Improvement of kynurenine aminotransferase-II inhibitors guided by mimicking sulfate esters

Article abstract of DOI:10.1371/journal.pone.0196404

The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related isoforms that are pyridoxal 5’-phosphate-dependent, responsible for the irreversible transamination of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such as schizophrenia where it is found in elevated levels and consequently KAT-II, as the isoform predominantly responsible for kynurenic acid production in the brain, has been targeted for the development of specific inhibitors. One class of compounds that have also shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters. Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate makes a significant contribution to its potency. The work here demonstrates that the effect of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC₅₀: 315 μM), but the deliberate placement of a sulfonamide group significantly improved the potency of JN-01 (IC₅₀: 73.8 μM) and JN-02 (IC₅₀: 112.8 μM) in comparison to the parent compound. This 3–4 fold increase in potency shows the potential of these moieties to be accommodated in the KAT-II active site and the effect they can have on improving inhibitors, and the environments in the KAT-II have been suitably modelled using docking calculations.

Full text of DOI:10.1371/journal.pone.0196404

RESEARCH ARTICLE
Improvement of kynurenine
aminotransferase-II inhibitors guided by
mimicking sulfate esters
Gayan S. Jayawickrama, Alireza Nematollahi, Guanchen Sun¤, William Bret Church*
Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, The University of Sydney, Sydney,
New South Wales, Australia
¤ Current address: Fountain Biotech, Sydney, New South Wales, Australia
* bret.church@sydney.edu.au
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Abstract
The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related iso-
forms that are pyridoxal 5’-phosphate-dependent, responsible for the irreversible transami-
nation of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such
as schizophrenia where it is found in elevated levels and consequently KAT-II, as the iso-
form predominantly responsible for kynurenic acid production in the brain, has been tar-
geted for the development of specific inhibitors. One class of compounds that have also
shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters.
Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate
makes a significant contribution to its potency. The work here demonstrates that the effect
of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two
novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC50: 315 μM),
but the deliberate placement of a sulfonamide group significantly improved the potency of
JN-01 (IC50: 73.8 μM) and JN-02 (IC50: 112.8 μM) in comparison to the parent compound.
This 3–4 fold increase in potency shows the potential of these moieties to be accommodated
in the KAT-II active site and the effect they can have on improving inhibitors, and the envi-
ronments in the KAT-II have been suitably modelled using docking calculations.
OPEN ACCESS
Citation: Jayawickrama GS, Nematollahi A, Sun G,
Church WB (2018) Improvement of kynurenine
aminotransferase-II inhibitors guided by mimicking
sulfate esters. PLoS ONE 13(4): e0196404. https://
doi.org/10.1371/journal.pone.0196404
Editor: Gilles J. Guillemin, Macquarie University,
AUSTRALIA
Received: January 31, 2018
Accepted: April 12, 2018
Published: April 24, 2018
Copyright: © 2018 Jayawickrama et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All HPLC assay files
and PDB files of docked molecules are available
from the Figshare repository (https://figshare.com/
projects/Design_of_novel_kynurenine_
Introduction
Kynurenic acid (KYNA) is a metabolite formed in the kynurenine pathway of tryptophan
catabolism (Fig 1), produced when kynurenine is irreversibly transaminated into KYNA by
the kynurenine aminotransferase (KAT) enzymes [1]. KYNA is an antagonist of the glycine
and glutamate binding sites of NMDA receptors [2, 3], the α-amino-3-hydroxy-5-methyl-
4-isoxazole propionic acid (AMPA) receptor [4], and kainate receptors [5]. By inhibiting the
activity of these glutamatergic receptors and preventing excitotoxic attacks, as well as diverting
the pathway from the formation of neurotoxic metabolites (such as 3-hydroxykynurenine and
quinolinic acid), KYNA can be considered neuroprotective. KYNA also may have antagonistic
aminotransferase-II_inhibitors/28713).
Funding: Authors A.N., G.S., G.S.J. received
support from the University of Sydney and
Australian Postgraduate Research Awards, and W.
