Crystal structure-based selective targeting of the pyridoxal 5?-phosphate dependent enzyme kynurenine aminotransferase II for cognitive enhancement

Article abstract of DOI:10.1021/jm100464k

Fluctuations in the brain levels of the neuromodulator kynurenic acid may control cognitive processes and play a causative role in several catastrophic brain diseases. Elimination of the pyridoxal 5?-phosphate dependent enzyme kynurenine aminotransferase II reduces cerebral kynurenic acid synthesis and has procognitive effects. The present description of the crystal structure of human kynurenine aminotransferase II in complex with its potent and specific primary amine-bearing fluoroquinolone inhibitor (S)-(?)-9-(4-aminopiperazin-1-yl)-8- fluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3a-azaphenalene-5-carboxylic acid (BFF-122) should facilitate the structure-based development of cognition-enhancing drugs. From a medicinal chemistry perspective our results demonstrate that the issue of inhibitor specificity for highly conserved PLP-dependent enzymes could be successfully addressed.

Full text of DOI:10.1021/jm100464k

5684 J. Med. Chem. 2010, 53, 5684–5689
DOI: 10.1021/jm100464k
Crystal Structure-Based Selective Targeting of the Pyridoxal 50-Phosphate Dependent Enzyme
Kynurenine Aminotransferase II for Cognitive Enhancement†
Franca Rossi,‡ Casazza Valentina,‡ Silvia Garavaglia,‡ Korrapati V. Sathyasaikumar,§ Robert Schwarcz,§ Shin-ichi Kojima,
Keisuke Okuwaki, Shin-ichiro Ono, Yasushi Kajii, and Menico Rizzi*,‡
‡DiSCAFF, University of Piemonte Orientale “Amedeo Avogadro”, 28100 Novara, Italy, §Maryland Psychiatric Research Center,
University of Maryland School of Medicine, Baltimore, Maryland 21228, and Research Division, Mitsubishi Tanabe Pharma Corporation,
Aoba-ku, Yokohama 227-0033, Japan
Received April 15, 2010
Fluctuations in the brain levels of the neuromodulator kynurenic acid may control cognitive processes and
play a causative role in several catastrophic brain diseases. Elimination of the pyridoxal 50-phosphate depen-
dent enzyme kynurenine aminotransferase II reduces cerebral kynurenic acid synthesis and has procognitive
effects. The present description of the crystal structure of human kynurenine aminotransferase II in complex
with its potent and specific primary amine-bearing fluoroquinolone inhibitor (S)-(-)-9-(4-aminopiperazin-
1-yl)-8-fluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3a-azaphenalene-5-carboxylic acid (BFF-122) should
facilitate the structure-based development of cognition-enhancing drugs. From a medicinal chemistry
perspective our results demonstrate that the issue of inhibitor specificity for highly conserved PLP-dependent
enzymes could be successfully addressed.
Introduction
that, once reprotonated, yields the corresponding ketimine.
The hydration of the iminic double bond by a water molecule
leads to the production of a second complex formed by the
ketoacid and the enzyme in its pyridoxamine 50-phosphate-
Kynurenic acid (KYNAa) is a neuroinhibitory metabolite
of the kynurenine pathway (KP), the principal mode of
tryptophan degradation in mammals.1 KYNA has several
unusual neurobiological characteristics, most remarkably in-
cluding antagonism of two abundant ionotropic receptors
involved in learning and memory, i.e., the R7 nicotinic acetyl-
choline and the NMDA receptor.2,3 Since these effects are
seenatKYNA concentrations found in the mammalian brain,
elevations in brain KYNA might cause cognitive impair-
ments. This mechanism not only may operate under normal
physiological conditions but also could play a role in the
cognitive deficits seen in catastrophic neurological and psy-
chiatric diseases such as Alzheimer’s disease and schizophre-
nia, both of which present with abnormally high brain KYNA
levels.4,5 In the brain as elsewhere, KYNA is formed enzyma-
tically by the irreversible transamination of the pivotal KP
metabolite L-kynurenine (L-KYN) (Scheme 1). This reaction
is catalyzed by pyridoxal 50-phosphate (PLP) dependent
aminotransferases by the two-step mechanism described for
this class of enzymes.6 Thus, in the first semireaction, an
external aldimine is formed between the C40 position of the
cofactor and the R-amino group of the substrate, L-KYN,
which substitutes in Schiff base linkage the ε-amino group of
the conserved catalytic lysine. The abstraction of a proton
from the substrate CR produces the quinonoid intermediate
bound form (E PMP). The details of the irreversible intra-
3
molecular condensation of the resulting 4-(2-aminophenyl)-
2,4-dioxobutanoic acid intermediate, yielding the final pro-
duct KYNA, have not been elucidated so far. Finally, the
internal aldimine form of the enzyme (E PLP) is regenerated
3
by the use of a R-ketoacid as the amino group acceptor.
