identical mode in the active
site. [25] Considering that, we have interrogated the co-crystal structures of homo sapiens
lysyl-tRNA synthetase (HsKRS) with ATP (PDB ID- 3BJU) [26] and cladosporin (PDB ID-
4YCU) [25] to dissect structural reasons for cladosporin’s affinity for its target in context
of ATP (Supplementary Figure 3 and 4).
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For the present study, cladosporin was selected for chemical optimization and the
rationale for the design of this study was based on the already published research studies
and the co-crystal structure of cladosporin/ derivatives-bound Plasmodium falciparum
lysyl-tRNA synthetase. [13, 19, 22, 28,29] Rusch et al. incubated cladosporin with mouse liver
microsomes in vitro and pointed three major metabolically weak points- the THP frame,
the hydroxyl group and C-4 of the isochromenone within the molecule and derivatized a
structural cyclohexyl analog based on the findings.[22] Similarly, Zhou et al. focused on the
metabolically weak points and confirmed that THP could be replaced by a more stable
methylcyclohexane while maintaining the potency. [19] Recently, research from our group
(Babbar et al.) described a systematic structure activity relationship (SAR) of four sets of
analogues that were designed based on changes in the chemical scaffold of cladosporin
and reported a metabolically stable cladosporin derivative. [29] This work also concluded
that the isocoumarin moiety of cladosporin forms hydrogen bonds with the side chains
of Glu332 and Asn339 through its two hydroxyl groups while the carbonyl forms H-bond
with Arg559 by virtue of a highly coordinated water. [13,29] The π/cation ‘cage’ formed by
adjacent aromatic residues His338 and Phe342 are also a key feature and must remain
unchanged in order to maintain the electrostatic interactions between the protein and
ligand. [29] In line with the above reports, we designed a series focusing on C-4 of the
isochromenone and the THP frame of the cladosporin. The THP is replaced with the
piperidine ring as a base because of its low nucleophilicity and high solubility in organic
solvents. Piperidine ring tends to impart pharmacokinetic properties such as water
solubility and bioavailability. The new series (called Cla-series) consists of four pairs of
stereoisomers differing at absolute R and S configuration along with other derivatives
designed based on point changes/ functional group modifications (Figure 2). These
piperidine ring-containing cladosporin derivatives were tested in vitro against Pf and
HsKRS enzymes along with in vitro parasite inhibition testing. Surface plasma resonance
(SPR) studies of selected compounds was also done (Figure 3 and 4). Amongst the set,
three pairs -Cla-A/B, Cla-C/D, Cla-E/F differing at absolute R and S configuration on C14
chiral center proved to be potent (Figure 3 and Table 1). Compounds Cla-B and Cla-C were
further studied for interaction with its target PfKRS. Their co-crystal structure revealed
the importance of enzyme-drug interactions in determining the potency (Figure 5, 6 and
7). The overall work presented here provides a valuable understanding of chemical
optimization of the lead molecule cladosporin for the target protein PfKRS.
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Preparation of a focused new cladosporin series
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The THP ring of cladosporin has been replaced with a piperidine ring with functional
group variations at C13, C14 and C15 position. The isocoumarin moiety is conserved in
compounds Cla-A (1) to Cla-D (4) and Cla-G (7) to Cla-L (12). For Cla-E (5) and Cla-F (6)
there is a double bond incorporated between C3 and C4 position along with the
replacement of THP to piperidine ring thus modifying the isocoumarin moiety as well.
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The stereochemistry of cladosporin at C14 is preserved in Cla-A, Cla-C, Cla-E, Cla-G and
Cla-K.
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Figure 2- The chemical structures of synthesized compounds that form the Cla-series
are shown. Double arrow (red and yellow) defines each diastereomeric and
enantiomeric pair respectively. Green dotted circle indicates metabolically weak points
in cladosporin that were selected for chemical optimization.
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Synthesis of Cla-A–D (1–4) and Cla-G–L (7–12)
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Commercially available 2,4-dihydroxybenzoic acid was converted into dibenzyl ether 5.
