Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity and inhibition by LC-MS/MS method

Article abstract of DOI:10.1016/j.jpba.2019.05.025

In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-hydroxykynurenine is removed by a specific transaminase (3-hydroxyky

Full text of DOI:10.1016/j.jpba.2019.05.025

Accepted Manuscript
Title: Study of Anopheles gambiae 3-hydroxykynurenine
transaminase activity and inhibition by LC-MS/MS method
Authors: Rossana Canavesi, Riccardo Miggiano, Martina
Stella, Ubaldina Galli, Franca Rossi, Menico Rizzi, Erika Del
Grosso
PII:
DOI:
Reference:
S0731-7085(19)30073-1
https://doi.org/10.1016/j.jpba.2019.05.025
PBA 12657
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date:
Revised date:
Accepted date:
21 January 2019
8 May 2019
12 May 2019
Please cite this article as: Canavesi R, Miggiano R, Stella M, Galli U, Rossi F, Rizzi M,
Del Grosso E, Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity
and inhibition by LC-MS/MS method, Journal of Pharmaceutical and Biomedical
Analysis (2019), https://doi.org/10.1016/j.jpba.2019.05.025
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Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity and inhibition by
LC-MS/MS method
Rossana Canavesi, Riccardo Miggiano, Martina Stella, Ubaldina Galli, Franca Rossi, Menico Rizzi,
Erika Del Grosso*
Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2,
28100 Novara, Italy
*Corresponding author: - Department of Pharmaceutical Sciences, Università del Piemonte
Orientale, Largo Donegani 2, 28100 Novara, Italy Tel: +39-0321375758; fax: +39-0321375621 e-
mail address: erika.delgrosso@uniupo.it
Graphical abstract
1

Highlights
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-
-
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Enzymatic assay for the study of Anopheles gambiae 3-hydroxykynurenine transaminase
Development and evaluation of a new LC-MS/MS
Kinetic characterization of Ag-HKT with the new method
Applicability of the new LC-MS/MS method for the kinetic evaluation of rationally
synthesized inhibitors
Abstract
In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-
hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-
hydroxykynurenine is removed by a specific transaminase (3-hydroxykynurenine transaminase,
HKT) which converts the compound into the more stable xanthurenic acid. Interfering with 3-
hydroxykynurenine metabolism in A. gambiae is a potential approach for the development of
transmission-blocking drugs and insecticides. Hence, the aims of this work were to develop and
validate a new LC-MS/MS method for the evaluation of A. gambiae 3-hydroxykynurenine
transaminase (Ag-HKT) activity and the determination of the potency of inhibitors of the enzyme.
We set up a LC-MS/MS-based enzymatic assay for the determination of kinetic constants values of
the recombinant Ag-HKT enzyme and for the evaluation of Ag-HKT inhibition by a known protein
inhibitor used as reference and a newly synthesized compound. The chromatographic separation
was performed in a gradient mode on a Phenomenex Synergi Polar-RP (150 x 2.0, 4 µm) with
methanol and water containing both 0.2% formic acid. Mass spectrometric detection was achieved
with an ion trap equipped with an ESI source, in positive ionization scan, operating in SRM mode.
The LC-MS/MS method was validated in terms of selectivity, linearity, precision and accuracy.
Abbreviations
Ag-HKT: A. gambiae 3-hydroxykynurenine transaminase
2

KP: kynurenine pathway
3-HK: L-3-hydroxykynurenine
XA: xanthurenic acid
KYN: kynurenine
KA: kynurenic acid
INI: 4-(2-aminophenyl)-4-oxobutanoic acid
3-OH-INI: 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid
Keywords: LC-MS/MS, Ag-HKT, Anopheles gambiae, kynurenine pathway, enzyme assay
1. Introduction
Malaria is a vector-borne disease that is caused by apicomplexan parasites belonging to the genus
Plasmodium, including the Plasmodium falciparum species, which is the predominant etiological
agent of both uncomplicated and severe malaria cases in humans [1]. The P. falciparum natural
infection is efficiently spread through the human population by the bite of parasite-infected female
anopheline mosquitoes, mainly by members of the Anopheles gambiae complex, which therefore
represent the deadliest malaria vectors world-wide [2]. In spite of the recent success in reducing the
malaria global burden and the advances in the development of an effective malaria vaccine (RTS,S)
[3], there is still an urgent need for novel approaches to fight the alarming emergence of
Plasmodium strains that gained resistance to all of the currently available anti-malarial drugs, and to
contain the expansion of insecticide-resistant Anopheles mosquito populations [4,5]. In this
scenario, the molecular study of central metabolic pathways, such as the kynurenine pathway (KP)
for the oxidative degradation of tryptophan, could provide useful information for the identification
3