At least four aminotransferases can utilize L-KYN as the
amino donor of the transamination reaction in the mamma-
lian brain.7,8 However, only one of them, kynurenine amino-
transferase II (KAT II, E.C. 2.6.1.7), recognizes L-KYN
unencumbered by abundant, competing amino acid sub-
strates. This explains why KAT II accounts for the majority
of cerebral KYNA synthesis in rat and human brain tissue.7
Notably, as revealed using mice with a genomic elimination of
KAT II, permanentreduction incerebral KATIIactivityleads
to decreased KYNA formation in vivo and significant cogni-
tive enhancement.9 Recent studies were designed to probe the
functional consequences of an acute disruption of cerebral
KYNA synthesis using specific KAT II inhibitors. Develop-
ment of isozyme-specific inhibitors is notoriously difficult,
especially in the case of PLP-dependent aminotransferases,
since the members ofthis familyshare significant conservation
of the active site.10 Early attempts were based on the structure
of L-KYN,11 eventually leading to the synthesis of [(S)-
4-(ethylsulfonyl)benzoylalanine, S-ESBA), which caused a
rapid decrease in KYNA formation in the rat forebrain upon
intracerebral application.12 Interestingly, this treatment also
promptly raised the extracellular levels of acetylcholine, glu-
tamate, and dopamine, three classic neurotransmitters with
well-established procognitive properties.13-15 Together with
†The atomic coordinates and structure factors have been deposited in
the Protein Data Bank (http://www.rcsb.org) with accession code 2xh1.
*To whom correspondence should be addressed. Phone: þ39-0321-
375712. Fax: þ39-0321-375821. E-mail: rizzi@pharm.unipmn.it.
a Abbreviations: hKAT II, human kynurenine aminotransferase II;
KAT, kynurenine aminotransferase; KYNA, kynurenic acid; L-KYN, L-
kynurenine; PLP, pyridoxal 50-phosphate; PMP, pyridoxamine 50-phos-
phate.
r
pubs.acs.org/jmc
Published on Web 07/19/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5685
Scheme 1. Mechanism of the First Half of the Reaction of the Transamination of L-KYN to KYNA Catalyzed by KATsa
a (a) PLP cofactor in the ligand-free form of the enzyme. The Schiff base linkage between the PLP C40 atom and the catalytic lysine ε-amino group is framed.
(b) Transaldimination with the amino acid substrate L-KYN, producing the external aldimine. (c) Abstraction of a proton (labeled by a blue star) from the
substrate CR, producing the quinonoid intermediate. (d) Reprotonation step leading to ketimine formation. (e) Hydrolysis of the ketimine intermediate
generating the L-KYN corresponding R-ketoacid [4-(2-aminophenyl)-2,4-dioxobutanoic acid]. The details of the intramolecular condensation of this last
molecule to give KYNA are currently unknown. (f) The release of KYNA leaves the enzyme in its PMP form. In the second half of the transamination reaction
(not shown), an amino acceptor oxoacid is used to regenerate the PLP-bound form of the enzyme (a) by catalyzing the same set of reverse reactions.
Figure 1. Inhibition of human KAT II by 1. (a) Chemical structure of 1. The asymmetric carbon atom is labeled by an asterisk. (b) Both 1
(circles) and its stereoisomer (1*, triangles) inhibit the activity of recombinant human KAT II in a dose-dependent manner (control activity =
20.4 (nmol/h)/mg protein). Data are the mean of duplicate assays. (c) Inhibition of KAT II by 1 determined in human brain tissue homogenate.
