Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase

Article abstract of DOI:10.1021/jacs.1c02456

The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

Full text of DOI:10.1021/jacs.1c02456

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Turnover and Inactivation Mechanisms for (S)‑3-Amino-4,4-
difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-
Based Inactivator of Human Ornithine Aminotransferase
Sida Shen,∇ Arseniy Butrin,∇ Peter F. Doubleday, Rafael D. Melani, Brett A. Beaupre,
Mauricio T. Tavares, Glaucio M. Ferreira, Neil L. Kelleher, Graham R. Moran, Dali Liu,*
and Richard B. Silverman*
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ABSTRACT: The inhibition of human ornithine δ-aminotransferase
(hOAT) is a potential therapeutic approach to treat hepatocellular
carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecar-
boxylic acid (SS-1-148, 6) was identified as a potent mechanism-
based inactivator of hOAT while showing excellent selectivity over
other related aminotransferases (e.g., GABA-AT). An integrated
mechanistic study was performed to investigate the turnover and
inactivation mechanisms of 6. A monofluorinated ketone (M10) was
identified as the primary metabolite of 6 in hOAT. By soaking hOAT
holoenzyme crystals with 6, a precursor to M10 was successfully
captured. This gem-diamine intermediate, covalently bound to
Lys292, observed for the first time in hOAT/ligand crystals, validates
the turnover mechanism proposed for 6. Co-crystallization yielded
hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was
utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and
the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass
spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never
been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second
external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a
quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography
employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual
turnover and inactivation pathways.
correlated with poor prognosis in HCC.3 In addition,
INTRODUCTION
■
glutamine synthetase (GS) catalyzes L-Glu’s conversion to L-
Human ornithine δ-aminotransferase (hOAT; EC2.6.1.13) is a
pyridoxal-5′-phosphate (PLP)-dependent enzyme that cata-
lyzes two coupled half-reactions, converting L-ornithine (L-
Orn) in the first half-reaction to L-glutamate-γ-semialdehyde
(L-GSA) and generating L-glutamate (L-Glu) from α-
glutamine (L-Gln).4 L-Gln is required by cancer cells to support
the abnormally elevated anabolic processes, thus promoting
cellular proliferation.
HCC is the predominant liver malignancy and ranks among
the most common causes of cancer-associated mortality
ketoglutarate (α-KG) in the second half-reaction (Figure
worldwide.5 Our prior DNA microarray analyses identified
1A).1 The product, L-GSA, is in equilibrium with Δ1-pyrroline-
the OAT gene as one of seven overexpressed genes in the
spontaneous HCC-developing livers from Psammomys obesus
5-carboxylate (P5C), which is converted to L-proline by P5C
reductases (PYCRs).2 The generated L-Glu can also be
converted to P5C by pyrroline-5-carboxylate synthase
Received: March 4, 2021
Published: June 7, 2021
(P5CS), therefore also participating in proline metabolism
(Figure 1A).2 Proline biosynthesis was identified as the most
substantially altered amino acid metabolism in human tumor
tissues of hepatocellular carcinoma (HCC), featured by
accelerated proline consumption, hydroxyproline accumula-
tion, and increased α-fetoprotein (AFP) levels, which are
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The optimization of reversible inhibitors mainly focuses on
improving the binding affinity (Ki) or IC50 values. In contrast,
kinact and KI values are two critical kinetic parameters for the
development of MBIs.11 The kinact value is the maximal rate
constant for enzyme inactivation. The KI value, the
concentration of inactivator that gives the half-maximal
inactivation rate, is the inhibition constant, which represents
the ability of an MBI to initially bind to the active site of the
target enzyme and compete with the substrate. The ratio kinact
/
KI is used to evaluate the inactivation efficiency of MBIs.11
A major challenge for discovering a selective MBI of hOAT
is to overcome the irreversible inhibition of other amino-
transferases,7 especially γ-aminobutyric acid aminotransferase
(GABA-AT), which has a high structural similarity with
hOAT.1 There are only two significant differences in the active
site pocket of their homodimer structures: Tyr85 and Tyr55 in
hOAT are replaced by Ile72 and Phe351* (asterisk denotes
arising from the adjacent subunit) in GABA-AT, respectively
(Figure 2).1 Moreover, Ile72 and Phe351* are responsible for
the slightly narrower and more hydrophobic active site of
GABA-AT relative to hOAT. In contrast, the hydroxyl group of
Tyr55 serves as a hydrogen bond acceptor to interact with the
charged C-2 amino group of substrates, while Tyr85 is a
significant determinant of substrate specificity and conforma-
tional flexibility to adopt bulky substrates.1
Because of the high similarity between these two amino-
transferases, a preliminary screening against hOAT was carried
Figure 1. (A) Transamination reactions of hOAT and related
metabolic pathways. (B) Structures of hOAT mechanism-based
inactivators 1−3.
out previously using our stock GABA-AT inhibitors.6
A
cyclopentane-based analogue, termed BisCF3 (1, Figure 1B),
bearing a bis(trifluoromethyl) group as its warhead, was
identified to be a selective MBI of hOAT while only showing
millimolar reversible inhibition of GABA-AT. Recent mecha-
nistic studies have revealed that one of its trifluoromethyl
groups undergoes fluoride ion elimination, leading the ligand
to covalently modify the catalytic Lys292 residue by conjugate
addition (Scheme 1 A).9,12 It is considered that the sterically
bulky bis(trifluoromethyl) group may not access the more
narrow pocket of GABA-AT as readily, influencing the initial
binding pose between the ligand and the enzyme, which may
be responsible for its reversible inhibition of this enzyme.
Compound 1 has been demonstrated to be effective in vivo6
and is being investigated further in HCC patient-derived
xenograft (PDX) models. On the basis of a similar strategy, we
enlarged the ring system and further developed a cyclohexene-
(sand rat).6 Moreover, the treatment (at 0.1 and 1.0 mg/kg;
po) of selective hOAT mechanism-based inactivator (MBI)
BisCF3 (1) remarkably decreased the serum AFP levels and
inhibited tumor growth in a human-derived HCC mouse
model,6 underscoring the antitumor effects of selective hOAT
inhibition. A MBI is a molecule that initially acts as an
alternative substrate for the target enzyme and is converted by
this enzyme to a species that inactivates that enzyme.7−9 MBIs
are typically unreactive before the initial binding with the
active site of the target enzyme and usually exhibit significant
target specificity and selectivity.10 Overall, hOAT is a potential
therapeutic target for HCC, and selectively inactivating hOAT
may provide a novel opportunity to discover an effective HCC
treatment.