of potential targets of innovative insecticides and malaria transmission-blocking drugs. In all animal
species examined thus far, most of the metabolites produced along the KP (Fig. 1) behave as
bioactive compounds, playing central roles in fundamental aspects of the organism biology. In
insects, the actions of KP metabolites range from acting as substrates for eggs, body and eye
pigments synthesis [6], to triggering tissue remodelling during metamorphosis [7], to modulating
complex behaviours [8]. Within the KP, L-3-hydroxykynurenine (3-HK) is endowed with a toxic
potential, due to spontaneous oxidation that produces reactive oxygen species displaying cytotoxic
and pro-apoptotic properties [7,9].
In mammals 3-HK could be metabolized by two main routes: i) by direct transformation into
xanthurenic acid (XA), through an irreversible transamination reaction catalysed by a limited
number of pyridoxal-5'-phosphate (PLP)-dependent aminotransferases (collectively indicated as
kynurenine aminotransferases or KATs); or ii) by kynureninase-dependent hydrolysis, that yields 3-
hydroxyanthranilic acid, which is fluxed in the main branch of the KP, leading to the de novo
nicotinamide adenine dinucleotide (NAD) synthesis [10]. In insects, this latter mechanism is
precluded by the absence of a kynureninase function. Consequently, the 3-HK aminotransferase (3-
HKT)-dependent conversion of 3-HK to XA is needed to contrast the harmful accumulation of 3-
HK exceeding the ommochrome synthesis requirements [11]. XA plays multiple roles in the
physiology of mosquitoes that are vectors of human diseases [reviewed in 11]. In particular, XA is
reported as a main heme-iron chelator, therefore contributing to neutralize the pro-oxidant potential
associated to the insect hematophagous behaviour [12]. Moreover, mosquito-synthesized XA is a
trigger of the Plasmodium "exflagellation", a spectacular phenomenon that brings the parasite male
gametes to full maturation, and that takes place in the midgut of the female mosquito, upon a
parasite-infected blood meal [13]. For these reasons, the unbalance of XA homeostasis, through the
inhibition of enzymes in the KP -and of 3-HKT in particular-, can be considered a promising
approach to impact the mosquito vector physiology per se, and the capability of the Plasmodium-
infected female individuals to sustain optimal parasite replication. Two main in vivo observations
4

corroborate this hypothesis. The siRNA-mediated silencing of the 3-HKT coding gene in Anopheles
stephensi larvae -which has been obtained by feeding the mosquito larval stages with transgenic
microalgae-, results in their impaired development or death [14]. In addition, Plasmodium berghei
oocysts formation is severely inhibited in the midgut of a XA-deficient Anopheles stephensi strain,
which has been obtained by knocking-out the kynurenine 3-monooxygenase gene, leading to a
reduced number of sporozoites in the mosquito salivary glands [15]. Although very promising,
these genetics approaches are extremely challenging, and the pursuing of innovative small-molecule
insecticides to fight the mosquito vectors of human pathogens still represent a research priority.
The determination of the crystal structures of Anopheles gambiae 3-HKT (Ag-HKT) both in its
PLP-bound form, and in complex with an inhibitor that mimics the kynurenine (KYN) substrate,
but lacks the α-amino group (compound INI in Fig. 2a) [16], allowed the description of the enzyme
molecular determinants involved in substrate recognition and binding. Of outmost interest, a class
of isoxazoles [17] and 1,2,4-oxadiazoles [18], which display potent activity in vivo against the
dengue vector Aedes aegypti larvae, have been developed, also basing on molecular docking
experiments using the Ag-HKT:INI complex atomic coordinates as the template, therefore pointing
to Ag-HKT as a possible target.
The current efforts aimed at the selection and/or the structure-based rational design of Ag-HKT-
specific inhibitors should be paralleled by analytical methods able to evaluate any potential
candidate in terms of potency, affinity and mechanism of action. Several analytical techniques, such
as liquid chromatography (LC) coupled with ultraviolet (UV), electrochemical (EC), or mass
spectrometry (MS) detectors, have been applied to quantify tryptophan and its metabolites in
various biological matrix [19-21]. To date, an HPLC-ECD method was reported for the
determination of 3-HK and XA levels in A. aegypti and D. melanogaster larvae extracts, as well as
to detect 3-HK transaminase activities [22]. Furthermore, an HPLC-UV method was adopted for the
kinetic characterization of the A. gambiae 3HKT [23], and the Aedes aegypti functionally
equivalent enzyme [24], towards substrates as D,L-3-HK, D,L-KYN and L-KYN.
5