B.C. was supported by funding from the Australian
Research Council and The Rebecca L. Cooper
Medical Research Foundation. GS is currently
employed by Fountain Biotech. Fountain Biotech
PLOS ONE | https://doi.org/10.1371/journal.pone.0196404 April 24, 2018
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Design of novel KAT-II inhibitors
provides support for author GS, but did not have
any additional role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. The specific role of
this author is articulated in the ‘author
contributions’ section.
Competing interests: GS is now employed by
Fountain Biotech [this employment commenced
after the completion of the study]. WBC is
associated with the following patent application
relating to small peptides and related compounds
in the treatment of human disease [Entitled:
Method of screening for inhibitors of sPLA2-IIA,
International Patent Application PCT/AU2011/
000781]. Fountain Biotech holds no patents, but
has products Syneregen and Fetopro (as listed at
https://www.fountainbiotech.com.au/innovita/).
Vman is a product coming soon. There are no
further patents, products in development or
marketed products to declare. This does not alter
our adherence to all the PLOS ONE policies on
sharing data and materials.
Fig 1. Tryptophan metabolism through the kynurenine pathway. Tryptophan metabolism proceeds through
kynurenine in multiple branches, which includes the formation of KYNA by the KAT enzymes.
https://doi.org/10.1371/journal.pone.0196404.g001
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Design of novel KAT-II inhibitors
effects on the α7-nicotonic acetylcholine receptors [6], which can play a role in glutamate and
dopamine modulation, and also displays antioxidant [7] and anticonvulsive [8, 9] capabilities.
Despite this ability to be neuroprotective, elevated KYNA levels have been observed in the
prefrontal cortex [10] and cerebrospinal fluid [11] of patients with schizophrenia, suggesting
that a balance is necessary to prevent adverse events, particularly with regards to cognitive
function. Animal studies have shown that lowering KYNA levels by application of KAT-II
inhibitors increases glutamate [12, 13], acetylcholine [14], dopamine [15] and GABA [16] lev-
els, for which roles in cognition have been established, and improves the performance of mem-
ory and spatial learning in rats and non-human primates [12, 13, 17]. A similar profile, with
increased glutamate release and cognitive improvements, was also observed in KAT-II knock-
out mice [18]. These pro-cognitive effects from KAT-II inhibition was neutralised by the
administration of KYNA [13].
There are four isoforms of KAT, differing in structure and substrate specificities. KAT-I
and KAT-III contain an aromatic crown present in the active site that enables the transamina-
tion of relatively hydrophobic substrates. In comparison, KAT-II and KAT-IV contain more
neutral and polar amino acids, and KAT-II in particular is able to accommodate larger struc-
tures in the active site due to its flexible N-terminal region which helps construct the active site
of the opposite subunit [19]. Individual KAT activity may also contrast greatly between species
of animals, as for example the liver and kidney KAT activity of rats were significantly higher
than the corresponding activity in cats [20].
The KAT isoforms are widely distributed in tissues, including the liver, kidney and brain
[21]. KAT-II is thought to be the isoform predominantly responsible for the synthesis of
KYNA in the brain, preferentially within astrocytes [22, 23]. KAT-I is relatively versatile with
broader substrate recognition, making its activity less specific to KYNA synthesis [24]. By
these considerations, KAT-II is targeted for the design of inhibitors to reduce brain KYNA lev-
els. KAT-II is a homodimer of the fold type 1 family of pyridoxal 5’-phosphate (PLP)-depen-
dent enzymes [25] and each of the subunits contain a large domain which has the PLP-binding
site, a small domain containing the C-terminus, and an N-terminal arm [1]. The interface of
the subunits houses an active site and the PLP-binding site is nearby for each subunit [1, 26].
PLP is covalently attached to Lys-263 of the enzyme by a Schiff base linkage [27]. When
kynurenine enters the active site, this link is broken and a new aldimine bond is formed
between PLP and the α-amine group of kynurenine [28]. This α-amino group is transferred
onto the PLP, forming pyridoxamine phosphate (PMP), and KYNA is also produced from the
remains of the substrate by spontaneous closure of its ring. PMP then transfers the α-amino
group to an α-keto acid co-substrate to regenerate into the PLP form.