Figure 2. Active site comparison of (A) hOAT (PDB entry 1OAT) and (B) GABA-AT (PDB entry 1OHV).
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Scheme 1. Inactivation Mechanisms of hOAT by 1−3
based analogue, WZ-2-051 (2, Figure 1B), bearing a difluoro
group.13 Compound 2 exhibited a 23-fold improvement in
inactivation efficiency (defined by the kinact/KI ratio) against
hOAT compared to 1 while showing 13.3-fold selectivity over
GABA-AT. The subsequent mechanistic studies disclosed that
2 undergoes a two-step fluoride ion elimination and inactivates
hOAT through an addition-aromatization mechanism (Scheme
1 B).13 An additional example of a selective hOAT inactivator
is 5-fluoromethylornithine (5-FMOrn, 3) inspired by the
structure of hOAT substrate L-Orn and related to the structure
of nonselective GABA-AT inactivator (S)-4-amino-5-fluoro-
pentanoic acid (AFPA).14 This molecule inactivates hOAT via
an enamine pathway by forming a ternary adduct (Scheme
1C).15
It should be noted that the α-amino group of 5-FMOrn
forms a strong hydrogen bond with the phenol group of Tyr55
in the hOAT crystal complex (PDB entry 2OAT).16 Moreover,
we also observed hydrogen bonds between the carboxylate
groups of 1 (PDB entry 6OIA) and 2 (PDB entry 6V8C) and
Tyr55 in hOAT crystal complexes.12,13 Among the published
hOAT inactivators, only 1 demonstrates (a) promising hOAT
selectivity through potent irreversible inhibition of hOAT, (b)
weak, reversible inhibition of GABA-AT (Ki = 4.2 mM), and
(c) no inhibition of either aspartate aminotransferase (Asp-
AT) or alanine aminotransferase (Ala-AT) (up to 4 mM).6
In 2000, (1R,4S)-4-amino-3,3-difluorocyclopentanecarbox-
ylic acid (4, Figure 3) was found to be a reversible inhibitor
Figure 3. Structures of hOAT inactivators 4−6.
against GABA-AT (Ki = 0.19 mM).17 Fifteen years later, it was
further demonstrated to be an hOAT inactivator.6 However, 4
exhibits poor binding affinity (KI = 7.8 mM) and has a low
maximum rate of inactivation (kinact = 0.02 min−1) against
hOAT, only yielding a modest inactivation efficiency (kinact/KI
= 0.003 min−1 mM−1).
In this work, we developed a novel cyclopentene-based
analogue 6 by incorporating an additional double bond into
the cyclopentane ring system of 4, which was demonstrated to
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a
Scheme 2. Synthetic Route to 4 and 5
a
Reagents and conditions. (a) (i) p-anisyl alcohol, conc. HCl, rt; (ii) NaH, tetra-n-butylammonium iodide (TBAI), THF/DMF (10:1), 0 °C−rt;
(b) 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), Ac2O, rt; (c) K2CO3, MeOH/H2O, rt; (d) (COCl)2, DMSO, TEA, THF, −78 °C−rt; (e)
Bu3SnH, azobis(isobutyronitrile) (AIBN), benzene, reflux; (f) Deoxo-Fluor (2.7 M in toluene), THF, 120 °C (MW); (g) ceric ammonium nitrate,
CH3CN/H2O, rt; (h) 4 N HCl, AcOH, 70 °C; (i) (i) HCl in EtOH (1.2 M), 70 °C; (ii) Boc2O, TEA, DCM, rt; (j) PhSeCl, KHMDS (3.0 equiv.,
0.5 M in toluene), −78 °C−rt; (k) 4 N HCl, AcOH, 70 °C.
a
Scheme 3. Synthetic Route to 6
a
Reagents and conditions. (a) (COCl)2, DMSO, TEA, THF, −78 °C−rt; (b) Deoxo-Fluor (2.7 M in toluene), THF, 120 °C (MW); (c) ceric
ammonium nitrate, CH3CN/H2O, rt; (d) (i) HCl (1.2 M in MeOH), 85 °C, seal; (ii) Boc2O, MeOH, rt; (e) Burgess reagent, THF, 70 °C; (f) 4 N
HCl, AcOH, 70 °C.
be a potent and selective hOAT inactivator. Furthermore, we
performed mechanistic studies utilizing protein crystallogra-
phy, multiple modes of mass spectrometry, transient-state
spectrophotometric measurements, and computational simu-
lations to reveal a novel noncovalent inactivation mechanism
for 6.
the configuration of the initial external aldimine and the
adjunct proton’s acidity resulting from the α,β-unsaturated
carboxylate.18,19 Therefore, we designed cyclopentene-based
analogues 5 and 6 bearing a gem-difluoro group on the basis of
the structure of parent compound 4 (Figure 3) with the intent
to develop more potent hOAT inactivators.
The synthetic route to prepare compound 5 initiated from
the enantiopure Vince lactam20 (7; (1R)-(−)-2-azabicyclo-
[2.2.1hept-5-en-3-one; CAS#: 79200-56-9) to afford the key
bicyclic intermediate 9 according to the procedure developed
previously (Scheme 2).17 The acetyl group of 9 was then
hydrolyzed under acid conditions followed by Swern oxidation,
yielding ketone intermediate 11.
RESULTS AND DISCUSSION
■
Synthesis of Cyclopentene Analogues 5 and 6
Bearing a gem-Difluoro Group. On the basis of our
previous experience with the discovery of GABA-AT
inactivators, the incorporation of a double bond into a
cyclopentane ring has been demonstrated to be an effective
strategy for improving inactivation efficiency, which influences
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a
Table 1. Kinetic Constants for the Inactivation of hOAT and Reversible Inhibition of GABA-AT by 4−6
a
kinact and KI values were determined by the equation: kobs = kinact × [I]/(KI + [I]) and presented as means and standard errors. Ki values were
calculated by the Cheng-Prusoff equation: Ki = IC50/(1 + [S]/Km) and shown as means and standard errors. IC50 values were obtained using
nonlinear regression analysis in GraphPad Prism 8 of a 9-point enzymatic assay with a 2-fold serial dilution against GABA-AT. The partition ratios
b
were determined under conditions in the presence of α-KG, while fluoride ion release results were determined in the absence of α-KG. Data were
c
extracted from ref 12. Enzyme activity remaining was measured as a function of the number of equivalents of 4−6 relative to enzyme
concentration. Linear regression analysis was used on the linear portion of the curves to obtain the x-intercept, which was the turnover number
d
(partition ratio = turnover number − 1). Data are shown as the means with standard errors. The fluoride ion release experiments were performed
in triplicate. Data are presented as the means with standard deviations.