[20]
Palladium (II)-catalyzed C-H alkylation
of 5 with (S)-glycidol afforded
dihydroisocoumarin 6, though a lot of degradation of starting materials occurred. After
triflation of 6, substitution reaction by (R)-3-methylpiperidine produced 7a, reductive
debenzylation of which provided Cla-A (1) (Scheme 1). Substitution by several types of
substituted piperidines instead of (R)-3-methylpiperidine afforded 3’-epimer of 1, Cla-B
(2), and 3-ethyl derivatives, Cla-C (3) and Cla-D (4), (R)-3-methylmorphonine derivative,
Cla-G (7), 4-methyl derivative, Cla-H (8), 2-methyl derivatives, Cla-I (9) and Cla-J (10),
and (S)-3-carboxy derivative, Cla-K (11), respectively.
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On the other hand, 3-methylene derivative, Cla-L (12) was difficult to be synthesized
through the synthetic route demonstrated in Scheme 1, because the exo-olefin moiety of
Cla-L (12) is easily hydrogenated in the process of Pd(OH) -catalyzed debenzylation.
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Thus, the benzyl groups of intermediate 14 were replaced by the MOM groups to afford
compound 18 (Scheme 2). After triflation of 18, substitution reaction by 3-
methylenepiperidine produced 19, acidic hydrolysis of MOM groups of which provided
Cla-L (12).
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Scheme 1. Synthesis of Cla-A-D (1-4) and Cla-G–K (7–11).
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Scheme 2. Synthesis of Cla-L (12).
Synthesis of Cla-E (5) and Cla-F (6)
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For compounds Cla-E (5) and Cla-F (6), palladium (II)-catalyzed retro-aldol/α-arylation
[21]
of methyl 2-iodo-4,6-dimethoxybenzoate with 4-hydroxy-4-methylpentane-2-one
provided 8, which was converted into 9 by demethylation and acetylation. After benzylic
bromination of 9, substitution reaction by (R)-3-methylpiperidine and deacetylation
produced Cla-E (5) (Scheme 3). Cla-F (6), 3’-epimer of 5, was also synthesized by the use
of (S)-3-methylpiperidine (Scheme 3).
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Scheme 3. Synthesis of Cla-E (5) and Cla-F (6).
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Results and Discussion
Evaluation of biological potency of the series
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Thermal shift assays were performed to assess the binding of analogs with both PfKRS
and HsKRS in the presence of L-lysine. Cladosporin was used as a control. Our data
indicate that Cla-A to Cla-F analogs-PfKRS complexes showed a thermal melting (T
m
) shift
in the range of ~8.75 to 14.6°C. The ΔT for Cladosporin-PfKRS complex is of 17.3° C
m
(Figure 3a). Cla-A to Cla-F provided less thermal stability to PfKRS when compared to
PfKRS-cladosporin complex. Rest of the cladologs (Cla-G to Cla-L) provided almost
insignificant thermal shift (Supplementary table 2). Based on drug-binding
characteristics, the series was divided into two sets- Set 1(Cla-A to Cla-F) as moderate
binders while Set 2 (Cla- G to Cla-L) as non-binders. When identical data were collected
on HsKRS, revealed no binding indicating that the series retained their overall selectivity
against the HsKRS (Supplementary Figure 1, Table 1 and Supplementary table 2).
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We then tested the enzymatic activity of recombinant proteins PfKRS and HsKRS in the
presence of Cla-series by performing aminoacylation assays. First part of the
aminoacylation reaction where aminoacyl-adenylate complex is formed was studied in
the presence of these analogs. Here, the aminoacyl-adenylate complex is formed with the
release of pyrophosphate (PPi) which can be indirectly measured by a malachite green
assay that uses pyrophosphatase-induced conversion of PPi into inorganic phosphate
(Pi). For PfKRS enzyme inhibition assay, concentration ranges of 100 to 0.01 μM were
used for the Cla-series and 10 to 0.01 µM was used for cladosporin. The evaluation of the
half-maximal inhibitory concentration (IC50) for PfKRS revealed the Set 1(Cla-A to Cla-F)
to be moderate inhibitors with a range of ~ 5.0 to 9.5 μM as compared to cladosporin with
an IC50 of 0.06 μM (Figure 3b and c). To determine specificity, we assessed the IC50 values
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for HsKRS alongside and all compounds were found to be inactive against the same (Table
1 and Supplementary table 2).