Arg-20 is another notable residue in the active site, with its side chain having pi-cation
interactions with the aromatic ring of kynurenine (Fig 2) [26]. This region of the N-terminal
residues allows the entry of the substrate into the active site by undergoing a conformational
change. Asn-202 and Gly-39 forms hydrogen bonds with the amine group, and Arg-399 forms
a salt bridge with the carboxyl group in kynurenine, and in doing so these residues help anchor
the substrate in the necessary orientation within the active site [26].
Previous KAT-II inhibitors include S-ESBA, BFF-122, PF-04859989, BFF-816 and NS-1502
(S1 Fig). S-ESBA was designed by using kynurenine as a scaffold and adding an ethylsulfonyl
substituent, which confers selectivity by increasing steric bulk and disqualifying it from occu-
pying the relatively inflexible KAT-I [24]. The phenyl amino group was also removed from the
scaffold, which eliminated the ability of KAT-II to transaminate it, rendering it inert and
therefore inhibitory [30]. BFF-122, which shares a high similarity to the fluoroquinolone, levo-
floxacin, may also derive its selectivity from steric bulk that is accommodated by KAT-II [31].
Both BFF-122 and PF-04859989 feature heterocyclic ring systems and a primary amine group,
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Design of novel KAT-II inhibitors
Fig 2. Kynurenine in the KAT-II active site. The amino acids within 5.0 Å of kynurenine (green) were chosen for display, with PLP in magenta and
residue Tyr-142 removed for clarity. Depicted is the aromatic ring forming pi-pi interactions (green dashes) with Tyr-74 and pi-cation interactions
(blue dashes) with Arg-20. A hydrogen bond (yellow dashes) between the amine group and Asn-202, and a salt bridge (yellow dashes) formed between
the carboxyl and Arg-399 helps anchor kynurenine in the correct conformation for transamination. Image generated with PyMOL [29].
https://doi.org/10.1371/journal.pone.0196404.g002
the latter of which confers a mechanism of irreversible inhibition as it is observed to form a
covalent adduct with PLP [31, 32]. NS-1502 combined features of these three inhibitors by
incorporating a phthalimide core to resemble BFF-122 and PF-04859989, and adding an aro-
matic amino acid branch which resembled S-ESBA to improve the potency further [33].
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Design of novel KAT-II inhibitors
Notably NS-1502 was designed to be reversible by the omission of the primary amine group
present in BFF-122 and PF-04859989. Although irreversible inhibitors may be suitable in
many instances, as PLP is a cofactor used by hundreds of enzymes, there is rationale in creat-
ing reversible inhibitors that specifically target KAT-II as opposed to PLP. This approach may
be important to avoid the prospects of adverse events caused by depleting the cofactor or
inhibiting off-target enzymes. Such concerns appear to be borne out by the use of carbidopa in
patients with Parkinson’s disease, as it is able to permanently deactivate PLP, and is associated
with the development of dyskinesias and increasing death rates [34, 35].
Steroid compounds, such as estrogen and sulfate esters of estrogen, have also been shown
to inhibit a variety of PLP-dependent enzymes [36], including the KAT enzymes. Typically it
has been reported that unconjugated steroids such as estradiol and estrone have a lower
potency in inhibiting KAT from rat kidney in comparison to the sulfate ester forms [37]. The
disulfate esters of estrogens, such as estradiol disulfate and diethylstilbestrol disulfate, were the
most potent of these, while estrone sulfate was inhibitory at higher concentrations [37]. Similar
results were found with phosphate esters, with estradiol diphosphate being very inhibitory
towards rat kidney KAT [38]. The steroid phosphate compounds were also tested on another
PLP-dependent enzyme, aspartate aminotransferase, with the phosphate groups appearing on
different positions in the steroid structure. From this work, it was found that the phosphate
group on the 17-position, as opposed to the 3-position, yielded the compound inhibiting with
the greatest potency [38]. Similar results have been inferred in our lab, where a sulfate in the
17-position, instead of the 3-position, appears to increase the potency of the sulfated estrogen
compounds inhibiting KAT-II [39]. From molecular docking, it is predicted that the increased
potency may derive from the 17-sulfate forming hydrogen bonds with key residues in the
active site, including, Asn-202 and Lys-263 [39].