Subsequently, the reduction of the bromo group on the
bridgehead of 11 to form 12 was carried out with Bu3SnH/
AIBN by a published procedure.17 Intermediate 12 was treated
with Deoxo-Fluor reagent under microwave conditions to
afford difluoro intermediate 13. The PMB protecting group of
13 was removed using ceric ammonium nitrate (CAM) to
produce lactam 14. The treatment of 14 with HCl/EtOH
under reflux conditions followed by Boc protection yielded 15.
Unexpectantly, when we attempted to selenate 15 at the α-
position of the ethyl ester with KHMDS (3.0 equiv) and
PhSeCl for the follow-up α-elimination reaction,18 intermedi-
ate 16, bearing an α,ε-conjugated carboxylate group, was
produced directly as the sole product. Because of the strong
electron-withdrawing effect of the gem-difluorines, the
theoretical pKa values of the hydrogens at the Cε position
are considerably decreased, which are further decreased by the
incorporation of the phenylselenyl group (Scheme S1). This
should facilitate deprotonation in the presence of excess
KHMDS, thereby causing the elimination of the phenylselenyl
group. 1D and 2D NMR spectra were obtained to validate the
structure of 16. Cyclopentene-based analogue 5 was generated
after deprotection under acidic conditions. The parent
cyclopentane-based analogue (4) was also prepared from
intermediate 14 by acid hydrolysis and was evaluated together
with new analogues and 1 in subsequent kinetic studies.
The synthetic route to 6 started from the preparation of the
critical bicyclic intermediate 17 from PMB-protected Vince
lactam 8 following our recently published procedure (Scheme
3).19 The hydroxyl group of 17 was converted to ketone 18 by
Swern oxidation. Difluoro intermediate 19 was obtained
through the same fluorination conditions described in Scheme
2. The PMB group of 19 was removed by ceric ammonium
nitrate followed by lactam hydrolysis and Boc protection,
yielding cyclopentane intermediate 21. The hydroxyl group of
21 was dehydrated using Burgess reagent21,22 under reflux,
forming cyclopentene intermediate 22. The final product (6)
was afforded using the deprotection conditions in Scheme 2.
Kinetic Studies of Analogues 4−6. The kinetic results in
Table 1 indicate that all three difluoro-based compounds 4−6
are irreversible inhibitors of hOAT but reversible inhibitors of
GABA-AT. Whereas inactivators 4 and 5 are weak binding
inactivators (KI = 4.0 and 2.0 mM, respectively) of hOAT, 6
exhibited a significantly higher affinity (KI = 0.06 mM). The
partition ratio is defined as the ratio of the number of
equivalents of inactivator consumed as a substrate per active
site compared to each equivalent of inactivator leading to
inactivation.18,23 The fluoride ion release result is determined
as the equivalents of fluoride ions released per active site for
each inactivation event, which can be measured with a fluoride
ion-selective electrode.23 The partition ratio determination and
fluoride ion release results in Table 1 revealed that the majority
of reactions of 4 and 5 involve an alternative turnover pathway,
resulting in the release of a large fraction of fluoride ions.
Compound 6 showed the highest maximal rate constant of
inactivation (kinact) relative to 4 and 5, thereby leading to
superior inactivation efficiency (kinact/KI) (4 vs 6, 0.003 vs
1.300 min−1 mM−1, a 400-fold improvement). Furthermore,
the inhibitory activities of cyclopentene-based 5 (Ki = 1.40
mM) and 6 (Ki = 1.10 mM) toward GABA-AT are about 10-
times weaker than that of cyclopentane-based 4 (KI = 0.10
mM). It should be noted that, compared to parent cyclo-
pentane 4 (OAT, KI = 4 mM; GABA-AT, KI = 0.10 mM),
newly developed cyclopentene 6 displayed a significantly
higher binding affinity for OAT but much lower binding
affinity for GABA-AT (OAT, KI = 0.06 mM; GABA-AT, Ki =
1.10 mM), indicating 6 has an improved ability to more
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Scheme 4. Possible Inactivation Mechanisms for 6
Figure 4. Primary metabolites of 6 in (A) hOAT and (B) GABA-AT.
selectively bind with OAT relative to GABA-AT. Compound 6,
also called SS-1-148, exhibited comparable inactivation
efficiency against hOAT while retaining the reversible
inhibition of GABA-AT similar to that of the preclinical
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Scheme 5. Plausible Turnover Mechanisms of 6 with hOAT and GABA-AT
Figure 5. Crystal structures of hOAT resulting from the (A) soaking experiment (PDB entry 7LK1) and (B) co-crystallization (PDB entry 7LK0)
with 6. The 6 soaking structure is shown in two alternate conformations (beige): one in which the carboxylate group interacts with Tyr55
(conformation A) and the other in which the carboxylate forms a salt bridge with Arg413 (conformation B). For this specific chain, the refined
occupancies of conformers are 0.51 (conformation A) and 0.49 (conformation B). hOAT residues are in stick representation with carbon atoms in
the residues colored gray, nitrogen in blue, and oxygen in red; the water molecule is shown as a red sphere. Hydrogen bonding distances between
atoms are in Ångstroms (Å) and are shown as black dashed lines.
compound 1. It also did not show a noticeable inhibition of
Asp-AT or Ala-AT at concentrations up to 10 mM, motivating
interest in the elucidation of the inactivation and turnover
mechanisms of 6 with hOAT.
Proposed Inactivation Mechanism Pathways of 6. On
the basis of our previous mechanistic studies of other related
GABA-AT/hOAT inactivators,8,13 we propose three potential
pathways (Scheme 4). Initially, transimination with 6 would
form the external aldimine M1. M1 would then undergo
deprotonation to form the quinonoid species M2 followed by
fluoride ion elimination, affording the intermediate (M3) that
can branch into three different pathways.