In the present work, a new selective and specific LC-MS/MS method was developed and validated
with the aim of studying new potential inhibitors of the enzyme, using the recombinant Ag-HKT
enzyme and L-KYN and KA for the LC-MS/MS quantification method. The method was validated
for selectivity, linearity, precision, and accuracy. A further advantage of the LC-MS/MS-based
approach is represented by the direct measurement of substrates and products with greater
sensitivity and selectivity. The robustness and reliability of the method for its future use in
screening procedures for the identification of potent and selective Ag-HKT inhibitors, was
evaluated in the analysis of a newly synthesized compound (3-OH-INI, Fig. 2b), which differs from
the previously described INI molecule (Fig. 2a, see above) for the presence of a hydroxyl group at
position 3 of the anthranilic ring, therefore more closely mimicking the 3-HK physiologic substrate,
and in principle better exploiting the ligand binding potential of the Ag-HKT ligand binding cavity
with respect to INI [16].
2. Experimental
2.1 Materials and methods
Methanol (HPLC grade), formic acid, L-kynurenine (MW 208.21; L-KYN), kynurenic acid (MW
189.2 ; KA), xanthurenic acid (MW 205.17 ; XA), 3-hydroxy-DL-kynurenine (MW 224.21 ; D,L-3-
HK), pyridoxal 5′-phosphate (PLP), sodium glyoxylate monohydrate, dimethyl sulfoxide (DMSO)
were supplied by Sigma-Aldrich (St. Louis, MO, USA). Water (HPLC grade) was obtained from
Milli-Q reverse osmosis system (Millipore Co., Billerica MA, USA). Petroleum ether (PE), ethyl
acetate (EtOAc), glacial acetic acid (AcOH), ethanol (EtOH), dichloromethane (DCM) and
commercially available reagents were purchased from Aldrich or Alfa Aesar, and were used without
further purification. 4-(2-aminophenyl)-4-oxobutanoic acid (INI) was kindly gifted by CAGE
Chemicals (Novara, Italy). Flash column chromatography was performed on silica gel (Merck
Kieselgel 60, 230–400 mesh ASTM). Thin layer chromatography (TLC) was carried out on 5x10
cm plates with a layer thickness of 0.25 mm (Merck Silica gel 60 F254); when necessary, they were
6

developed with KMnO4 reagent. The recombinant Ag-HKT enzyme was expressed and purified as
previously described [23].
2.2 Spectroscopic analyses and chemical characterization
Melting points were determined in open glass capillary with a Stuart scientific SMP3 apparatus and
1
are uncorrected. All compounds were checked by IR (FT-IR THERMO-NICOLET AVATAR), H
and 13C APT (JEOL ECP 300 MHz spectrometer), and mass spectrometry (Thermo Finningan
LCQ-deca XP-plus), equipped with an ESI source and an ion trap detector.
2.3 Instrumentation and chromatographic conditions
2.3.1 LC-MS/MS analyses
A Thermo Finningan LCQ Deca XP Plus ion trap mass spectrometer equipped with a quaternary
pump, a Surveyor AS autosampler and a vacuum degasser was used for LC-MS analysis (Thermo
Electron Corporation, Waltham, MA, USA). The chromatographic separation was performed using
a Phenomenex Synergi™ 4 µm Polar-RP 80 Å (150 x 2.0 mm I.D) column with a Polar
SecurityGuard column (Phenomenex, Torrance, CA, USA) maintained at 25 °C. The injection
volume was 5 μl. A mobile phase (flow rate 200 µl/min) was used consisting of water with 0.2%
formic acid (solvent A) and methanol with 0.2% formic acid (solvent B). A gradient elution was set
as follows: 25-90% solvent B in 7 min, up to 95% solvent B in 0.1 min, held at 95% solvent B for
5.0 min, then column reconditioning at 25% solvent B to 18 min. The eluate was injected into the
electrospray ion source between 2.0 and 17.90 minutes whereas the eluate was sent to waste before
and after the run time. The operating conditions of the mass spectrometer in positive mode were the
following: source voltage, 4.00 kV; source current, 80 μA; capillary temperature, 350 °C; capillary
voltage, 44.00 V; tube lens offset, 5.00 V; multipole 1 offset, -4.75 V; multipole 2 offset, -11.00 V;
sheath gas flow (N2) 45,00 AU; sweep gas flow 4,00 AU. Data were acquired both in selected
reaction monitoring (SRM) and in full-scan in electrospray positive ion mode. The SRM transitions
7