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Following this, we then investigated the cell-based parasite growth inhibition assays to
calculate the half-maximal effective concentration (EC50) for the series. The
concentration ranges from 10 to 0.078 µM for the analogs and 1 to 0.0078 μM for
cladosporin. The observed EC50 values were consistent with the observations from
enzyme inhibition assays and ranged from ~3.7 to 8.6 μM for Set 1(Cla-A to Cla-F).
Cladosporin inhibited the parasite strongly with an EC50 of 0.08 μM (Figure 3b and c). Set
2 (Cla- G to Cla-L) were inert with no enzyme and parasite inhibition (supplementary
Table 2) Hence, together these results allowed us to identify cladosporin inspired Cla-A
to Cla-F analogs as moderate inhibitors with the least potent compound (Cla-C) that
suffers from an 80-fold reduction in enzymatic activity as well as parasite growth
inhibition when compared to the parent molecule cladosporin (Figure 3b and c).
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Binding of Cla-B to PfKRS as measured by Surface Plasma Resonance (SPR)
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We also performed surface plasmon resonance (SPR) experiments and calculated the K
D
using the ratio of the dissociation rate constant (k ) to the association rate constant (k )
d
a
for Cla-B with PfKRS. The binding kinetics were determined using BIAcore T200 SPR (GE
Healthcare). PfKRS was chemically immobilized on a BIAcore CM5 sensor chip at pH 4.5
according to the immobilization kit (GE Healthcare). The sensogram was obtained by
using a different concentration of Cla-B ranging from 50 to 0.5 μM. The kinetic fitting of
the data was done using 1:1 protein/compound interaction model. Sensogram analysis
−
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for the interactions of Cla-B with immobilized PfKRS resulted in k
a
105 M ·s and k
d
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-4 −1
ₓ10 s with K
D
7.5 μM. This is of the same order of magnitude as of the IC50 (6.2 μM) and
EC50 (7.5 μM). SPR experiment validated the enzymatic data results for the compound to
have moderate binding affinity (Figure 4).
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Figure 3- (a) Protein thermal shift profiles (T
m
) of PfKRS (2 μM) in the presence of 2 mM
L-lysine with inhibitors cladosporin (20 μM) or piperidine ring inspired clado-analogs
(20 μM). (b) Aminoacylation activity inhibition assays and blood stage Plasmodium
falciparum growth inhibition assays in presence of cladosporin. For aminoacylation, the
concentration ranges from 10 to 0.01 μM and for parasite inhibition assay it ranges from
1 to 0.0078 μM. (c) Aminoacylation activity inhibition assays and blood stage Plasmodium
falciparum growth inhibition assays for Cla- series. For aminoacylation, the concentration
for compounds ranges from 100 to 0.01 μM and for parasite inhibition assay it ranges
from 10 to 0.078 μM.
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Figure 4 – BIAcore surface plasma resonance (SPR) binding analyses of compound Cla-B
binding to PfKRS (a) Surface plasmon resonance (SPR) Sensogram for the interaction of
Cla-B with immobilized PfKRS on CM5 chip. The Sensogram was obtained by using a
different concentration of ligands (ranging from 50 to 0.5 μM) from top to bottom. (b)
The binding curves for the interaction of compound CLA-B with PfKRS were fit using a
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1:1 kinetic model to generate a K of 7.5 μM. The black lines show the fitting curves.
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Table 1- Compiled data for Set 1 analogues from the Cla-series. The data consists of
thermal shift profile (T
m
), in vitro aminoacylation (IC50) and parasite inhibition assay
(EC50).