Pathway a is proposed on the basis of our recent findings
with the cyclohexene-based analogue 2.13 The electrophilic Cδ
position of intermediate M3 could undergo conjugate addition
by Lys292, forming a covalent bond (M4). Quinonoid species
M4 is also subject to a second fluoride ion elimination to give
final adduct M5. Pathway b is inspired by the inactivation
mechanisms of CPP-11523 and OV32918 with GABA-AT,
which are achieved through a water-mediated mechanism,
resulting in tight electrostatic interactions between their
carboxylates and arginine residues in the active site. Lys292
would then activate a water molecule that attacks the
electrophilic Cδ position of intermediate M3 followed by a
further fluoride ion elimination that would yield enol/carbonyl
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Figure 6. (A) Denaturing and (B) native intact protein mass spectrometry of hOAT untreated and inactivated by 6.
species M7 rather than establishing a covalent bond with the
catalytic lysine residue in hOAT. Pathway c is proposed
according to a typical enamine mechanism.19 Lys292 attacks
the C4′ position of the aldimine instead of the Cδ position
while releasing enamine intermediate M8, which attacks the
imine linkage of the internal aldimine (PLP-Lys292) and
produces covalent adduct M9.
hOAT structure was solved by molecular replacement from a
previously reported structure (PDB entry 1OAT). The space
group for the 6 soaking structure was found to be P3221, and
the structure contains three copies of the protein monomer in
one asymmetric unit. The crystal structure (PDB entry 7LK1)
shown in Figure 5A and Figure S3 indicates that PLP is
covalently linked to 6, and a covalent bond between Lys292
and 6 tethers the compound to the enzyme. The covalent bond
between Lys292 and 6 represents a stable gem-diamine species
that has not been observed in any previous hOAT/ligand
crystals.9,12,13,24 This observation further validated the gem-
diamine precursors (M12 or M13, Scheme 5) of enamine
intermediate M8. Moreover, two alternate conformations of
this intermediate were observed, which differ in the position of
the carboxylate group derived from 6. The first conformation
forms a hydrogen bond between Tyr55 and the carboxylate of
6, while the second forms a salt bridge with Arg413. The
interpretation that there are two alternate conformations for
the intermediate structure was based on the positive density in
proximity to Arg413 and Tyr55 as well as the relatively high B-
factors for any single conformation. An alternative explanation
could include two different, yet structurally similar, inter-
mediate species that could interact with the protein active site
in different ways.
Plausible Turnover Mechanisms of 6 with hOAT and
GABA-AT. Compound 6 exhibited a relatively high partition
ratio (34-fold, Table 1 and Figure S1), indicating that 35 equiv
of 6 are turned over per active site for each equivalent of
compound leading to inactivation. Moreover, 34 1 equiv of
fluoride ion (Table 1) are released per inactivation event,
indicating that the primary turnover pathway only involves a
single fluoride ion elimination step. Previously, we carried out
electrostatic potential (ESP) charge calculations to demon-
strate that the fluorine atom decreases the nucleophilicity of
the enamine intermediate (similar to the structure of M8),13
which may prevent enamine addition. A mass spectrometry
(MS)-based analysis of 6 with hOAT (Figure 4A) showed that
the molecular weight and fragmentations of the primary
metabolite match the structure of M10 in Scheme 5, the
hydrolyzed product of enamine intermediate M8. Conversely,
we also identified the primary metabolite of 6 in GABA-AT
(Figure 4B). The molecular weight and fragmentations suggest
that 6 acts as a substrate with this enzyme to yield ketone M11,
bearing a difluoro group (Scheme 5).
The dominant turnover mechanisms for 6 in hOAT and
GABA-AT are proposed in Scheme 5. After capturing the PLP
coenzyme from Lys292, M1 undergoes deprotonation to give
quinonoid M2 that causes the elimination of a fluoride ion
(Pathway a; Scheme 5) in hOAT to produce aldimine M3
bearing a single fluorine atom. The majority of M3 is attacked
In an attempt to capture the primary intermediate of the
noninactivation pathway, hOAT holoenzyme crystals were
utilized to perform one-hour soaking experiments with 6. The
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Scheme 6. Possible Inactivation Mechanisms of 6 with hOAT
by Lys292 at its C4′ position, forming the first gem-diamine
(M12), followed by proton transfer,25,26 which results in the
second gem-diamine (M13), which is further converted to
enamine metabolite M8 and the internal aldimine. M8
undergoes hydrolysis to ketone M10 as the primary metabolite
of 6 in hOAT. In contrast, quinonoid intermediate M2 only
undergoes electron transfer to yield ketimine M14, which
hydrolyzes to release PMP and M11 as the primary metabolites
in the GABA-AT reaction. The behavior of 6 in GABA-AT
mimics a canonical transamination reaction without eliminat-
ing fluoride ions, consistent with its reversible inhibition of
GABA-AT. Given the structural differences between hOAT
and GABA-AT, the known selective hOAT inactivators (1−3)
described above contain a bulky moiety or α-amino group to
improve their selectivity. However, the selectivity of 6 for
hOAT over GABA-AT occurs because of its competition with
substrate GABA.
evaluated with a 48 h time-dependent dialysis experiment
against a buffer containing excess PLP and α-KG (Figure S2).
The residual activity of hOAT was unchanged, demonstrating
that 6 is an irreversible inhibitor of this enzyme. To reveal the
structure of the final product of the inactivation reaction, we
co-crystallized hOAT in the presence of excess 6. The crystal
structure (PDB entry 7LK0) was solved using the same
procedure described above. The space group for the 6 co-
crystal (Figure 5B and Figure S4) was found to be P3112 and
contained three subunits per asymmetric unit. Similar to the
soaking crystal structure shown in Figure 5A and Figure S3,
PLP is covalently linked to the 6 moiety in the co-crystal
structure. However, no covalent bond is observed between
Lys292 and the final product. Moreover, in the published
crystal structure of the native enzyme (holo-hOAT; PDB entry
1OAT), Arg413 typically forms a salt bridge with Glu235,
which was observed for several co-crystals of hOAT with
different inactivators.16,24,27 In the hOAT/6 soaking structure
(Figure 5A), the salt bridge of Arg413-Glu235 is broken
Plausible Inactivation Mechanisms of 6 with hOAT.