In our current study, our goal was to use NS-1502 with a mind to improve its inhibitory
capability further using detailed structural knowledge about the manner in which sulfated
estrogens derive their potencies. Using an inhibition assay, we were able to evaluate the
potency of two novel, reversible KAT II inhibitors, JN-01 and JN-02.
Results and discussion
For the design approach to improving this inhibitor, we took the core of NS-1502 and strategi-
cally mimicked sulfated estrogens when synthesising JN-01. Due to the considerations from
the inhibitor synthesis protocol, the sulfate moiety was replaced by a sulphonamide group, and
the carboxyl present in NS-1502 was omitted. Evidence suggested that in the inhibitor estradiol
disulfate, the sulfate on the 17-position, located on the end opposite to its aromatic ring (S2
Fig), was most important for its potency. Molecular docking predicted that the phthalimide
core of NS-1502 (Fig 3) corresponded to the location of the aromatic ring of both kynurenine
(Fig 2) and estradiol disulfate (S3 Fig). Therefore in designing JN-01, the rationale was to
introduce a sulfonamide group onto the end opposite to the phthalimide core of NS-1502, so
that it occupies the same location as the 17-sulfate of estradiol disulfate, where it can similarly
exploit interactions with important nearby residues such as Asn-202 and Lys-263. Overall, JN-
01 was also designed to ensure that it retained a length comparable to estradiol disulfate (Fig
4), with the difference coming from the relative sizes of the sulfonamide and the sulfate group.
JN-02 was designed with a similar concept in mind to JN-01, however the length of the
compound was further increased, by an ethyl chain, which better reflected the size of NS-1502,
while still incorporating the other features of JN-01 and estradiol disulfate. Although it is
unquestionably larger than estradiol disulfate, JN-02 is flexible and can be accommodated to
interact with similar active site residues.
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Design of novel KAT-II inhibitors
Fig 3. NS-1502 docked into the KAT-II active site. The amino acids within 5.0 Å of NS-1502 (green) were chosen for display. Residues Ser-143, Gly-
144, and Gln-289 were removed for clarity. The carboxyl group forms hydrogen bonds (yellow dashes) with Asn-202 and Arg-399, and the aromatic
ring has pi-pi interactions (green dashes) with Tyr-142 and pi-cation interactions (blue dashes) with Lys-263. Image generated with PyMOL [29].
https://doi.org/10.1371/journal.pone.0196404.g003
The inhibition assay was previously performed on KAT-II with racemic NS-1502, BFF-122,
and PF-04859989, which determined their IC50 to be 315 μM, 15–20 μM and 1–3 μM, respec-
tively [33]. By varying the concentration of PLP in the assay conditions, NS-1502 was found to
be reversible. When PLP concentration was increased, the inhibition caused by NS-1502
decreased as it competed with it. This contrasts with the irreversible inhibitor PF-04859989,
which displayed similar inhibition as PLP concentration changed [33].
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Design of novel KAT-II inhibitors
Fig 4. Comparative lengths of the structures when fully extended. Distances measured in the 3D conformer of the compounds, generated from
LigPrep at the time of preparation for docking, from the first carbon atom (A) in each compound to the carbon from which the carboxyl branches off
(B), the furthest carbon atom (C), or the sulfur atom in the sulfate/sulfonamide groups (D).
https://doi.org/10.1371/journal.pone.0196404.g004
Relative to NS-1502, JN-01 and JN-02 displayed improved potency towards inhibiting
KAT-II, with an IC50 of 73.8 μM (Fig 5A) and 112.8 μM (Fig 5B), respectively. The IC50 of
both inhibitors for KAT-I in this assay is beyond the maximal concentration tested (2 mM),
therefore they are considered to be selective towards KAT-II. Additionally, both inhibitors
were found to be reversible. This reaffirms what was seen previously with estradiol disulfate
and estradiol diphosphate where the 17- sulfate or 17-phosphate groups improved the inhibi-
tory potency of the molecule, as these moieties were mirrored in JN-01 and JN-02 with the use
of a sulfonamide group and similarly improved inhibition greatly from the parent compound,
NS-1502.