and the corresponding collision energies (CE) were as follow: L-KYN (m/z 209 → m/z 192; CE =
20%), KA (m/z 190 → m/z 172; CE = 35%), D,L-3-HK (m/z 225 → m/z 208 ; CE = 20%), XA (m/z
206 → m/z 188 ; CE = 35%). The acquisition in full scan was performed using mass scan range m/z
50 to 500. MS spectra were acquired and processed using Xcalibur® software (Thermo Fisher
Scientific)
2.3.2 Synthesis of novel inhibitor 3-OH-INI
2.3.2.1 3ˈ-Hydroxy-2ˈ-nitroacetophenone (2)
To a solution of 3-hydroxyacetophenone (1) (15.00 g, 0.1103 mol) in EtOAc (230 mL) was slowly
added nitric acid 90% v/v (8.0 mL) at 0 °C and the mixture was stirred at room temperature
overnight. The reaction was monitored by TLC with PE/EtOAc 8:2 (Rf 0.22), then the mixture was
diluted with EtOAc and washed successively with water (x1), sat. aq. NaHCO3 (x1) and brine (x1).
The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude
product was purified by two sequential column chromatography using DCM followed by PE/EtOAc
9:1 and PE/EtOAc 5:5 as eluents to give 5.0 g of product as a pale yellow solid, yield 25%. The
compound was crystallized with EtOH/PE to afford an off white solid, mp 137-138 °C, (lit. [25]
131-132 °C; lit. [26] 134-136 °C); IR (KBr) 3142-2710 (br), 1668, 1583, 1532, 1473, 1376, 1292,
-1
1
799 cm ; H-NMR (300 MHz, CDCl3) δ 10.51 (s, 1-H), 7.59 (t, Ј = 7.6 Hz, 1-H), 7.21 (dd, J =
13
8.2/1.2 Hz, 1-H), 6.83 (dd, Ј = 7.3/1.2 Hz, 1-H), 2.51 (s, 3-H) ppm; C-NMR (75 MHz, CDCl3) δ
199.6, 155.3, 140.8 (2-C), 137.1, 121.3, 118.3, 30.5 ppm. MS (ESI) m/z 180 (M-H)-.
(E)-4-(3-Hydroxy-2-nitrophenyl)-4-oxobut-2-enoic acid (3)
A mixture of 3ˈ-Hydroxy-2ˈ-nitroacetophenone (2) (2.10 g, 11.60 mmol, 1 equiv) and glyoxylic
acid monohydrate (1.07 g, 11.60 mmol, 1 equiv) in 20 mL glacial acetic acid and 2 mL conc. HCl
was heated under reflux. After 5 h glyoxylic acid monohydrate (1.07 g, 11.60 mmol, 1 equiv) was
added and the mixture was further heated under reflux overnight. The reaction progress was
monitored by TLC with PE/EtOAc 5:5 + 1% AcOH, (Rf 0.3). The solvents were removed under
8

reduced pressure and the residue was purified by column chromatography using DCM + 1% AcOH
and successively PE/EtOAc 7:3 + 1% AcOH as eluants to give 1.0 g of product as a yellow solid,
yield 36%, mp 159-160 °C (lit. [25] 158 °C; lit. [26] 158-159 °C); IR (KBr) 3296-2521 (br), 1704,
1677, 1602, 1519, 1432, 1274, 1169, 977 cm -1; 1H-NMR (300 MHz, CD3OD) δ 7.54 (t, Ј = 8.3 Hz,
1-H), 7.43 (dd, J = 15.9/0.9 Hz, 1-H), 7.26 (d, Ј = 8.3/0.9 Hz, 1-H) ppm; 13C-NMR (75 MHz,
CD3OD) δ 191.3, 167.9, 152.7, 138.9, 138.0, 135.1, 134.4, 134.0, 123.1, 121.1 ppm. MS (ESI) m/z
238 (M+H)+.
2.3.2.2 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid (3-OH-INI)
To a solution of (E)-4-(3-Hydroxy-2-nitrophenyl)-4-oxobut-2-enoic acid (3) (0.61 g, 2.58 mmol, 1
equiv) in EtOAc (62 mL) and AcOH (1 mL, 18.07 mmol, 7 equiv) Pd/C 5% (95 mg) was added as
catalyst. The mixture was stirred under hydrogen atmosphere (1 atm) at 25 °C in the dark for 18 h.
The reaction progress was monitored by TLC with PE/EtOAc 5:5 + 1% AcOH, (Rf 0.42). The
resulting suspension was filtered through a pad of Celite and the solvent was removed in vacuo. The
residue was purified by column chromatography using PE/ EtOAc 7:3 + 1% AcOH as eluant to give
0.25 g of product as a light brown solid, yield 46%, mp 134-135 °C (lit. [26] 134-135 °C); IR (KBr)
-1
1
3504, 3382, 3278, 2929, 1717, 1677, 1643, 1543, 1273, 1175, 739 cm ; H-NMR (300 MHz,
CD3OD) δ 7.35 (d, Ј = 8.2 Hz, 1-H), 6.79 (d, J = 7.6 Hz, 1-H), 6.46 (t, Ј = 8.0 Hz, 1-H), 3.24 (t, Ј =
13
6.4 Hz, 2-H), 2.64 (t, Ј = 6.4 Hz, 2-H) ppm; C-NMR (75 MHz, CD3OD) δ 201.9, 177.1, 146.2,
142.0, 122.5, 118.8, 117.8, 115.6, 35.0, 29.2 ppm. MS (ESI) m/z 210 (M+H)+.
2.4 Preparation of solutions
Stock solutions were prepared by dissolving KA in MeOH to achieve a final concentration of 500
µM. Working solutions of KA were prepared by diluting stock solutions in a solvent mixture
constituted by 50% of water with 0.2% formic acid (solvent A) and 50% of methanol with 0.2%
formic acid (solvent B) to reach the appropriate concentrations. IS stock solution was prepared in
buffer phosphate (200 mM, pH 7.0) at 2 mM and then diluted to 10 µM with (solvent A)/ (solvent
9