The irreversibility of the inhibition of hOAT by 6 was
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because of the formation of an alternative salt bridge between
the carboxylate of one intermediate and Arg413. Interestingly,
the Arg413−Glu235 salt bridge is also found to be broken in
the hOAT/6 co-crystal structure, while no direct interaction is
observed between Arg413 and the ligand. Instead, Arg413,
Gln266, and the final product are hydrogen-bonded to the
same water molecule. Additionally, the carbonyl oxygen of the
ligand shows a hydrogen bond with Glu235 (3.0 Å) (Figure
5B), which may contribute to stabilizing the ligand in the
hOAT pocket. The ligand structure in the hOAT/6 co-crystal
structure indicates that the inactivation may be attributed to
Pathway b in Scheme 4, generating a final product that
resembles M7 (theoretical mass: 370.06 Da).
We previously have applied denaturing intact protein mass
spectrometry (denaturing MS) to probe covalent inactivation
mechanisms of hOAT inactivators.12,13 Here, through
denaturing MS using hOAT fully inactivated by 6, only
∼16% of hOAT was found to be covalently modified, leading
to a mass increase of 370.07 Da (Figure 6A, right). The
majority of hOAT remained unmodified (Figure 6A, right) like
the untreated hOAT used as a control (Figure 6A, left). The
observed mass addition (370.07 0.82 Da) corresponds to the
theoretical mass of gem-diamine M15 (370.06 Da; Scheme 6)
that could be in equilibrium with the noncovalent form M7.
However, we would expect a higher abundance of the
covalently bound enzyme under these conditions where the
enzyme is fully inactivated. This finding supports a non-
covalent inactivation mechanism between hOAT and 6.
We further employed native mass spectrometry (native MS)
to identify solution-state, noncovalent protein interactions that
are preserved in the gas phase and characterized the binding of
the 6 product in hOAT. The results shown in Figure 6B
observed at 45% relative abundance (observed: 92 275 4 Da;
theoretical: 92 278 Da), and a second mass of 92 597 5 Da
was observed at 100% relative abundance. Compared to the
previously observed dimer mass, which is consistent with each
active site bound by M7 and one ammonium cation, this
species has a 457 Da mass shift. Interestingly, the high
abundance mass shift cannot be explained by the loss of a
single M7 NH4+ adduct from the protein dimer (theoretical
mass for M7-hOAT: 92 648 Da; theoretical mass for M7-
+
hOAT+ NH4 : 92 666 Da). The mass shift observed through
HCD activation, however, can be explained by the loss of PLP
from both active sites, while the 6 moiety with one ammonium
cation was apparently retained (observed: 457 Da; theoretical:
460 Da). This finding is surprising, given that the same
collisional energy does not eject PLP from the untreated
protein dimer (Figure S5).
As no apoenzyme is observed by native MS and
approximately 84% of hOAT was in an apoenzyme state
under denaturing MS conditions at pH 2.5, these results
indicate that noncovalent M7 is the primary form after
inactivation, while covalently bound M15 is a minor form that
is in equilibrium with M7. Therefore, M7 generated from the
water-mediated Pathway b (Scheme 4) seems to be the final
product of 6 after inactivation. However, given its highly
conjugated structure, M7 readily tautomerizes. A Gibb’s free
energy calculation28 was performed on MOPAC to assess the
stabilities of M7 and the related tautomeric forms in the
hOAT’s active site. The results shown in Figure S6 suggest
that, compared with enol form M7 (Tautomer 1; ΔG° =
−26.36 kcal mol−1), the corresponding ketone (Tautomer 5,
ΔG° = −48.07 kcal mol−1) and two potential ketimines
(Tautomer 8, ΔG° = −49.89 kcal mol−1 and Tautomer 9, ΔG°
= −49.07 kcal mol−1) are relatively more stable. Moreover, a
species that shows absorbance at ∼275 nm was determined as
the final product in the subsequent transient-state spectropho-
tometric measurements (Figure 8C), thereby ruling out all
external aldimines (e.g., M7 and Tautomer 5) that would
display absorbance maxima at ∼420 nm. The results above
indicate that the final state is more likely a gem-diamine or
ketimine that typically has an absorbance maxima ∼330−340
nm.25,29 According to the literature, in comparison with the
ketoenamine moiety, containing a protonated aldimine and
deprotonated hydroxyl, the neutral enolimine of the hydroxyl
and aldimine groups exhibit shifted the absorption maximum
(410 vs 330 nm).30 Taken together, we believe that gem-
diamine M15 (Scheme 6) is in equilibrium with M7, and
ketimine M18 is tautomerized from M7; both M15 and M18
may then undergo additional electron transfer to form M16
and M19, respectively. Because of the neutral states of gem-
diamine M16 and ketimine M19, their absorption transitions
shift from 330 to 275 nm, consistent with the absorbance of
the final species observed in the transient-state measurements.
Proposed inactivation pathways for 6 with hOAT are
summarized in Scheme 6. The initial external aldimine M1
undergoes deprotonation, catalyzed by Lys292, and forms the
first quinonoid (M2). The elimination of a single fluoride ion
follows to yield monofluoro aldimine M3 from M2. The C4′
position of the majority of M3 (∼97%; determined by its
partition ratio) is attacked by Lys292, which releases an
enamine metabolite that hydrolyzes to ketone M10 as the
primary metabolite (Pathway a; Schemes 5 and 6).
indicate that untreated hOAT appears as a dimer (92,737
2
Da) that is 459 Da more than the mass of the apo-hOAT
dimer. This mass shift is consistent with two PLP-bound
internal aldimines that reside in the active sites of the dimer
(459 Da shift observed; 460 Da theoretical). A proteolytic
proteoform of hOAT was also observed, but only under
untreated conditions (Figure 6B, left (*)). In contrast, native,
dimeric hOAT fully inactivated by 6 was observed with two
high-abundance masses of 93 020 4 Da and 93 056 3 Da,
corresponding to a mass addition of 742 and 778 Da,
respectively (Figure 6B, middle). Several additional masses
were observed and can be attributed to salt adducts (Figure
6B, middle (*)). In addition, neither unmodified apo-hOAT
nor PLP-bound hOAT was observed. The observed 93,020
4
Da mass is consistent with the predicted mass of M7 (370.06
Da) bound in both active sites (theoretical: 93 018 Da). As the
hOAT samples were desalted by dialyzing against 100 mM
NH4OAc solution for 1 week, we think that the 93 056 3 Da
observed mass is the predicted mass of M7 complexed with
one ammonium cation ([M + NH4]+ = 388.09 Da) in both
protein chains (theoretical: 93 054 Da).
