Structure-based design of irreversible human KAT II inhibitors: Discovery of new potency-enhancing interactions

Article abstract of DOI:10.1021/ml300237v

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structu

Full text of DOI:10.1021/ml300237v

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Design of Irreversible Human KAT II Inhibitors:
Discovery of New Potency-Enhancing Interactions
Jamison B. Tuttle,*,† Marie Anderson, Bruce M. Bechle, Brian M. Campbell, Cheng Chang,
Amy B. Dounay, Edelweiss Evrard, Kari R. Fonseca, Xinmin Gan, Somraj Ghosh, Weldon Horner,
Larry C. James, Ji-Young Kim, Laura A. McAllister, Jayvardhan Pandit, Vinod D. Parikh, Brian J. Rago,
Michelle A. Salafia, Christine A. Strick, Laura E. Zawadzke, and Patrick R. Verhoest
Pfizer Worldwide Research and Development, Neuroscience Medicinal Chemistry, Eastern Point Road, Groton, Connecticut 06340,
United States
S
* Supporting Information
ABSTRACT: A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino
transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The
utilization of structure−activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of
hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The
use of kinact/Ki to assess potency was critical for understanding the SAR in this series and for identifying compounds with
improved pharmacodynamic profiles.
KEYWORDS: kynurenine amino transferase, kynurenic acid, aryl hydrocarbon receptor, irreversible inhibition, hydroxamic acid,
dose response modeling, in vivo microdialysis, schizophrenia
ship (SAR) indicated that substituents were better tolerated on
C6 and C7, whereas potency losses were observed with
substitution on C5 and C8 (Figure 1). This SAR was supported
he kynurenine amino transferase (KAT) class of pyridoxal
Tphosphate (PLP)-dependent transaminases catalyzes the
cyclization of L-kynurenine to kynurenic acid (KYNA) in the L-
tryptophan metabolic pathway.1,2 Recently, KYNA has been
shown to agonize the arylhydrocarbon receptor (AHR), a
nuclear protein involved in gene transcription and other cellular
regulatory functions.3 Additionally, a burgeoning body of
research implicates KYNA in a range of neurological disorders
due to its antagonism of the α7 nicotinic acetylcholine receptor
and the N-methyl-D-aspartate (NMDA) receptor.4,5 Elevated
levels of KYNA have been found in the cerebral spinal fluid
(CSF) and postmortem brain tissue of schizophrenics.6−8
Furthermore, individuals with bipolar disorder have also been
found to have increased levels of KYNA in the CSF.9
Decreasing central KYNA levels in afflicted individuals may
therefore provide therapeutic benefit for treating these and
other psychiatric and neurological disorders.10,11
Among the four homologous members of the KAT family,
KAT II is largely responsible for centrally produced KYNA,
thereby emerging as an attractive target for medicinal
chemists.12 As such, recent articles have reported both
reversible and irreversible KAT II inhibitors.13−16 One useful
scaffold is hydroxamate 1, a centrally active and irreversible
inhibitor of KAT II.14,17 Preliminary structure−activity relation-
Figure 1. SAR trends on the aryl hydroxamate scaffold.
by the crystal structure, which showed that positions 6 and 7
are oriented toward a solvent-exposed region in the enzyme.14
In contrast, positions 5 and 8 are directed toward the walls of
the binding pocket. As a consequence of these findings, a broad
range of analogues were designed to assess the SAR of this
series.14,15 This communication focuses on key analogues that
led to the discovery of substantial potency-enhancing
interactions with human KAT II.
Received: August 11, 2012
Accepted: October 24, 2012
Published: October 24, 2012
© 2012 American Chemical Society
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Letter
At the outset, a reliable method for assessing potency of
irreversible inhibition was required. For example, analysis of
IC50 values does not discriminate potency differences among
these KAT II inhibitors (Tables 1 and 2, entries 1−4), and
Table 1. Key SAR of Hydroxamate Derivatives
Figure 2. Superposition of the crystal structures of 1 and 2 with KAT
II, showing the additional van der Waals interactions made by the
methoxy substituent at C-7. The entire helix at the entrance of the
binding pocket, consisting of residues Ile-19 to Gly-29, is disordered in
the structure of PLP-bound 1 (yellow), whereas it is ordered in the
structure of 2 (light blue).
a
Values represent the geometric mean of at least three experiments.
Values represent the arithmetic mean of at least three experiments.
b
c
Additional statistical details are provided in the Supporting
Information.
Table 2. Activity of Compound 4
a
Values represent the geometric mean of at least three experiments.
Values represent the arithmetic mean of at least three experiments.
b
Additional statistical details are provided in the Supporting
Information.
consistent with earlier accounts, kinact/Ki proved to be a critical
measure that differentiates these compounds (Table 1).18,19
Indeed, these data led to the selection of 2 and 3 for X-ray
analysis, which subsequently led to the design of 4.
Figure 3. Crystal structure of 3 with KAT II. The C-6 phenoxy
substituent of 3 orients into a shallow pocket at the mouth of the
binding site, which is bounded on one face by Leu-293.
Cocrystal structures of PLP bound to hydroxamates 2 and 3
in the KAT II active site show intriguing changes to the protein.
In the case of PLP adduct of compound 2 bound to KAT II, the
overlay with a crystal structure of 1 bound to the same
components highlights discrete differences (Figure 2), in
particular with a domain consisting of Ile-19 to Gly-29, located
at the entrance of the binding pocket.20 In the structure
containing hydroxamate 1, this domain is highly disordered. In
contrast, the crystal structure containing 2 shows a highly