B) 50/50 v/v to obtain the corresponding working solution. Working solutions obtained were used
for the preparation of matrix-based calibration standards, quality control, blank and zero samples.
Calibration standards (final volume, 1 mL) were prepared by diluting appropriate volumes of KA
working solutions in the presence of 10 µL of matrix obtaining eight calibration standards for KA at
concentration ranging from 0.25 to 50 µM in the presence of IS (XA 2.5µM). A mixture of all
components involved in the enzymatic assay constitutes the matrix (i.e. DMSO; Ag-HKT 0.4
µg/µL; buffer phosphate 200 mM, pH 7.0; formic acid 0.8 M: the matrix was vortexed, centrifuged
at 12.000 rpm for 5 min: under these conditions, the enzyme is inactive). Quality controls (QC)
samples were prepared from a separate stock solution in the same way as the calibration standards.
Blank samples were prepared in the mobile phase containing the matrix without the IS. Zero
samples were obtained by adding the IS to the mobile phase containing the matrix.
2.5 LC-MS/MS method validation
The LC-MS/MS method was validated in terms of selectivity, linearity, accuracy, precision,
recovery, matrix effect and stability. Selectivity was assessed by the analysis of blank enzymatic
assay mixture (a), blank enzymatic assay mixture spiked with standard compounds (b), enzymatic
assay mixture after reaction (c). Extracted ion chromatograms for each analyte were used to
evaluate the presence of potential interfering substances at the corresponding retention time.
Linearity was determined for KA at eight concentrations levels ranging from 0.25 µM to 50 µM, in
the presence of IS used at the fixed concentration of 2.5 µM. Calibration of the method was
performed by using standards prepared as described above on three non-consecutive days. The
peak-area ratio between the analyte and internal standard was used after quantitative integration by
Xcalibur® software. The calibration curves were obtained by plotting the peak-area ratio with the
analyte concentration of the standard KA using 1/x weighted linear regression. Moreover, LLOQ
was determined on signal-to-noise ratio by establishing the minimum concentration at which the
10

analyte is characterized to have at least a signal to noise ratio of 5:1 compared to blank response.
Accuracy and precision of the analytical method were measured for QC samples at four
concentration levels containing known amounts of the analyte, by repeating five times the analysis
per concentration. The mean value should be within 15% of the actual value except at LLOQ, where
it should not deviate by more than 20%. Intra-batch and inter-batch precisions were determined by
using the relative standard deviation (RSD %) of the replicate samples between three non-
consecutive days. The precision determined at each concentration level should not exceed 15% of
the RSD except for the LLOQ, where it should not exceed 20% of the RSD. The matrix effect was
determined at low, medium, and high concentrations in triplicates by comparing the response of the
analyte spiked in extracted matrix with the response of analyte obtained from solvent solutions.
Recovery was calculated at the same concentrations by comparing the response of extracted QC
samples with the extracted matrix spiked with the analyte. The stability assay was performed by
analyzing QC samples after storage at room temperature for 12 h.
2.6 Ag-HKT activity assay
The activity of Ag-HKT was assessed by quantitatively determining the KA production, using L-
KYN as the amino group donor and glyoxylate as the amino group acceptor in the transamination
reaction, according to the published procedure with minor modifications [23]. In details, the
standard incubation mixture (50 μl final volume) was assembled in 200 mM potassium phosphate
buffer (pH 7.0) containing 40 µM PLP, 5 mM glyoxylate, 1.5 mM L-KYN. Reactions were initiated
by the addition of 4 µg of Ag-HKT. The solutions were then shaken for 10 minutes at 50 °C in a
Thermomixer equipment (Eppendorf, Milan, Italy). Blank incubations were carried out by omitting
either the PLP co-factor, or the substrate or the Ag-HKT enzyme in the reaction mixtures
assembling. Each incubation was stopped by adding 45 µl of formic acid (0.8 M) and 5 µL of IS
solution (2 mM), vortexed and centrifuged at 13,000 rpm for 10 min. The supernatants (15 µL)
were diluted in the mobile solvent (585 µl) and analysed by LC-MS/MS. In order to ensure
11