Data are the mean ( SEM (control activity = 1.9 ( 0.4 (pmol/h)/mg tissue; N = 4).
the demonstration of increased memory function in S-ESBA-
treated rats [Potter, M. C. Kynurenic Acid, Learning and
Memory: The Glutamate Connection. Ph.D. Dissertation,
University of Maryland, Baltimore, MD, 2008], these results
validated both the role of KYNA as an endogenous modu-
lator of cognitive processes and the enthusiasm for KAT II as
a novel target for procognitive interventions.
function in the rat brain in vivo, the compound turned out to
be a very poor inhibitor of the human orthologue of KAT II
(hKAT II), assessed both in brain tissue homogenate and
against recombinant hKAT II.16 By screening an in-house
random chemical library on recombinant hKAT II, we recently
found that the primary-amine-bearing fluoroquinolone 1(BFF-
122)17 (Figure 1a), a compound with a structure shared by
classic antimicrobial agents,18 is a potent inhibitor of partially
purified rat brain KAT II (IC50 ≈ 1 μM) and can also reduce
KYNA synthesis in the rat brain in vivo.19 In contrast to S-
ESBA and suggestive of a distinct mechanism of action, how-
ever, 1 displayed equally high activity against recombinant
hKAT II (IC50 = 0.91 and 1.5 μM for the two enantiomers)
Results and Discussion
Characterization of (S)-(-)-9-(4-Aminopiperazin-1-yl)-8-
fluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3a-azaphenalene-
5-carboxylic Acid (BFF-122) Specific Inhibition of Human KAT
II. Although S-ESBA can be effectively used to probe KYNA

5686 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 1. Data Collection, Processing, and Refinement Statisticsa
Rossi et al.
hKATII 1
3
Data Collection
space group
cell dimensions
P21212
˚
a, b, c (A)
98.32, 152.86, 60.80
90.00, 90.00, 90.00
82.76-2.10 (2.21-2.10)
0.115 (0.398)
R, β, γ (deg)
˚
resolution (A)
Rmerge
I/σ(I)
completeness (%)
redundancy
5.1 (1.8)
100.0 (100.0)
6.9 (7.0)
Refinement
˚
resolution (A)
2.1
53172
19.0/24.5
no. reflections
b
R
work/Rfree
Figure 2. Molecular architecture of hKAT II in complex with 1.
The two chains forming the functional dimer are colored white and
purple. The 1 molecule occupying the active site in both monomers
is drawn as sticks and colored yellow.
no. atoms
protein
ligand/ion
6466
83
water
B factors (A )
424
2
˚
protein
ligand/ion
water
28.0
37.7
30.7
rms deviation
˚
bond length (A)
bond angle (deg)
0.020
1.812
b
a Values in parentheses are for highest-resolution shell. Rwork
|Fo - Fc|/ Fo. Rfree
omitted from refinement.
=
P
P
P
P
=
|Fo - Fc|/ Fo, based on 1096 reflections
(Figure 1b). Attesting to its pharmacological specificity, 1,
tested in cortical tissue preparations obtained from three to four
human donors, was far more potent as an inhibitor of KAT II
(IC50 ≈ 1 μM, Figure 1c) than of KAT I (E.C. 2.6.1.64) (IC50
>
30 μM; control = 0.4 (pmol/h)/mg tissue). In agreement, the
IC50 of 1 against recombinant hKAT I, too, was >30 μM
(control = 7.6 (nmol/h)/mg protein).
To further evaluate the specificity of 1, we measured the
activity of two other human KP enzymes that use L-KYN as
their substrate and observed no significant inhibition. Thus, 100
μM 1 inhibited kynurenine 3-monooxygenase activity by 8.3 (
7.3% (control = 2.1 (pmol/h)/mg tissue) and kynureninase
activity by 5.1 ( 2.9% (control = 117.9 (fmol/h)/mg tissue),
respectively (averages ( SEM, P > 0.05 each, Student’s t test).
It is noted that kynureninase is indeed a PLP-dependent enzyme
that does not belong to the aminotransferase family; therefore, 1
emerges as a high specific hKAT II inhibitor with no appreciable
activity against other enzymes that use the PLP cofactor as an
essential catalytic component.
Crystal Structure of hKAT II in Complex with 1. In order to
elucidate the molecular basis of the mechanism of inhibition of
hKAT II by 1, we determined the crystal structure of the enzyme/
˚
inhibitor complex (hKAT II 1) at2.1Aresolution (Table 1). The
3
Figure 3. Active site of hKAT II in complex with 1. (a) Chain A
(white) and chain B (purple) of the functional hKAT II dimer are
represented as a cartoon. The PLP and compound 1 carbon atoms
are depicted as sticks and colored white and yellow, respectively.