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Structural aspects of Cla-B and Cla-C binding to target enzyme
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To further understand the structure-activity relationships, Cla-B and Cla-C were co-
crystallized with PfKRS and L-lysine. Their crystals diffracted to ∼3 Å and 2.8 Å resolution
respectively (Supplementary Table1). Both the crystal structures were solved by
molecular replacement using chain A of PfKRS-L-lysine-Cladosporin (PDB ID: 4PG3) as a
template. The asymmetric unit in PfKRS-L-lysine-Cla-B/C crystals contain two PfKRS
molecules that form a typical class II synthetase homodimer. Clear electron densities for
L-lysine and Cla-B/Cla-C were observed in each of the adenosine pockets of the
protomers indicating good binding (Figure 5b and Supplementary Figure 2). Structures
of Cla-B/C and cladosporin bound PfKRS enzymes are essentially identical with a root
mean-square deviation (RMSD) between 0.6-0.7 Å. The structures have a well-defined
electron density for a disulfide bond (Cys517–Cys540) (Figure 5a and 5b) similar to the
cladosporin-bound PfKRS. (13,19,29) However, the orientation of the piperidine ring is
different from that of the THP ring of the cladosporin (Figure 7). The similar orientation
change was observed for the cladosporin derivative, CLD 1 (PDB ID 6KA6) from Zhou et
al. (compound with the methylcyclohexane ring instead of the tetrahydropyran
moiety).[19] An additional water molecule was found for the active site of cladosporin
(CLD) (PDB ID- 4PG3) and CLD 1 (PDB ID-6KA6) bound PfKRS structures. This water
molecule is absent for Cla-B (PDB ID- 6L4Q) and Cla-C (PDB ID- 6L3Y) bound PfKRS
structures and this may be due to displacement from ensuing drug-protein interactions
(Figure 6). The isocoumarin moiety of Cla-B and Cla-C interacts with the side chains of
His338, Phe342, Asn503 and Glu500 (Figure 6a.3 and a.4) in exact accordance to
previously reported studies (PDB ID- 4PG3 and 6KA6) (Figure 6a.1 and a.2) [13,19]. The
two hydroxyl groups on the isocoumarin moiety form hydrogen bonds with the side chain
of Glu332 and Asn339 in comparison to cladosporin and CLD 1. By virtue of the missing
water molecule, a water mediated H-bond is not found to exist with the carbonyl group
of isocoumarin ring – which was the case in cladosporin (Figure 6).[13] Cladosporin had a
tetrahydropyran moiety which is contrasted by both the compounds (PDB ID-6KA6 and
6L3Y) having a piperidine ring instead and it was noted that the nitrogen atom in the ring
interacts with Glu500 (Figure 6a.3 and a.4). The piperidine ring of Cla-B/C accommodates
the ribose recognizing pocket formed by the side chains of Arg330, Ser344, and Asn503.
The structures of Cla-B, Cla-C bound PfKRS were superimposed on cladosporin and it was
observed that loop1 (residues 278-289) and loop 2 (residues 330-340) undergo key
rearrangements in case of Cla-C bound PfKRS. The bulkier ethyl group at C14 of the
piperidine ring in case of Cla-C leads to destabilization of loop 1. It was also noted that
the engagement of Cla-C within the active site leads to opening of loop 2 when compared
to cladosporin and Cla-B bound structures (Figure 7).
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Cla-B with an R-configuration of methyl moiety on C14 in piperidine ring differs from Cla-
C having an S-configuration of ethyl moiety on C14. When the two compounds were
superimposed on each other as well on cladosporin (S-configuration methyl moiety on
C14 in THP ring) (Supplementary Figure 2a), it was evident that the methyl moiety of Cla-
B points downward while ethyl moiety of Cla-C points upwards, similar to cladosporin.
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Figure 5: (a) The molecular surface representation for the ternary complex structure of
PfKRS-Cla-B- L-Lys. The bound ligands Cla-B (pink), L-lysine (green) and the disulphide
bond (Cys517-Cys540, yellow) were highlighted with stick representation. (b) Simulated
annealing omit (SA-omit) (orange) and experimental 2F
o
-2F map (blue) countered at
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1.0σ level for Cla-B, L-lysine and the disulphide bond (Cys517-Cys540). (c) The surface
view showing the buried ligands in the PfKRS active site.
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Figure 6: (a) The zoomed views show the PfKRS active site bound ligands cladosporin
(CLD, green), cladosporin derivative 1 (CLD 1, blue), Cla-B (pink) and Cla-C (sandy
brown). The water molecules involved in the interaction is shown as orange sphere. The
hydrogen bonding interactions are shown in dashed lines and the interaction distances
(Å) were also marked. The water mediated hydrogen bonds are marked with orange
circle and the interaction between the N atom of piperidine ring in Cla-B and Cla-C and
protein residue Glu 500 is shown by black circle.