reproducibility, all assays were performed in duplicate and analysed twice. The protocol described
above was used to determine the kinetic constants (KM and Vmax) of the Ag-HKT-dependent L-
KYN to KA transamination reaction, by measuring the formation of KA in the presence of L-KYN
at a concentration ranging from 0.3 to 5.6 mM, followed by fitting the experimental data to the
Michaelis-Menten equation (eq 1) using GraphPad Prism software 5.0 (GraphPad Software Inc.,
San Diego, CA)
[ ]
× 푆
푉
푚푎푥
(1) 푣 =
[ ]
+ 푆
퐾
푀
where v is the velocity of the transamination reaction (µmol/min), Vmax is the maximal velocity
(µmol/min), and KM is the Michaelis-Menten constant (mM), [S] is the substrate concentration
(mM).
2.7 Ag-HKT inhibition studies
Stock 15 mM solutions of INI and 3-OH-INI were prepared in DMSO, and were tested at three
different concentration (150, 300 and 750 µM) in the presence of fixed concentration of L-KYN.
The DMSO final concentration in the control assays -i.e. in the absence of the inhibitor- has been
kept at 10%. All the assays were performed following the procedure reported in the paragraph “Ag-
HKT activity assay”. After analysis in LC-MS/MS and quantitative integration by the Xcalibur®
software, data have been input in a Microsoft Excel® spreadsheet. The percentage of inhibition
value was calculated by using the formula [100-(CI/C0 *100)] where CI and C0 are the mean
concentration of KA formed in the presence or in the absence of inhibitor, respectively.
2.8 Determination of inhibition constants (Ki) and inhibitors mechanism of action by LC-MS/MS
The Ki were determined for both INI and 3-OH-INI by performing the activity assays in the
presence of increasing concentrations of inhibitor (ranging from 0.06 to 1.5 mM) in the presence of
L-KYN at concentration between 0.3 to 5.6 mM. Reaction rates were calculated with respect to the
12

calibration curve. The values obtained (expressed as µmol min-1) were used to calculated the Ki
value, by plotting the inhibitor concentration (mM) to the substrate concentrations (mM), by using
GraphPad Prism and Sigma Plot software. Inhibition constants for compounds INI and 3-OH-INI
were calculated by fitting the data to competitive inhibition equation (eq. 2)
퐾
× [퐼]
푀
(2) 퐾푖 =
퐾
− 퐾
푀
푀 푎푝푝
where Ki is the dissociation constant for the inhibitor-enzyme complex, KM is the Michaelis-Menten
constant, [I] is the inhibitor concentration in mM, and KM app is the apparent KM value.
3. Results and discussion
3.1 LC-MS/MS method
At present, a general limit to analytical procedures based on 3-HK as the amino-group donor
substrate in transamination reactions is that pure L-3-HK (the natural substrate) is not commercially
available. Therefore, the published Ag-HKT enzyme kinetics characterization has been performed
by comparing different substrates, highlighting that Ag-HKT displays comparable affinity and
catalytic efficiency both for the the racemic mixture of D,L-3-HK and for the L-KYN substrate
[23]. Moreover, D,L-3HK shows poor solubility in the reaction buffers, as previuosly described by
Li et al. [22], and high instability [27], making its use not suitable for routinely screening of Ag-
HKT inhibitors. Therefore, since the Ag-HKT enzyme can use also L-KYN as the amino-donor
substrate with comparable efficiency, we developed a method for measuring the levels of the
transamination product KA.
In the development of the LC-MS/MS method for the analysis of L-KYN and KA, the structural
relationship between KA and XA was exploited in the optimization of the LC-MS/MS procedure, as
XA was used as the internal standard, although deuterated standard are usually preferred.
13