The portion of the 2Fo - Fc electron density map covering the
resulting overall structure consists of a functional dimer (Figure 2)
displaying the peculiar swapping of the N-terminal regions
(residues 13-46) previously described in the 3D structures of
both the ligand-free and L-KYN-bound forms of the enzyme.20,21
PLP 1 molecule is shown in gray and contoured at 1σ. Residues
A close-up view of the active site of the hKAT II 1
3
3
providing major interactions to the PLP 1 complex are drawn as
sticks. Protein segment 20-32 has been omitted from the final
complex (Figure 3a) showed that in each monomer the
inhibitor forms an hydrazone adduct with the PLP molecule
by establishing a covalent bond that involves the primary
amine group of 1 and the C40 position of the cofactor
(Figure 3a and Figure 3b). Since 1 lacks a proton on the
N2 atom that occupies a position structurally equivalent to
3
model. (b) Schematic representation of the PLP 1 complex shown
in (a). (c) Side view of the hKAT II 1 complex showing the inhibitor
3
3
carboxylic group exposed to the enzyme surface and highlighting
the significant hydrophobic environment (white color) featuring the
ligand binding pocket.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5687
the CR atom of the physiological amino acid substrate, the
mandatory proton abstraction step in the transamination
process6 cannot take place, and the enzyme is potently and
irreversibly inactivated. In order to verify that 1 behaves as
an irreversible hKAT II inhibitor also in solution, we pre-
incubated the enzyme with 1 in a 1:1 molar ratio; after
extensive dialysis (Supporting Experimental Section in Sup-
porting Information), hKAT II turned out to be completely
inactive. Moreover, we titrated hKAT II in solution with 1
and recorded the corresponding UV-visible spectra (Figure 1
in Supporting Information). Spectroscopic analysis demon-
strated that hKAT II reactivity with 1 does not result in the
formation of the PMP form of the enzyme, as signaled by the
absence of the diagnostic peak around 330 nm, while a 1
concentration dependent increase in the 364 peak was re-
corded. Finally, by comparing the UV-visible spectra of 1,
free PLP, and a 1:1 mixture of the two molecules, we
observed no spectral variation (data not shown), confirming
that the covalent adduct does not form in the absence of
hKAT II.
The entire PLP 1 group is firmly anchored at the bottom
3
of the catalytic cavity through a specific and strictly con-
served network of contacts that engages the PLP moiety.20 In
turn, the 1 moiety is kept in place by the π-stacking of its
heterotricyclic ring to Tyr74 and, on the opposite side, by an
electrostatic contact involving the inhibitor fluorine and the
carbonyl carbon of Gly39 (Figure 3a). The conformation
adopted by the 1 molecule bound to hKAT II revealed that
the 3-methyl group does not interact with the protein envir-
onment, thus explaining why both 1 stereoisomers act as
powerful inhibitors of the enzyme (Figure 1b).
Figure 4. Structural basis of hKAT II inhibition by 1. The upper
images show the active site of hKAT II (a) in its ligand-free form (green,
PDB code 2QLR20), (b) in complex with L-KYN (yellow, PDB code
2R2N21), and (c) in complex with 1 (purple). In (b) and (c), the ligand
molecules are colored dark gray. (d) Superposition of the structures of
hKAT II shown in (a), (b), and (c). For clarity, only the PLP 1molecule
3
and the Arg20 are drawn as sticks. The arrows indicate the direction of
the movements of the N-terminal loop and of the Arg20 side chain
during catalysis (same color scheme as in (a), (b), and (c)).
The bulky inhibitor molecule occupies the significantly hy-
drophobic ligand-binding pocket with the carboxylic group
exposed on the enzyme surface (Figure 3c). The observed 1
binding mode interferes with the conformational transition
observed in hKAT II along the catalytic cycle that was proposed
to control substrate access to the enzyme’s active site.20-23 L-
KYN binding triggers a structural rearrangement of the hKAT
II N-terminal region (residues 16-32) characterized by a pro-
nounced movement of the loop encompassing residues 16-21
(Figure 4a and Figure 4b). In particular this loop becomes
displaced from the ligand binding pocket, changing the enzyme
from a closed to an open conformation and allowing Arg20 to
rigid to host a bulky molecule such as 1. This is in contrast to
the conformational plasticity of the catalytically important
N-terminal motif in hKAT II, which appears to be the key
determinant of the observed selectivity of 1.