ordered region.21 The 2-fold kinact/Ki enhancement of derivative
2 as compared to parent may be attributed to an additional van
der Waals interaction with Ile-19.
A previously unknown lipophilic interaction was discovered
by introducing a phenoxy side chain at C-6. In the crystal
structure containing compound 3 with KAT II, the aryl group
rotates into a hydrophobic pocket found at the entrance to the
active site and bounded on one face by Leu-293 (Figure 3),
leading to a 1.5-fold boost in potency over compound 1 (Figure
3).22
ment in potency was observed in the kinact/Ki, an improvement
not evidenced by the IC50 value of 4.
In the crystal structure of 4 bound to KAT II, in analogy to
the structure of compound 3, the benzyl group is oriented into
a lipophilic pocket framed by Leu-293.23 Interestingly, an
apparent cation−π interaction formed with Arg-20, the residue
adjacent to Ile-19, and the phenyl ring (3.5 Å,24 Figure 4). This
type of interaction is well-known in proteins, where it plays a
role in secondary and tertiary structure stabilization.25,26 The
previously disordered region now acts as a clamp that anchors
the hydroxamic acid−PLP complex into the active site and, as a
result, enhances potency.21
It is intriguing to speculate that mechanistically, the 6-OMe
may form lipophilic and/or hydrogen bond interactions with
Ile-19 and Arg-20 on the α-helix that, upon orienting Arg-20
into a coplanar interaction with the benzyl group, is further
stabilized by a hydrogen bond with the oxygen of the methoxy
group. This bidentate interaction is only possible when both
interactions are present, as this clamped architecture is not
observed in the KAT II structures with 2 and 3 (Figure 5,
compare to Figure 3). Additionally, this suggests that enthalpic
These unique interactions inspired the design of a
disubstituted analogue that combined both key elements from
compounds 2 and 3. To avoid the potential formation of a
reactive metabolite characteristic of catechols, hydroxamate 4
was synthesized (Table 2). Interestingly, a 4−5-fold enhance-
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ACS Medicinal Chemistry Letters
Letter
each compound at 10 mg/kg results in a 9-fold lower free brain
exposure of 3 as compared to 1 (65 ng h/g vs 584 ng h/g), due
in part to the high nonspecific binding of 3 to brain tissue
(Table 3, 99% bound). Nevertheless, compound 3 reduces
Table 3. PK of Compounds 1 and 3
brain KYNA levels for a significantly longer period of time and
to a greater degree than compound 1, at the same dose, as a
result of its improved rat KAT II kinact/Ki. (Figure 6).
Figure 4. Crystal structure of 4 with KAT II. The combination of 7-
OMe and 6-Bn substituents results in additional interactions with Arg-
20. The arrow marks a potential cation−π interaction between the
guanidium group in Arg-20 and the phenyl ring.
stabilization overrides the entropic loss and leads to this greatly
improved potency.
Figure 6. Prefrontal cortex extracellular levels of KYNA in freely
moving rats measured using microdialysis. Compounds 1 and 3 were
administered at 10 mg/kg sc at t = 0. Data are expressed as a
percentage of preinjection baseline and are means SEMs (n = 4).
The lowering of KYNA is attributed to the selective
inhibition of KAT II as both compounds are weakly potent
at KAT I and KAT III isoforms. Inhibitors 2 and 4 exhibited a
similar selectivity profile.27 While compound 4 was equipotent
at rat KAT II (kinact/Ki = 763 M−1 s−1) as compared to
compound 1, KYNA levels were not lowered at any dose due to
high clearance. Currently, follow-up designs are focusing on
lowering the clearance of compound 4, as the poor
pharmacokinetics has a deleterious effect on in vivo efficacy.28
In summary, X-ray crystallography and a kinact/Ki assay led to
the rational design of irreversible KAT II inhibitor 4. The
crystal structure of this compound with KAT II led to the
discovery of a novel interaction with a region previously found
to be disordered. Ongoing efforts to optimize ligand
interactions with the enzyme and improve pharmacokinetic
properties will be reported in due course.
Figure 5. Overlay of crystal structures generated from 2 (green) and 4
(yellow). Two binding elements are necessary to interact with Arg-20.
Further analysis of the individual kinact and Ki values provided
insight into the nature of the enhanced potency. In the overall
scheme of enzyme (E) inhibition by an irreversible inhibitor
(I), the first step is reversible binding characterized by
equilibrium binding dissociation constant Ki followed by the
irreversible covalent bond forming event characterized by first-
order inactivation rate constant kinact (Scheme 1).20
Scheme 1. Schematic of Irreversible Inhibition
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental data for the synthesis and characterization of
compounds 3 and 4, assay protocols, and X-ray crystallography
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
For these analogues, kinact values fall within a close range from
0.0108 to 0.0156 min−1, indicating that the rates of covalent
bond formation with PLP are similar. The difference lies in Ki,
where there is a large discrepancy in values, indicating that the
inhibition for these compounds is largely Ki driven (Tables 1
and 2, compare entries 1−4). In the event, analogue 4 binds to
KAT II 5× more efficiently than 2 and 3.
Comparing entries 1 and 3 by in vivo microdialysis
experiments illustrates the impact that increased potency has
on lowering central KYNA. Subcutaneous administration of
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-395-0701. E-mail: jamison.b.tuttle@pfizer.com.
■
Present Address
†700 Main Street, Cambridge, Massachusetts 02139, United
States.
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dx.doi.org/10.1021/ml300237v | ACS Med. Chem. Lett. 2013, 4, 37−40