XA was chosen as internal standard because the 8-OH in the XA structure can be exploited on the
one hand for the chromatographic separation due to the similar interaction to KA with the stationary
phase, and on the other hand for the similar ionization and fragmentation pattern, as shown in
Supplemental 1. The chromatographic separation of analytes was achieved on a Synergy Polar
column allowing the use of a simple mobile phase. The gradient elution was optimized to achieve
the elution of analytes within 8 minutes of the gradient. Retention times of L-KYN, KA and IS
were 3.94, 7.11 and 6.82 minutes respectively as reported in Fig. 3.
KYN, KA and IS were ionized in ESI positive mode to generate protonated molecular ions [M+H]+
at m/z 209, 190, 206 respectively. When MS/MS experiments were performed on these ions the
product ions obtained (m/z 192, 172 and 188 respectively) were directly used as specific transitions
to increase specificity and detection responses; finally, the detection was operated in selected
reaction monitoring mode (SRM). It is worth of mention that the analytical method was suitable
also for the analysis of D,L-3HK compound that could be monitored following SRM transition at
m/z 225→208 and retention time of 3.16 min, ensuring the chromatographic separation of all
analytes, as shown in Supplemental 2. This evidence suggest that the method could be applied for
the analysis of enzyme assay performed on 3-HK, depending on the availability of pure isomers.
3.2 LC-MS/MS validation
The selectivity of the LC-MS/MS method was assessed by analysing the blank samples, prepared as
reported in the experimental part. Extracted ion chromatograms for each analyte ruled out the
presence of interferences at the corresponding retention time. The representative SRM
chromatograms are shown in Supplemental 3. The calibration curves obtained from analyte/IS peak
area ratios versus the nominal concentration were linear using weighted (1/x) regression over the
concentration range, with correlation coefficient R2 ≥ 0.99. The calibration equations were as
follows: y = 0.4042x - 0.02271 (R2 = 0.9982); y = 0.3261x + 0.005754 (R2 = 0.9974); y = 0.3619x +
0.001683 (R2 = 0.9988). The lower limit of quantification (LLOQ) was determined by injecting
14

lower concentration solutions of KA. The LLOQ value was 0.25 μM, and the representative
chromatogram is reported in the Supplemental 4. Accuracy and precision were determined by
measuring four concentration levels (LLOQ, low, medium and high concentration), covering the
entire range of the calibration curve (data are provided in Table 1). The accuracy was between
88.21 and 111.21%. The mean intra-assay precision was below 14% and below 11% for the inter-
assay precision for LLOQ, the values of intra-assay and inter-assay precision for the remaining
levels were below 10.5% and 9.5% respectively. The matrix effect values were found to vary
between 105% and 111% suggesting a negligible effect to ion suppression or enhancement caused
by co-eluting compounds. Recovery values were found at 110%, 104% and 101% at low, medium
and high concentration respectively. Stability of the standard solutions of KA and IS were studied at
the benchtop at room temperature over 12 h. The concentrations of freshly prepared solutions and
those aged for 12 h were determined and the difference between them was found to be not
significant (RSD < 2 %). The results obtained proved that the samples solutions were stable for the
specified duration.
3.3 3-OH-INI Synthesis
4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid (3-OH-INI) was prepared in a three-step
synthesis starting from the commercially available 3-hydroxyacetophenone (1), as shown in Fig. 2b.
Nitration of 3-hydroxyacetophenone (1) was directly carried out with HNO3 in ethyl acetate at 25
°C to give 3ˈ-hydroxy-2ˈ-nitroacetophenone (2) in 25% yield. Methyl ketone 2 was condensed with
2 equiv of glyoxylic acid monohydrate in glacial acetic acid and conc. HCl under reflux for 12 h to
afford (E)-4-(3-hydroxy-2-nitrophenyl)-4-oxobut-2-enoic acid (3) in 36% yield. Our synthetic
procedure is easier to set up than previously reported Mizdrak’s method [26] which required the use
of 11.8 equiv of glyoxylic acid monohydrate and heating at 120 °C without solvent, under vacuum
for 24 h. Finally, hydrogenation of 3 in ethyl acetate with glacial acetic acid, catalysed by Pd/C,
afforded 3-OH-INI in 46% yield. Acetic acid was used to assist protonation of the amino group in
order to prevent the formation of Michael adduct.
15

3.4 Ag-HKT activity assay
Previous works demonstrated that the recombinant form of Ag-HKT displays comparable affinity
and catalytic efficiency towards both D,L-3-HK and L-KYN, in the PLP-dependent transamination
to XA and KA, respectively, by using the α-ketoacid glyoxylate as the amino group acceptor [23].
The new LC-MS-based analytical method was therefore used to quantify the KA produced in
standard Ag-HKT activity assays for the determination of the enzyme kinetics. The calculated KM
value (1.4 ± 0.18 mM) for the Ag-HKT catalyzed transamination of L-KYN to KA is in agreement
with the one reported in literature (1.0 ± 0.4 mM) [23].
By slightly modifying the published procedure, DMSO was added to the incubation mixtures in
order to ensure the solubility of the tested inhibitors. The influence of DMSO on the enzyme
activity was evaluated in the 1% to 20% v/v concentration range (data not shown). The presence of
DMSO at 10% final concentration did not affect the product formation, allowing us to test inhibitors
at a wide range of concentrations while keeping the compounds soluble. Moreover, the linearity of
KA production was investigated and the optimal incubation time was found at 10 minutes. This
evidence demonstrated the good reliability of the enzyme assay coupled to the LC-MS/MS
analytical method, and its exploitability for the in vitro analysis of Ag-HKT inhibition.
The method was applied for the analysis of 3-OH-INI, a newly synthesized compound (Fig. 2)
which is different from INI for the presence of a hydroxyl group at position 3 of the anthranilic
group; therefore, by more closely mimicking the 3-HK physiologic substrate compared to INI, 3-
OH-INI could represent a more efficient inhibitor of the Ag-HKT enzyme. Interestingly, the
manual modelling of 3-OH-INI inside the ligand binding cavity of the crystal structure of the Ag-
HKT:INI complex [16], revealed that the 3-OH group of the modelled molecule occupies an ideal
position to established a strong hydrogen bond with the side chain oxygen atom of a serine residue
(Ser43) (Supplemental figure 5), which is also involved in the coordination of the amino group of
16