Estradiol disulfate and estradiol has also been tested using this assay in these conditions and
inhibits KAT-II with an IC50 of 26.3 μM and >2 mM, respectively [39]. The apparent increase
in potency for KAT-II inhibition is 100-fold comparing estradiol disulfate and estradiol,
whereas it is approximately 3–4 times for JN-01 and JN-02 in comparison to NS-1502. The
absolute value for the IC50 is also lower for estradiol disulfate than it is for JN-01 and JN-02,
suggesting that there also remains ample area for improvement in these new inhibitors. Two
such areas that have been identified includes the lack of the carboxyl that was present in NS-
1502 but not the new inhibitors, and also the sulfate present in estradiol disulfate being substi-
tuted by a sulfonamide moiety in the new inhibitors. The reintroduction of the carboxyl group
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Design of novel KAT-II inhibitors
Fig 5. Dose-dependent inhibition of KAT-II. Inhibition assays with 0.5 μg KAT-II incubated for 10 min at 37 ˚C in a 50 μL reaction mixture
containing 50 μM PLP, 5 mM α-ketoglutarate, 5 mM L-kynurenine and the inhibitor studied. The reaction was terminated with 0.8 M formic acid and
analysed using HPLC. Experiments were performed as independent replicates, and figures were produced using GraphPad Prism v7.02 [40] (A) JN-01
inhibition (IC50: 73.8 (95% CI: 66.3–81.8) μM, R2: 0.91). (B) JN-02 inhibition (IC50: 112.8 (95% CI: 100.8–126.1) μM, R2: 0.92).
https://doi.org/10.1371/journal.pone.0196404.g005
and the replacement of the sulfonamide with the sulfate could maximise the number and
strength of the hydrogen bonds formed with the residues in the active site. Molecular docking
of NS-1502, JN-01, JN-02, and NS-1502 with a sulfate (JN-03) reinforces this.
Docking into KAT-II suggests that the best binding pose for NS-1502 has the phthalimide
core situated near Arg-20, with the carboxyl forming hydrogen bonds with Arg-399 and Asn-
202, and the lone six-membered aromatic ring forming pi-pi interactions with Tyr-142 and pi-
cation interactions with Lys-263 (Fig 3). When JN-01 is docked, the phthalimide is orientated
in a similar location, and the sulfonamide group has hydrogen bonding interactions with Asn-
202 and Lys-263 (Fig 6). The whole of JN-02 docks in a similar pose to NS-1502, while lacking
the carboxyl interactions but gaining an additional hydrogen bond between the sulfonamide
and Gln-118 (Fig 7). Both JN-01 and JN-02 displayed the potential of this binding pocket, near
Lys-263, in accommodating the sulfonamide moieties.
Considering NS-1502 and estradiol disulfate, the key interactions these two inhibitors have
with the active site are with Arg-399, Asn-202 and Lys-263. A carboxyl on JN-02, branching
off the same location as it does in NS-1502, would retain the positioning required to interact
with Arg-399 and Asn-202, whilst the flexibility of JN-02 allows the sulfonamide group to also
interact with residues near Lys-263. The docking of JN-03 was performed for the purpose of
considering all these interactions (Fig 8). For JN-03, as with the others, the phthalimide core is
oriented next to Arg-20. The carboxyl group forms hydrogen bonds with Arg-399 and Asn-
202, just as it does in NS-1502, a feature that was not prevalent in either JN-01 or JN-02. In
place of the sulfonamide, the sulfate forms many hydrogen bonding interactions with the
nearby residues including that of Tyr-74, Gln-118, Lys-263, and Arg-270.