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Structural comparison of ATP and cladosporin bound HsKRS
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The drug-enzyme interactions were evaluated using protein ligand interaction profiler
[23]
and Chimera.[24] The ATP (PDB ID 3BJU) [26] and cladosporin (PDB ID 4YCU)
(PLIP)
[25]
bound HsKRS structures were superimposed and it was noted that cladosporin
engagement with the HsKRS causes significant local perturbations in side chains of
Glu325, Arg323, Glu487, Asp445, Arg553, and Glu494 within the active site, leading to its
chemically robust steric and stereochemical accommodation (Supplementary Figure 3).
The isocoumarin moiety that mimics the adenine portion of ATP is stabilized by pi(π)
stacking interactions with the benzene ring of Phe335 and His331 while adenine is
recognized only by Phe335. It was noted that the isocoumarin moiety makes
comparatively stronger H-bond with side chains of Glu325, Asn332, and Arg553
(Supplementary Figure 4). [25,27] The THP moiety of cladosporin is stabilized by
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hydrophobic interactions with the side chain of Thr337, Glu494 and Asn497. These
hydrophobic interactions are absent in the case of ribose, and instead it forms an H-bond
with the side chain of Glu494 (Supplementary Figure 4). These few subtle differences
likely structurally underpin the tight binding of cladosporin when compared to the
natural ligand ATP.
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Overall, the biological evaluation and structural exploration for compounds Cla-B and
Cla-C confirms our previous finding[18] that both R and S configuration are allowed at C14
position. Moreover, it appears to avoid any bulkier group at C14 since it was noted that
the addition of ethyl group at C14 in case of Cla-C leads to key readjustments of loops
within the active site. It was noted that the substitution of carboxylic (Cla-K) or a
methylene (Cla-L) functional group at C14 leads to non-active analogs. Diastereomeric
pair Cla-I and Cla-J with methyl at C15 position too were inactive. Similarly, Cla-G and H
were non potent with no enzyme or parasite growth inhibition. Hence it is noteworthy
that replacing the THP moiety with similar cyclic six membered piperidine decreases the
potency of the chemical scaffold, cladosporin.
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Figure 7: Superimposed structure of Cla-B (pink) and Cla-C (sandy brown) onto
cladosporin (green) highlighting changes in the loop1 and 2 around the active site
(marked in circle). The bulkier ethyl group of Cla-C is marked with a square.
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In this study we investigated how the cladosporin scaffold provides a robust anchoring
moiety for chemical optimization through substitution of its THP ring to a piperidine. This
was done to see whether the THP moiety as a whole is crucial for anti-malarial potency
of the compound. This study highlights a strategy for synthesizing piperidine ring scaffold
inspired clado- analogs. The planned analogs helped us to decipher whether the nitrogen
atom in the piperidine contributes to any interaction with the active site residues. The
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series was synthesized and validated with enzyme-based binding and in-vitro inhibition
assays that were complemented with cell-based in vitro parasite inhibition assays and
surface plasma resonance (SPR) studies. Cla-B and Cla-C structures in complex with
PfKRS and L-lysine revealed the key interactions that differ from the parent molecule,
cladosporin, in complex with PfKRS and L-lysine. We note the failure of the carbonyl
group forming H-bond with Arg559 due to the absence of a highly coordinated water.
Also, the nitrogen in the piperidine was found to interact with the side chain of Glu500.
Interestingly, loop 1 is destabilized in Cla-C bound PfKRS structure (PDB ID 6L3Y) due to
the presence of a bulkier ethyl group at C14 of the piperidine ring. The biological
evaluation for set 1 (Cla-A to Cla-F) indicate equivalence in binding and inhibition activity
of the isomeric pairs regardless of the stereoisomeric orientation of the methyl/ ethyl
group of the piperidine ring (Table 1). The current study suggests that replacing the THP
ring to the piperidine moiety leads to a decrease in potency against the parasite and the
PfKRS enzyme as compared to cladosporin. The SPR binding study for Cla-B validated the
enzymatic data and parasite inhibition assay results. Compounds Cla-E and Cla-F with
double bond between C3-C4 position are potent compared to Cla- A to D where there is
no optimization on the isocoumarin ring (Table 1). This indicates that further
modification of the aromatic isocoumarin moiety could potentially be viable in obtaining
a better drug candidate. Hence, this study highlights a strategy for further structural
derivatization at the THP ring or chemical optimization of these piperidine ring inspired
inhibitors to improve bio-availability as well as the potency of the compounds.