From a drug discovery perspective, our data indicate that
crucial conformational changes occurring during catalysis
can be successfully exploited for developing highly specific
inhibitors targeting kynurenine aminotransferases. In this
respect, the mechanism of enzyme inhibition by 1 resembles
the one reported for the specific human Abl tyrosine kinase
inhibitor STI571 (Gleevec/imatinib).26
engage the bound ligand.22 In the structure of the hKAT II 1
3
complex, no electron density for the majority of such a N-term-
inal region (residues 20-32; omitted from the final model) is
visible, revealing that this structural motif is affected by a
significant conformational disorder (Figure 4c). This demon-
strates a high conformational plasticity of this N-terminal
structural element in hKAT II. In other words, this element
not only exists in two alternative conformations, i.e., open and
closed, occurring during catalysis, but can be further displaced
to accommodate bulkier molecules, such as 1, in the enzyme’s
active site (Figure 4d).
Conclusions
In conclusion, our results demonstrate that the synthesis of
specific inhibitors of PLP-dependent enzymes, which has long
posed a formidable challenge in drug development, can be
successfully achieved. In view of the recently uncovered func-
tional link between brain KYNA and cognitive processes,
KAT II appears to be an especially attractive target for
exploiting this concept. Thus, it should be possible to use
the conformational plasticity of the N-terminal region of the
enzyme for structure-based lead optimization to produce
novel cognition-enhancing drugs.
Our structural model allows us to explain both the potency
and the selectivity of 1 as an inhibitor of hKAT II. While the
former can be largely attributed to the formation of a
covalent adduct with the PLP cofactor, which permanently
inactivates the enzyme, the selectivity of the compound is
mainly the result of conformational principles. Indeed, the
protein architectures of both hKAT I24and mitochondrial
aspartate aminotransferase25 (the third kynurenine amino-
transferase present in the brain) are determined by largely
preformed ligand binding sites that appear too narrow and
Experimental Section
Chemicals. Chemicals used in the experiments were obtained
from Sigma-Aldrich (St. Louis, MO). The synthesis, chemical
characterization, and purity of 1 have been reported elsewhere.17
Briefly, the synthetic strategy adopted for the chemical synthesis of
1 followed the one used for quinolones that are readily prepared

5688 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Rossi et al.
starting from a fluoroquinolone core and a nucleophilic amine.27
The hydrazine can be formed using a two-step procedure, i.e.,
nitrosylation of the amine with a nitrosylating agent (for example,
sodium nitrite in aqueous acidic solution), followed by reduction
with zinc (Figure 2 of Supporting Information).
200, Waltham, MA) using an excitation wavelength of 340 nm and
an emission wavelength of 410 nm. The retention time of anthranilic
acid was ∼7 min.
Kynurenine Aminotransferases I and II (KAT I and KAT II).
For the determination of KAT I activity, the original tissue
homogenate was diluted (1:1, v/v) in 5 mM Tris-acetate buffer
(pH 8.0) containing 10 mM 2-mercaptoethanol and 50 μM PLP
and then dialyzed overnight at 4 °C against 4 L of the same
buffer to remove competing amino acid substrates. The reaction
mixture contained 150 mM Tris-acetate buffer (pH 7.4), 100 μM
L-KYN, 1 mM pyruvate, 80 μM PLP, 10 mM L-2- aminoadipic
acid (to block KAT II), 10 mM aspartic acid (to block mito-
chondrial aspartate aminotransferase), and 50 μL of the dia-
lyzed tissue preparation in a total volume of 200 μL. KAT II
activity was determined in the original tissue homogenate, which
was further diluted (1:1, v/v) in 5 mM Tris-acetate buffer (pH
8.0) containing 10 mM 2-mercaptoethanol and 50 μM PLP. The
reaction mixture contained 150 Tris-acetate buffer (pH 7.4),
100 μM L-KYN, 1 mM pyruvate, 80 μM PLP, and 50 μL of the
tissue preparation in a total volume of 200 μL. The effects of
1 were compared to those of the specific KAT II inhibitor
L-2-aminoadipate (10 mM).