It has been argued that the ability of sulfated estrogens to inhibit KAT is derived from a
reversible reaction with the apoenzyme form of the protein, and it competes with PLP for the
binding to the apoenzyme [37]. As mentioned previously, NS-1502, JN-01 and JN-02 displayed
a similar competition with PLP to that seen for the estrogens, as they had reduced inhibition
when PLP concentration increased. This work demonstrates that the potency of these types of
inhibitors can be improved by introducing moieties, such as the sulfonamide or sulfate groups,
to form stronger bonds within the active site and out-compete PLP for the apoenzyme. The
notion that these moieties could be accommodated in the inhibitor binding region was proven
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Design of novel KAT-II inhibitors
Fig 6. JN-01 docked into the KAT-II active site. The amino acids within 5.0 Å of JN-01 (green) were chosen for display. Residues Ser-143, Gly-144,
and Gln-289 were removed for clarity. The oxygen atoms in the sulfonamide form hydrogen bonds (yellow dashes) with Asn-202 and Lys-263, and the
aromatic ring has pi-pi interactions (green dashes) with Tyr-74. Image generated with PyMOL [29].
https://doi.org/10.1371/journal.pone.0196404.g006
to be correct by the assay results, although the docking of the compounds suggest that the
intricate detail in which they bind can vary depending on the flexibility of each structure.
Materials and methods
General procedures
Commercially available reagents were used without additional purification unless otherwise
stated. 4,5-dichlorophthalic anhydride, 4-aminobenzenesulfonamide, and 4-(2-aminoethyl)
benzenesulfonamide were purchased from Sigma-Aldrich (Sydney, Australia). 1H-NMR (400
MHz) and 13C-NMR (101 MHz) spectroscopy was obtained using a Gemini 400-MR NMR
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Design of novel KAT-II inhibitors
Fig 7. JN-02 docked into the KAT-II active site. The amino acids within 5.0 Å of JN-02 (green) were chosen for display. Residues Ser-143, Gly-144,
and Gln-289 were removed for clarity. The amine in the sulfonamide forms hydrogen bonds (yellow dashes) with Gln-118, and the carbonyl in the
phthalimide rings forms a hydrogen bond with Tyr-142. The aromatic ring has pi-pi interactions (green dashes) with Tyr-142 and pi-cation interactions
(blue dashes) with Lys-263. Image generated with PyMOL [29].
https://doi.org/10.1371/journal.pone.0196404.g007
spectrometer (Varian, Palo Alto, CA, USA), using DMSO-d6 as NMR solvent. Low-resolution
ESI-MS was measured on an amaZon SL (Bruker Daltonics, Bremen, Germany). High resolu-
tion ESI-MS was performed on an Apex Ultra Fourier Transform Ion Cyclotron Resonance 7
T Mass Spectrometer (Bruker Daltonics, Bremen, Germany). The mass spectra were obtained
by direct infusion electrospray ionization and are reported as mass to charge ratio (m/z) and
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Design of novel KAT-II inhibitors
Fig 8. JN-03 docked into the KAT-II active site. The amino acids within 5.0 Å of JN-03 (green) were chosen for display. Residues Ser-143, Gly-144,
and Gln-289 were removed for clarity. The sulfate forms hydrogen bonds with Tyr-74, Gln-118, Lys-263, and Arg-270, and the carboxyl moiety forms
hydrogen bonds with Asn-202 and Arg-399. The aromatic ring has pi-cation interactions (blue dashes) with Lys-263. Image generated with PyMOL
[29].
https://doi.org/10.1371/journal.pone.0196404.g008
relative intensity (%). The estimated purity for the synthesised inhibitors is > 93%, with the
final yield approximately 70%. HPLC was performed using a CBM-20A Shimadzu HPLC sys-
tem (Kyoto, Japan), with an Ascentis C18 HPLC column (particle size: 10 μm, length: 25 cm,
internal diameter: 4.6 mm) purchased from Sigma-Aldrich (Sydney, Australia). The inhibition
assay data was modelled with a nonlinear regression fit of the log[inhibitor] vs. normalised
response (using a variable slope) in GraphPad Prism v7.02 software [40], and the IC50 is
reported with 95% confidence intervals.