the phenyl ring of the ligand. Such an analysis suggested that 3-OH-INI could behave as a more
potent Ag-HKT inhibitor when compared to INI.
Preliminary Ag-HKT inhibition assays were performed by using 3-OH-INI, or the known reference
molecule INI, at three fixed concentrations. The obtained data revealed that the inhibitory potency
of 3-OH-INI is higher than the one of INI at all the concentrations studied and it was considered
worthy of further investigation. Based on the results of the activity assays/LC-MS/MS analyses
described above, we determined the Ki value of INI and 3-OH-INI towards the L-KYN to KA
transamination reaction. The Ki in the high micromolar range revealed for INI (740 µM) is in
agreement with what has been previously reported (300 µM) [16], confirming the accuracy and the
reliability of the LC-MS/MS developed procedure. Noticeably, the lowering of the Ki value
revealed for 3-OH-INI (325 µM), highlighted that the addition of the hydroxyl-group at position 3
of the INI reference molecule improves its potency, in line with what has been hypothesized on the
basis of Ag-HKT structural analyses [16]. As expected, the Lineaweaver-Burk reciprocal plot
analysis of the inhibition assays results (Fig. 4) indicates that 3-OH-INI acts as a competitive
inhibitor of the Ag-HKT enzyme.
4. Conclusion
In this work, a new LC-MS/MS method was developed and its applicability was confirmed by the
determination of the kinetic constants of the Ag-HKT-catalysed transamination of L-KYN to KA,
and the re-evaluation of INI, a previously describe weak inhibitor of the enzyme. Moreover, a new
compound 3-OH-INI was synthesized and analysed, showing an improved inhibition potency. The
greater specificity and sensibility of the LC-MS/MS method over conventional HPLC-UV methods
suggests that it can be applied for the screening of further and potential Ag-HKT inhibitors, also
including new compounds that should be rationally designed based on the 3-OH-INI scaffold.
Acknowledgments and Foundings
17

This research is original and it has been supported by Fondazione Cariplo (grant n° 2015-2246) and
by University of Piemonte Orientale (grants: RSdelgrossoeRL15_ANOPHELES_GAMBIAE_16-
18 and RIVrossifRL16_TRIPTOFANO_17-19).
Conflict of interest
The authors declare that they have no conflict of interest.
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21

Figures and tables
Figure 1 The kynurenine pathway in the Anopheles gambiae mosquito. Enzymes involved are
abbreviated as follows: TDO, tryptophan 2,3-dioxygenase; KMO, Kynurenine monooxygenase;
KAT, Kynurenine aminotransferase; 3-HKT, 3-hydroxykynurenine transaminase
Figure 2 a. Chemical structures of INI and 3-OH-INI inhibitors; b. Scheme of 3-OH-INI synthesis
22

Figure 3 Representative extracted ion chromatograms corresponding to the PLP-dependent
transamination of L-KYN to KA catalysed by the recombinant form of Ag-HKT in the presence of
IS
23

Figure 4 Kinetic study of 3-OH-INI mechanism of Ag-HKT inhibition. Overlaid Lineweaver-Burk
reciprocal plots of 3-OH-INI compound
24

TABLE
Table 1 Accuracy and precision data of KA using internal standard
Intra-serie
Series 2
Accuracy
109,38
Analyte
KA
Inter-series
Series 1
Accuracy
111,21
88,21
Series 3
Accuracy
109,22
87,76
[µM]
0,25
1,0
Mean
0,28
RSD
13,34
6,73
4,45
8,88
Mean
0,27
RSD
7,87
5,31
7,21
3,23
Mean
0,27
RSD
13,96
4,67
2,22
2,39
Mean
0,27
Accuracy
109,94
90,03
RSD
10,94
9,24
5,10
6,02
0,88
0,98
97,97
0,88
0,90
10
9,05
90,50
9,04
90,36
9,48
94,84
9,19
91,90
50
47,35
94,70
51,32
102,65
49,61
99,22
49,43
98,86
Mean = mean of calculated concentration (µM); Accuracy = accuracy %; RSD = relative standard deviation (%)
25