Inhibition Studies Using Recombinant Kynurenine Amino-
transferases. Reverse transcription PCR products of human
KAT I (A/C X82224.1) and KAT II (A/C AF481738.1) from
HEK293 total RNA were cloned into pCR-Blunt II-TOPO
(Invitrogen, Carlsbad, CA) and then subcloned into commer-
cially available vectors to produce each recombinant protein
fused with maltose-binding protein (MBP) in E. coli BL21. The
resulting MBP-hKAT I and MBP-hKAT II were purified by
conventional methods. Conversion of L-KYN to KYNA was
assessed by incubating either 0.2 μg of MBP-hKAT I or 0.1 μg of
MBP-hKAT II at 37 °C for 1 h in a total volume of 50 μL of a
solution containing 10 μM PLP, 1 mM 2-oxoglutarate, 3 μM
L-KYN, and 150 mM Tris-acetate, pH 8.0, in the absence or
presence of 1 (30 μM for KAT I and 0.25-4 μM for KAT II).
The reaction was terminated by adding trichloroacetic acid at a
final concentration of 5%, and de novo produced KYNA was
analyzed as described below.
Enzyme Analyses Using Human Brain Tissue Homogenate.
Human brain tissues from four individuals [age ( SEM, 40 ( 8
years; average post-mortem interval, 22 ( 5 h] who had died
without evidence of neurological or psychiatric disease were
obtained from the Maryland Brain Collection (Baltimore, MD).
On the day of the assays, samples of the prefrontal cortex
(Brodmann area 9, stored at -80 °C) were thawed, homogenized
in 5 volumes of ultrapure water, and processed as detailed
below. For each tissue homogenate, all assays were performed
on the same day. The inhibitory potency of 1 was tested by
adding the compound in a small volume immediately before the
substrate at the beginning of the respective assay. The final
concentration of 1 in the incubation media was up to 100 μM.
Kynurenine 3-Monooxygenase. After dilution of the original
homogenate 1:5 (v/v) in 100 mM Tris-HCl buffer (pH 8.1) contain-
ing 10 mM KCl and 1 mM EDTA, an amount of 80 μL of the tissue
preparation was incubated for 40 min at 37 °C in a solution contain-
ing 1 mM NADPH, 3 mM glucose 6-phosphate, 1 U/mL glucose
6-phosphate dehydrogenase, 100 μM L-KYN, 10 mM KCl, and
1 mM EDTA in a total volume of 200 μL. The reaction was stopped
by the addition of 50 μL of 6% perchloric acid. Blanks were obtained
by including the specific enzyme inhibitor Ro 61-8048 (100 μM,
For the measurement of KAT I or KAT II, the reaction
mixture was incubated for 2 h at 37 °C, and the reaction was
terminated by the addition of 20 μL of 50% (w/v) trichloroacetic
acid and 1 mL of 0.1 M HCl. The reaction mixture was then
centrifuged to remove proteins (16000g, 10 min), and an amount
of 20 μL of the supernatant was applied to a 3 μm HPLC column
(HR-80, 80 mm ꢀ 4.6 mm, ESA), using a mobile phase consist-
ing of 250 mM zinc acetate, 50 mM sodium acetate, 3%
acetonitrile and a flow rate of 1.0 mL/min. In the eluate, KYNA
was detected fluorimetrically (Perkin-Elmer series 200) using an
excitation wavelength of 344 nm and emission wavelength of
398 nm. The retention time of KYNA was ∼7 min.
Crystallization, Data Collection, and Structure Determination.
Native crystals of recombinant human KAT II (hKAT II),
expressed and purified as described,20 were obtained by mixing
1 μL of a protein solution at 10 mg/mL with an equal volume of a
reservoir solution containing 20% PEG 3350, 0.2 M potassium
iodide, and 0.1 M potassium phosphate, pH 8.5, and equilibrat-
ing the resulting drop against 500 μL of the reservoir solution at
20 °C. Needle-shaped yellow crystals grew to a maximum
dimension of 0.7 mm ꢀ 0.2 mm ꢀ 0.05 mm in about 3 weeks.
The crystals of the hKAT II 1 complex were obtained by
3
soaking crystals of the PLP form in their crystallization solution
with the addition of 0.5 mM 1 for 24 h at 20 °C.