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Design of novel KAT-II inhibitors
Protein preparation
The recombinant KAT-II protein was expressed in E. coli Rosetta 2 cells with a hexa-histidine
tag, and purified using nickel nitrilotriacetic acid column chromatography in our lab as previ-
ously described [41]. Recombinant KAT-I was expressed in Spodoptera frugiperda insect cells,
precipitated using ammonium sulfate, and purified in a high salt ion exchange column as pre-
viously described [42].
Synthesis of JN-01
A solution of 4,5-dichlorophthalic anhydride (2.5 mM) and 4-aminobenzenesulfonamide (2.5
mM) in glacial acetic acid (7 mL) was stirred and heated under reflux for 4 h. The product, 4-
(5,6-dichloro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzenesulfonamide (JN-01), was
recovered by precipitation using cold water and ethanol, filtered and dried. 1H-NMR (400
MHz, DMSO-d6): δ 8.33 (s, 2H), 7.98 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 7.48 (s, 2H).
13C-NMR (101 MHz, DMSO-d6): δ 125.69 (C-14,17), 126.43 (C-8,12), 127.49 (C-9,11), 131.55
(C-3,4), 134.44 (C-7), 137.66 (C-15,16), 143.51 (C-10), 164.91 (C-2,5) (S1 Appendix); HRE-
SIMS: 424.97 m/z [C14H8N2O4S1Cl2 + CH3OH + Na]+ (Calcd. 424.974171) (S2 and S3
Appendices).
Synthesis of JN-02
A solution of 4,5-dichlorophthalic anhydride (2.5 mM) and 4-(2-aminoethyl)benzenesulfona-
mide (2.5 mM) in glacial acetic acid (7 mL) was stirred and heated under reflux for 4 h. The
product, 4-(5,6-dichloro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl benzenesulfonamide
JN-02, was recovered by precipitation using cold water and ethanol, filtered and dried.
1H-NMR (400 MHz, DMSO-d6): δ 8.17 (s, 2H), 7.71 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.1 Hz,
2H), 7.30 (s, 2H), 3.84 (t, J = 7.1 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H). 13C-NMR (101 MHz,
DMSO-d6): δ 33.30 (C-8), 38.89 (C-7), 125.28 (C-16,19), 125.76 (C-11,13), 129.19 (C-10,14),
131.43 (C-3,4), 137.32 (C-17,18), 142.32 (C-12), 142.37 (C-9), 165.85 (C-2,5) (S4 Appendix).
Inhibition studies using recombinant human KAT-I and KAT-II
0.5 μg of KAT-I or KAT-II was incubated at 37 ˚C for 10 min in a 50 μL reaction mixture con-
taining 50 μM PLP, 5 mM α-ketoglutarate, 5 mM L-kynurenine in PBS, pH 7.4, with the inhib-
itor being studied (1–2000 μM) dissolved in DMSO. Equal volume of formic acid (0.8 M) was
added to terminate the reaction, and 50 μL of this mixture was diluted to 1 mL. Kynurenine
and KYNA produced during the reaction was analyzed by HPLC using a C18 reverse-phase
column, with 50% (v/v) methanol and 50% (v/v) water used as the mobile phase, and with an
injection volume of 20 μL. The column temperature was 20 ˚C. Kynurenine and KYNA were
detected by UV detection at a wavelength of 330 nm. The retention time for kynurenine and
KYNA were 2.5 mins and 3.5 mins, respectively.