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Experimental Section
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Protein expressions and purifications:
PfKRS and HsKRS were produced in E. coli BL-21 strain in accordance with the methods
already published. [13,18] Highly pure enzymes, post-gel filtration chromatography, were
stored in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM βME at -80 °C.
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Thermal shift assays:
The TSAs were performed for both PfKRS and HsKRS in presence of compound (20 µM)
as per methods described earlier. [13,18] Briefly, purified proteins alone and/or with
different analogue were heated from 25 to 99 °C at a rate of 1 °C min and fluorescence
signals of the SYPRO® orange dye were monitored by StepOnePlus™ quantitative real-
time PCR system (Life Technologies). Data were analysed on Protein Thermal shift
software (Thermofisher). Mean value from triplicates are presented as points. No protein
controls along with buffers with only inhibitors were used as blanks and flat lines were
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observed for these fluorescence readings across the temperatures. The derivative T was
used for analysis. Binding constant calculations were done as reported earlier.[30]
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Aminoacylation assay:
The enzyme inhibition assay was done using Bio-Mol and the assay was performed
according to the earlier published report.[31] Briefly, the reaction was observed for 200
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µM ATP, 200 µM L-Lys and 100 nM recombinant KRS enzymes (Pf and Hs) in a buffer
containing 30 mM HEPES (pH 7.5), 150 mM, NaCl, 30 mM KCl, 50 mM, MgCl , 1 mM DTT
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and 2 U/ml E. coli inorganic pyrophosphatase (NEB) at 37 °C. Enzymatic reactions (100
μl total volume) were performed in a clear, flat-bottomed, 96-well plates (CoStar 96-well
standard microplates). The reaction mixture was incubated for 2 h at 37 °C. The reaction
was stopped by adding 100 μl of Bio-Mol solution to the reaction mixture and levels of
inorganic phosphate (Pi) were detected after incubation of 5 min at room temperature.
Absorbance was the measured at 620 nm using a Spectramax M2 (Molecular Devices).
Reactions without KRS enzyme were performed as background controls, values of which
were subtracted from the reactions with enzyme. For the standard, cladosporin (CLD),
were added to our aminoacylation assay reaction buffer in varying concentrations
ranging from 10 to 0 µM while rest of the Cla-series compounds from 100 to 0 µM. A non-
linear regression curve i.e., the percent enzyme activity versus different inhibitor
concentration (log10 scale) were plotted using Prism (GraphPad). The IC50 value for the
data is shown for three replicates as the mean ± SD.
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Parasite inhibition assay:
The strain 3D7 was cultured in O erythrocytes in RPMI 1640 (Invitrogen, USA)
+
supplemented with 4.5 mg mL−1 glucose (Sigma, USA), 0.1 mM hypoxanthine
(Invitrogen, USA), 25 mg mL−1 gentamicin (Invitrogen, USA) and 0.5% Alumax-I
(Invitrogen, USA), according to standard methods.[32] Synchronised culture was
maintained by treating ring stage parasite with sorbitol as described.[33] For inhibition
assays, 20 mM stocks were prepared of the compounds in DMSO (Sigma). Plasmodium
falciparum was cultured in 96-well plates and synchronized at ring stages. At ∼1%
parasitaemia and 4% haematocrit, inhibitor concentration ranges from 1 to 0.0078 µM
for Cladosporin, 10 to 0.078 µM for clado-series. They were incubated for 48 hours with
the parasites. Growth was assayed by SYBR green-I (Molecular probes) DNA staining
assays as described earlier.[34] Briefly, 100 μl SYBR green dye in 1× concentration in lysis
buffer supplemented with 0.1% saponin was added to each well. After 45 min incubation
at 37 °C fluorescence was estimated using multi-well plate reader (Victor 3, Perkin Elmer)
with excitation and emission wavelength bands cantered at 485 and 530 nm respectively.
Chloroquine was taken as a positive control and all experiments were done in triplicates.
The EC50 values were obtained by plotting a nonlinear regression curve as a function of
fluorescence values expressed in terms of percentage inhibition of parasite growth at
each inhibitor concentration using GraphPad Prism. All data are shown for three
replicates as means with standard errors.