ACS Medicinal Chemistry Letters
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Letter
irreversible kynurenine aminotransferase II inhibitors for schizophre-
nia. ACS Med. Chem. Lett. 2012, 3, 187−192.
(14) Claffey, M. M.; Dounay, A. B.; Gan, X.; Hayward, M. M.; Rong,
S.; Tuttle, J. B.; Verhoest, P. R. Bicyclic and Tricyclic Compounds as
KAT II inhibitors. WO2010146488.
Notes
(15) Pellicciari, R.; Rizzo, R. C.; Costantino, G.; Marinozzi, M.;
Amori, L.; Guidetti, P.; Wu, H.-Q.; Schwarcz, R. Modulators of the
kynurenine pathway of tryptophan metabolism: synthesis and
preliminary biological evaluation of (S)-4-(ethylsulfonyl)-
benzoylalanine, a potent and selective kynurenine aminotransferase
II (KAT II) inhibitor. ChemMedChem 2006, 1, 528−531.
(16) Schwarcz, R.; Kajii, Y.; Ono, S. Preparation of amino-
piperazinylquinolonecarboxylates as kynurenine-amino-transferase in-
hibitors. WO2009064836.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Scot Mente and Ann Aulabaugh for helpful
discussions. Katherine Brighty played a critical role in editing
this manuscript. Zoe Hughes provided key insights into the
analysis and depiction of the microdialysis data.
(17) In the event, when measuring KYNA in dialysates from rat
prefrontal cortex, a 10 mg/kg dose, sc, showed a 50% decrease from
basal levels that returned to baseline approximately 20 h postdose.
(18) Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.; Liimatta,
M.; Pabst, B.; Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt,
B. F.; Ahn, K.; Stevens, R. C. Structure-guided inhibitor design for
human FAAH by interspecies active site conversion. Proc. Natl. Acad.
Sci. U.S.A. 2008, 105, 12820−12824.
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=
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