To further probe the lability of the 6-hOAT interaction by
mass spectrometry, higher-energy collisional dissociation
(HCD) was applied to untreated and treated hOAT to
dissociate the protein−ligand interactions and eject ligands
from the enzyme complex. To eject adducts from protein
dimers, HCD was applied with normalized collisional energy
(NCE) ranging from 5 to 15%. No mass shift was observed for
untreated hOAT (Figure S5). Under these same conditions,
two additional masses were produced for 6-inactivated hOAT
(Figure 6B, right). One mass, consistent with apo-hOAT, was
The Cδ position of a small portion of M3 (∼3%) goes
through a water-mediated nucleophilic attack (Pathway b;
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Figure 7. Transient state absorption changes observed at 275, 420, and 560 nm for hOAT reacting with 6. OAT (12.7 μM final) was mixed with 6
(126, 251, 502, 1004, 2008, and 4016 μM), and CCD spectra were collected for the time frame 0.009−49.2 s. (A) Data observed at 420 nm fit to a
linear combination of three exponential terms according to eq S3 described in the Supporting Information. The arrow indicates the trend observed
in amplitude for increasing inhibitor concentration. (B) Observed rate constant dependence of the first phase observed at 420 nm fits eq S5
described in the Supporting Information. The values for k2 and k3 indicated are the average values obtained from the fit in Figure 7A. The fit is
shown by red dashes. (C) Data observed at 560 nm. The curved arrow indicates the trend observed in amplitude for increasing inhibitor
concentration. These data were fit to a linear combination of two (1004, 2008, and 4016 μM, blue traces) or three (126, 251, and 502 μM, black
traces) exponential terms according to eq S4 described in the Supporting Information. The fit is shown by red dashes. (D) Data observed at 275
nm over 2000 s obtained in the presence of 8032 μM 6 fit a linear combination of two exponential terms according to eq S4 in the Supporting
Information. The fit is shown by red dashes.
Scheme 6), generating the second quinonoid (M6). Inter-
mediate M6 undergoes another fluoride ion elimination to
form M7. Subsequently, a small fraction of M7 (∼16%) may
covalently bond to Lys292 at its C4′ position via a gem-diamine
form (M15) (Pathway c; Scheme 6), which is in equilibrium
with M7 and also facilitates further proton transfer to generate
the neutral gem-diamine (M16), which should be a more stable
form. However, the majority of M7 (∼84%) tautomerizes to a
more favorable ketimine (M18, the most stable tautomeric
form in Figure S6), which is followed by proton transfer to
afford ketimine M19 as the primary final product (Pathway d;
Scheme 6).
Transient-State Measurements of hOAT Inhibited by
6. As a variety of transient states are involved in the proposed
mechanisms, we performed rapid-mixing spectrophotometric
measurements in an attempt to capture the kinetics of the
inhibition of hOAT by 6. This approach takes advantage of the
conjugated species that accumulate sequentially in PLP-
dependent aminotransferase reactions.9 The inhibition reaction
that occurs with 6 was interpreted in combination with crystal
structures (mentioned above) acquired for different stages of
the reaction progression. The experimental data shown in
Figures 7 and 8 indicate that the reaction of 6 with hOAT is
complex. Four discernible phases were observed with the
evidence of at least two parallel reaction paths (Figure 8),
indicating that the mechanistic conclusions drawn are
necessarily from undetermined models. Within the deadtime
of the stopped-flow instrument, the reaction of 6 with hOAT
formed a spectrum signal (∼420 nm) consistent with an
external aldimine (red spectrum, Figure 8A,B). The titration of
hOAT with 6 modulated the rate and the extent of
accumulation of a second external aldimine that is presumably
additive with the aldimine formed in the deadtime (green
spectrum, Figure 8A,B). The second external aldimine forms
with a rate dependence that indicates a bimolecular reaction
(6.2 × 103 M−1 s−1) and suggests that 6 interacts with hOAT
in at least two ways, resulting in parallel reaction paths (Figure
7A,B). The intensity of the combined external aldimine spectra
decays partially with the accumulation of a spectral transition
characteristic for a quinonoid species (∼560 nm) at 0.4 s−1
(Figures 7C and 8A,B). The apparent quinonoid species is
formed with concomitant and partial decay of the external
aldimine transitions that are approximately equal in amplitude
to that gained with the second external aldimine accumulation.
This suggests that these species reside on the same reaction
pathway.
It has been reported that the deprotonation of initial external
aldimine M1 and stepwise fluoride elimination steps proposed
in Scheme 6 are typically considered as an E1cB elimination
mechanism.31,32 The electron-withdrawing effect of fluorine
and the protonated nitrogen of the aldimine may stabilize the
formed carbanion state during the elimination reaction.32,33
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Figure 8. Partial deconvolution by singular value decomposition (SVD) of transient state absorption changes observed for hOAT reacting with 6.
hOAT (6.94 μM; final concentration) was allowed to react in a stopped-flow spectrophotometer with 6 (1040 μM; final concentration) at 10 °C.
To obtain a time resolution sufficient to analyze kinetic rates spanning 4 orders of magnitude, a composite CCD absorbance data set was prepared
spanning 250−800 nm and 0.0137−9843 s by splicing together averaged short and long time frame data sets. These data were fit to a linear
irreversible four-step model in which the rate constants were constrained to those determined from single-wavelength analyses (Figure 7). (A)
Deconvoluted composite spectra were derived from SVD analysis. (B) Species concentration profile based on the rate constants was used to fit the
data set. (C) Three-dimensional depiction of a subset of spectra from the data set was analyzed.
Figure 9. (A) Theoretical pKa calculations of the hydrogen at the Cγ position using the DFT/B3LYP method and (B) electron density maps
colored coded to the electrostatic potential of intermediates and ESP charges of Cδ and C4′ positions.