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Crystallization:
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Highly pure PfKRS was stored at 15 mg ml -80 °C. Before crystallization, 0.5 mM
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inhibitors along with 2 mM L-Lys were added to 13 mg ml protein solutions and
incubated at 4 °C for 30 min. The PfKRS+ Cla-B/C+L-Lys crystals were obtained using
hanging-drop vapour diffusion method at 20 °C. Initial screenings were carried out using
nano-drop dispensing robot Mosquito (TTP Labtech) and the droplet size was a mixture
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of 100 nl of PfKRS+ Cla-B/C +L-Lys complex and 100 nl of well solutions from commercial
crystal screens (Hampton Research and Molecular Dimensions). The crystallization
drops were equilibrated against 75/100 µl well reservoir solutions using commercially
available Morpheus screening reagents from Molecular Dimensions Ltd.
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Data collection and structure determination:
X-ray diffraction data were collected using beam-line I03 at Diamond Light Source (DLS),
United Kingdom at a wavelength of 0.9795 Å. The data were processed by the xia2 auto-
processing pipeline using DIALS [35], for integration. The initial model for PfKRS-L-Lys-
Cla-B/C was determined by the molecular-replacement (MR) method using Phaser [36]
with PDB entry 4PG3 as the template. The structure was further refined by iterative
cycles of refinement with Phenix [37] and model building with COOT [38]. The stereo-
chemical quality of the model was analysed using MolProbity [39]. Statistics of data
collection and structure refinement are given in Supplementary Table 1. The atomic
coordinates and structural factors have been deposited into Protein Data Bank with
accession codes 6L4Q and 6L3Y. All structural superposition and preparation of figures
was done using UCSF Chimera and PyMOL (http://www.pymol.org).
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Surface Plasma Resonance (SPR) Assay:
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PfKRS was chemically immobilized on a BIAcore CM5 sensor chip at pH 4.5 according to
the immobilization kit (GE Healthcare). PfKRS (100 µg/µl) was captured for 7mins at a
flow of 5 µl/min. Affinity between PfKRS and Cla-B was measured by SPR on a BIAcore
◦
T200 (GE Healthcare) at 25 C with a running buffer (0.02 M phosphate buffer pH 7.4 and
5% DMSO). Cla-B dilutions were made to yield (dissolved in running buffer) at
concentrations of 50, 25, 10, 125 and 0.5 µM (association time: 60 s, dissociation time:
120 s, flow: 30 µl/min). Kinetic evaluation of the interaction between PfKRS and Cla-B
was performed by global fitting of the data to a 1:1 interaction model using BIAcore
Evaluation Software 3.1 (GE Healthcare).
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Author Contributions
A.S., H.K. and S.P. designed the study. M.S. produced cladologs. P.B. performed protein
purifications, all enzymatic and binding assays. P.B. crystallized PfKRS complexes and
froze the crystals along with M.Y. K.H. collected the data sets, and P.B. solved the crystal
structures. S.M. and S.G. assisted P.B. in structural analysis and parasite culture
maintenance respectively. P.B. drafted and edited the manuscript. All authors reviewed
the manuscript.
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ACKNOWLEDGMENT
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P. B. thanks UGC for research fellowship. AS is supported by JC Bose fellowship from DST
and DBT grant PR32713 for drug discovery against malaria parasite tRNA synthetases.
KEYWORDS: Analogues, Anti-malaria, Cladosporin, Diastereomers, Co-crystal, Drug
development, Medicinal chemistry, Structure activity relationship
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ABBREVIATIONS
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AARS- Amino-acyl tRNA synthetases, Pf- Plasmodium falciparum, PRS- Prolyl-tRNA
synthetase, KRS- Lysyl-tRNA synthetases, Hs- Homo sapiens, SPR- Surface plasma
resonance.
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ChemBioChem
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Table of Content
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The study focuses on a planned synthesis, biological and structural analysis of piperidine
ring inspired cladosporin inhibitors against PfKRS. The series retain selectivity towards
its human counterpart but it was observed that replacing the tetrahydropyran moiety to
piperidine, reduces the potency of the compound. Co-crystals of Cla-B and Cla-C with
PfKRS reveals key interaction and loops readjustment that allow drug binding and
inhibition of the enzyme.
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Keywords: Analogs, Anti-malaria, Cladosporin, Stereoisomers, Co-crystal
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