€
kindly provided by Dr. W. Frostl, Novartis, Basel, Switzerland) in
the incubation solution.28 After centrifugation (16000g, 15 min), a
total of 20 μL of the supernatant was applied to a 3 μm HPLC
column (HR-80, 80 mm ꢀ 4.6 mm, ESA, Chelmsford, MA), using a
mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine,
0.59% phosphoric acid, 0.27 mM EDTA, and 8.9 mM sodium
heptane sulfonic acid and a flow rate of 1.0 mL/min. In the eluate,
the reaction product 3-hydroxykynurenine was detected electroche-
mically using a HTEC 500 detector (Eicom Corp., San Diego, CA;
oxidation potential, þ0.5 V). The retention time of 3-hydroxyky-
nurenine was ∼11 min.
For X-ray diffaction experiments, hKAT II 1 crystals were
3
directly taken from the crystallization droplet, quickly equili-
brated in a solution containing the crystallization buffer and
15% glycerol as the cryoprotectant, and flash-frozen under a
liquid nitrogen stream. Diffraction data were collected at 100 K
˚
˚
up to 2.1 A resolution using synchrotron radiation (λ = 0.981 A)
at the ID14 EH1 beamline (European Synchrotron Radiation
Facility, Grenoble, France). Data processing was performed
with the programs of the CCP4 suite.29 The structure determi-
nation of the hKAT II 1 complex was carried out by molecular
3
Kynureninase. The original tissue homogenate was incubated
in 5 mM Tris-HCl (pH 8.4) containing 10 mM of 2-mercap-
toethanol and 50 μM PLP. An amount of 80 μL of the tissue
preparation was then incubated for 2 h at 37 °C in a solution
containing 90 mM Tris-HCl buffer (pH 8.4) and 100 μM L-KYN in
a total volume of 200 μL. The reaction was terminated by adding
50 μL of 6% perchloric acid. To obtain blanks, tissue homogenate
was added at the end of the incubation, i.e., immediately prior to the
denaturing acid. After centrifugation to remove the precipitate
(16000g, 15 min), an amount of 25 μL of the resulting supernatant
was applied to a 5 μmC18 reverse-phase HPLC column (Adsorbosil,
150 mm ꢀ 4.6 mm; Grace, Deerfield, IL) using a mobile phase
containing 100 mM sodium acetate (pH 5.8) and 1% acetonitrile at
a flow rate of 1.0 mL/min. In the eluate, the reaction product,
anthranilic acid, was detected fluorimetrically (Perkin-Elmer series
3 L-KYN com-
replacement, using the structure of the hKAT II
plex (PDB code 2r2n21) as the search model where both the
ligand and all solvent molecules were omitted. A clear solution
was obtained for both cross-rotation and translation functions
using the program AmoRe.30 The initial model was subjected to
iterative cycles of crystallographic refinement with the program
REFMAC,31 alternating with graphic sessions for model build-
ing using the program O.32 A random sample containing 1090
reflections was set apart for the calculation of the free R factor.33
The inhibitor molecule was modeled when the R factor dropped
to a value of around 0.25 at full resolution, based upon both the
2Fo - Fc and Fo - Fc electron density maps. Solvent molecules
were automatically added with ARP/wARP.34 The procedure
converged to an R factor and free R factor of 0.19 and 0.24, with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5689
ideal geometry. Data collection, processing, and refinement
statistics are provided in Table 1. Figures have been generated
with Pymol.35
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Acknowledgment. We thank Dr Giovanni Battista Gio-
venzana (University of Piemonte Orientale, Italy) for helpful
discussions and the European Synchrotron Radiation Facility
(ESFR, Grenoble, France) for data collection at beamline
ID14-EH1. This work was supported by grants from Regione
Piemonte and MIUR (PRIN 2007 and Interlink 2004) (to M.
R.) and by NIH Grant NS25296 (to R.S.).
(17) Schwarcz, R.; Kajii, Y.; Ono, S. I. PCT/US2008/083321 (original
Supporting Information Available: UV-visible spectra of
hKATII titrated with 1 and schematic representation of the
preparation of quinolone compounds with a primary amine.
This material is available free of charge via the Internet at http://
pubs.acs.org.
US patent, US 60/988,231), 2008.
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