Docking of inhibitors into KAT-II
A crystal structure of human KAT-II (PDB ID: 2R2N [27]) was downloaded from the Protein
Data Bank. 2R2N is a high resolution human KAT-II structure (1.95 Å) with the ligand, kynur-
enine, already occupying the active site. The PLP cofactor was removed from the active site of
the protein as there was evidence it competed with the ligands in the inhibition studies. Opti-
misation and minimisation of the protein was performed using the Impref utility in IMPACT
(Schrodinger LLC) and the OPLS-2005 [43] force field, and hydrogen atoms were also added
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Design of novel KAT-II inhibitors
and water molecules not involved in the reaction were removed, using the protein preparation
wizard in Maestro version 10.4.017 (Schrodinger LLC). The active site was determined by the
location of kynurenine in the crystal structure. The ligands were prepared using LigPrep [44],
using the OPLS-2005 force field, and were docked using Glide [45] with XP docking [46]. Both
enantiomers of NS-1502 and JN-03 were docked, with the best scoring pose represented in the
figures.
Conclusions
KAT-II inhibitors have been shown to reduce the production of KYNA, increase neurotrans-
mitter release and improve memory tasks in animals [12, 13, 17]. Other compounds, particu-
larly sulfate esters of estrogens, have also been shown to be very adept in inhibiting the KAT
enzymes [37]. For sulfated estrogens, the 17-sulfate position appears to be very important in
comparison to their unconjugated parent estrogens in this potency for inhibition, with dock-
ing also suggesting that it interacts with key active site residues [39].
Using the information derived from these estrogens, and the template of the previously
designed reversible inhibitor NS-1502, we have synthesised two new inhibitors, JN-01 and
JN-02. Notably, JN-01 and JN-02 differ from NS-1502 with the addition of a sulfonamide moi-
ety, which seems to improve inhibition by approximately 3–4 fold, with an IC50 of 73.8 ( 3.9)
μM and 112.8 ( 6.1) μM, respectively. This finding suggests that knowledge gleaned from sul-
fated estrogens binding can help improve the potency of current inhibitors. A sulfate (or like-
minded) moiety can be placed to mimic the interactions of the 17-sulfate in estradiol disulfate,
and greatly improve potential inhibition.
Supporting information
S1 Fig. Structures of existing KAT-II inhibitors.
(TIF)
S2 Fig. Structure of estradiol disulfate.
(TIF)
S3 Fig. Estradiol disulfate docked into the KAT-II active site. The amino acids within 5.0 Å
of estradiol disulfate (green) were chosen for display. Residues Tyr-142 and Gly-144 were
removed for clarity. The 3-sulfate forms hydrogen bonds (yellow dashes) with Arg-20 and the
17-sulfate forms hydrogen bonds with Asn-202 and Lys-263. Image generated with PyMOL
[29].
(TIF)
S1 Appendix. 1H-NMR and 13C-NMR results for JN-01.
(PDF)
S2 Appendix. HRESIMS results for JN-01.
(PDF)
S3 Appendix. HRESIMS results for JN-01.
(PDF)
S4 Appendix. 1H-NMR and 13C-NMR results for JN-02.
(PDF)
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Design of novel KAT-II inhibitors
Acknowledgments
The authors wish to acknowledge the Sydney Computational Drug Discovery Group for soft-
ware access, as well as Noushin Aminimoghadamfarouj, Faculty of Pharmacy, for assistance
performing the NMR and Nick Proschogo, Mass Spectrometry Facility, School of Chemistry,
for performing the mass spectrometry, both in the University of Sydney, and both also for
advice and discussions.
Author Contributions
Conceptualization: Gayan S. Jayawickrama, Alireza Nematollahi, Guanchen Sun, William
Bret Church.
Formal analysis: Gayan S. Jayawickrama.
Investigation: Gayan S. Jayawickrama, Alireza Nematollahi, Guanchen Sun.
Methodology: Gayan S. Jayawickrama, Alireza Nematollahi, Guanchen Sun.
Project administration: William Bret Church.
Resources: William Bret Church.
Supervision: William Bret Church.
Writing – original draft: Gayan S. Jayawickrama.
Writing – review & editing: Gayan S. Jayawickrama, William Bret Church.
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