Quinonoid transient state M2 is expected to form between the
first and second external aldimines (M1 and M3). However,
the stopped-flow experimental results suggest that the
antiperiplanar hydrogen and fluorine atoms in M1 undergo a
Lys292-assisted E2 mechanism,31 i.e., concerted loss of a
proton fluoride ion, with the formation of the alkene as a more
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favorable fluoride ion elimination pathway, affording the
second external aldimine (M3) directly as the single transient
state species (Pathway e; Scheme 6). This E2 elimination
pathway has never been observed experimentally in previous
mechanistic studies of other related PLP-dependent amino-
transferase inactivators.1,8
participate in the alternative turnover pathway rather than the
inactivation pathway, simultaneously releasing large amounts
of fluoride ions. The electron density maps and ESP charge
calculations13 (Figure 9B) indicate that the electrophilicity of
Cδ in the transient state M3 of 6 is much higher than that in
the corresponding transient states of 4 and 5 (M3′ for 4 and
M3″ for 5), suggesting that the Cδ position of M3 is more
reactive than in the other two intermediates. The C4′ positions
in the aldimine linkage of M3′ and M3″ display comparable
electrophilicity that is much greater than that of M3, which
demonstrates that M3′ and M3″ are easier to be attacked by
catalytic Lys292 to trigger their turnover pathways, eventually
resulting in significant partition ratios for 4 and 5.
Furthermore, additional MS-base analyses in hOAT revealed
that, similar to 6 in hOAT, cyclopentene analogue 5 generated
a ketone bearing a single fluorine atom as its primary
metabolite (M5-1, Figure S7B). In contrast, cyclopentane 4
not only formed a monofluorinated ketone (M4-1, Figure
S7A) but also generated a ketone metabolite bearing a
hydroxyl group (M4-2, Figure S7A), which was not detected in
the MS-base analyses of cyclopentene analogues. According to
the proposed mechanisms for 6 in Scheme 6, it is likely that
the incorporated double bond also plays a critical role in
stabilizing the conjugated system.
Assuming a quinonoid extinction coefficient of ∼30 mM−1
cm−1,9 the fractional accumulation of the quinonoid observed
is ∼20% of the total reacting species at 1 mM 6. The
quinonoid then decays at a rate of 0.09 s−1, while the residual
transitions assigned to the external aldimine species broaden
and persist (orange spectrum, Figure 8A,B). Quinonoid M6
seems to be a rare case of quinonoid species (∼560 nm) that
can be observed on the basis of the turnover (Pathway a;
Scheme 5) and inactivation (Pathway b; Scheme 6)
mechanisms. The final phase observed occurs with a rate
constant of 0.007 s−1; in this phase, the features of the external
aldimine decay with a pronounced increase in absorption
intensity at ∼275 nm, indicative of a loss of conjugation (blue
spectrum, Figure 8A,B), consistent with gem-diamine M16 and
ketimine M19, which are proposed as the final products in
Scheme 6.
Collectively, these data support a dominant pathway
composed of multiple distinct external aldimine species that
ultimately decay to a less conjugated product (turnover
mechanism; Pathways e then a to M10; Scheme 6) and a
second minor pathway that forms an initial external aldimine
more slowly but then proceeds through a quinonoid
intermediate and decays to also form a nonconjugated product
(inactivation mechanism; Pathways e-b-c and d to M16 and
M19, Scheme 6). The data shown in Figure 8C indicate that
the proportion of each pathway is dependent on the
concentration of 6. Higher concentrations of 6 diminish the
accumulation of the quinonoid species but do not alter the
observed rates of accumulation and decay, suggesting that the
more rapid and dominant pathway sequesters a larger fraction
of enzyme at higher 6 concentrations.
Significance of the Conjugated Alkene of 6. After
better understanding the inactivation and turnover mecha-
nisms of 6, we carried out computational calculations to
compare analogues 4−6. Our previous studies have revealed
that incorporating an extra double bond into the cyclopentane
ring system establishes an α,β-conjugated carboxylate and
facilitates the deprotonation step as a result of the increased
acidity of the adjunct proton, leading to an enhanced
inactivation rate constant.18,19 In this work, we performed
theoretical pKa calculations using the DFT/B3LYP method34
at 298 K to predict the proton’s acidity at the Cγ position of
difluoro analogues 4−6 (Figure 3). The results shown in
Figure 9A suggest that the hydrogen (highlighted with red) of
PLP-bound 6 (M1) with an α, β-conjugated carboxylate
system displays the lowest pKa value among the three
analogues, while the pKa value of the corresponding proton
in PLP-bound 5 (M1″) is not noticeably affected by the
introduced double bond at the Cα and Cε positions relative to
the parent cyclopentane 4 (M1′). Taken together with their
distinct kinact values (Table 1), our findings suggest that the
more acidic hydrogen at the Cγ position facilitates the first
external aldimine of 6 (M1), which initiates an E2 fluoride ion
elimination step rather than the typical E1cB elimination
reaction, thus contributing to its enhanced rate constant.
The partition ratio determination and fluoride ion release
results (Table 1) elucidated that 4 and 5 predominantly
CONCLUSION
■
Previously, we discovered (S)-3-amino-4,4-difluorocyclohex-1-
enecarboxylic acid (WZ-2-051, 2), which inactivates hOAT
through a covalent addition-aromatization mechanism
(Scheme 1 B). However, it also exhibited the apparent
inhibition of GABA-AT. In this work, (S)-3-amino-4,4-
difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) exhibited
comparable inactivation efficiency to that of preclinical stage
selective hOAT inactivator BisCF3 (1) but was demonstrated
to be a reversible inhibitor of GABA-AT. The kinetic studies
and computational calculations provide evidence to support
the notion that the conjugated alkene of 6 in its cyclopentene
ring is essential for retaining high hOAT inactivation efficiency.
A soaking experiment was performed to obtain a quasi-stable
gem-diamine intermediate covalently bound to Lys292 in the
soaked crystal, an intermediate that has never been captured in
other studies of related aminotransferase inactivators. The co-
crystallization of hOAT and 6 captured a stable noncovalent
final product in the hOAT co-crystal complex. The critical salt
bridge of Arg413-Glu235 in hOAT was found to be broken in
both crystal complexes. In addition, we have applied native
MS, for the first time, in studies of aminotransferase
inactivators to further support the noncovalent pathway as
the primary inactivation mechanism of 6. We also performed
intact MS to support a covalent modification observed as a
minor form, which appears to be a gem-diamine structure in
equilibrium with the noncovalent form. Using rapid-mixing
experiments, we observed, for the first time, that the first
external aldimine of 6 undergoes a lysine-assisted E2 fluoride
ion elimination instead of the typical E1cB elimination
mechanism, forming the second external aldimine as the single
transient state. Overall, we have carried out comprehensive
mechanistic studies to demonstrate that 6 mainly inactivates
hOAT through a noncovalent water-mediated mechanism.
However, it is still unclear why there are distinct inactivation
mechanisms for cyclopentene 6 and the corresponding
cyclohexene 2.
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Mauricio T. Tavares − Department of Molecular Medicine,
Scripps Research, Jupiter, Florida 33458, United States
Glaucio M. Ferreira − Department of Clinical and
Toxicological Analyses, School of Pharmaceutical Sciences,
̃ ̃
University of Sao Paulo, Sao Paulo, SP 05508-000, Brazil;
orcid.org/0000-0002-1952-9428
Neil L. Kelleher − Department of Chemistry, Center for
Molecular Innovation and Drug Discovery, and Center for
Developmental Therapeutics and Department of Molecular
Biosciences, Northwestern University, Evanston, Illinois
60208, United States; orcid.org/0000-0002-8815-3372
Graham R. Moran − Department of Chemistry and
Biochemistry, Loyola University Chicago, Chicago, Illinois
60660, United States; orcid.org/0000-0002-6807-1302
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02456.
■
sı
Figures of linear regression analysis, time-dependent
dialysis, Polder maps, MS spectra, Gibb’s free energy,
tautomeric forms, primary metabolites, NMR spectra,
and HRMS report, scheme of theoretical pKa calcu-
lations, tables of statistics of crystal structures, and
discussions of synthetic methods, expression and
purification of hOAT, aminotransferases and coenzymes
for kinetic studies, evaluation of compounds as time-
dependent inhibitors of hOAT, evaluation of com-
pounds as reversible inhibitors of GABA-AT, inhibition
of Asp-AT and Ala-AT by 6, dialysis assay, partition ratio
experiment, fluoride ion release assay, native protein
mass spectrometry, denaturing intact protein and small
molecule mass spectrometry, crystallization and crystal
soaking of hOAT with 6, transient state methods, Gibb’s
free energy calculation, theoretical pKa calculations, and
ESP charge calculation (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02456
Author Contributions
∇S.S. and A.B. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
Accession Codes
Atomic coordinates and corresponding structure factors for the
soaking result and co-crystal complex have been deposited at
the Protein Data Bank (PDB) as the 7LK1 and 7LK0 entries,
respectively. Authors will release the atomic coordinates upon
article publication.
ACKNOWLEDGMENTS
■
The authors are grateful to the National Institutes of Health
(Grant R01 DA030604 to R.B.S. and Grant P30 DA018310 to
N.L.K.) and National Science Foundation (Grant 2015210477
to P.F.D. and Grant 1904480 to G.R.M.) for financial support.
This work used the Extreme Science and Engineering
Discovery Environment (XSEDE) Comet Bridges Stampede2
through allocation TG-CHE190070, which is supported by
National Science Foundation grant number ACI-1548562.
This work made use of the IMSERC at Northwestern
University, which has received support from the Soft and
Hybrid Nanotechnology Experimental (SHyNE) Resource
(NSF NNCI-1542205), the State of Illinois, and the
International Institute for Nanotechnology (IIN). X-ray
diffraction data collection used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
financial support. Science User Facility was operated for the
DOE Office of Science by Argonne National Laboratory under
contract no. DEAC02-06CH11357. The use of LS-CAT Sector
21 was supported by the Michigan Economic Development
Corporation and the Michigan Technology Tri-Corridor
(grant 085P1000817). G.M.F. is a recipient of a fellowship
from FAPESP, Brazil. The authors would also like to thank
Drs. Wei Zhu and Pathum M. Weerawarna for constructive
comments and revision.
AUTHOR INFORMATION
Corresponding Authors
■
Dali Liu − Department of Chemistry and Biochemistry, Loyola
University Chicago, Chicago, Illinois 60660, United States;
orcid.org/0000-0002-7587-703X; Phone: +1-773-508-
3093; Email: dliu@luc.edu
Richard B. Silverman − Department of Chemistry, Center for
Molecular Innovation and Drug Discovery, and Center for
Developmental Therapeutics and Department of Molecular
Biosciences, Northwestern University, Evanston, Illinois
60208, United States; Department of Pharmacology,
Northwestern University, Chicago, Illinois 60611, United
States; orcid.org/0000-0001-9034-1084; Phone: +1-
847-491-5653; Email: r-silverman@northwestern.edu
Authors
Sida Shen − Department of Chemistry, Center for Molecular
Innovation and Drug Discovery, and Center for
Developmental Therapeutics, Northwestern University,
Evanston, Illinois 60208, United States; orcid.org/0000-
0002-0295-2545
Arseniy Butrin − Department of Chemistry and Biochemistry,
Loyola University Chicago, Chicago, Illinois 60660, United
States
Peter F. Doubleday − Department of Molecular Biosciences,
Northwestern University, Evanston, Illinois 60208, United
States; Present Address: P.F.D.: Institute of Molecular
Systems Biology, ETH Zurich, 8093 Zurich, Switzerland.
Rafael D. Melani − Department of Molecular Biosciences,
Northwestern University, Evanston, Illinois 60208, United
States
Brett A. Beaupre − Department of Chemistry and
Biochemistry, Loyola University Chicago, Chicago, Illinois
60660, United States
ABBREVIATIONS
■
AFPA, (S)-4-amino-5-fluoropentanoic acid; Boc2O, di-tert-
butyl dicarbonate; Deoxo-Fluor, bis(2-methoxyethyl)-
aminosulfur trifluoride; DMPK, drug metabolism and
pharmacokinetics; DIPEA, N,N-diisopropylethylamine;
DBDMH, 1,3-dibromo-5,5-dimethylhydantoin; DMF, dime-
thylformamide; DMSO, dimethyl sulfoxide; DCM, dichloro-
methane; KHMDS, potassium bis(trimethylsilyl)amide; PO,
per os administration; THF, tetrahydrofuran; TEA, triethyl-
amine; TBAI, tetra-n-butylammonium iodide
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gamma-aminobutyric acid aminotransferase. J. Med. Chem. 2000, 43,
706−720.
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