Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase

Article abstract of DOI:10.1021/acsmedchemlett.9b00672

Aminotransferases are pyridoxal 5′-phosphate-dependent enzymes that catalyze reversible transamination reactions between an amino acid and an α-keto acid, playing a critical role in cellular nitrogen metabolism. It is evident that γ-aminobutyric acid aminotransferase (GABA-AT), which balances the levels of inhibitory and excitatory neurotransmitters, has emerged as a promising therapeutic target for epilepsy and cocaine addiction based on mechanism-based inactivators (MBIs). In this work, we established an integrated approach using computational simulation, organic synthesis, biochemical evaluation, and mass spectrometry to facilitate our design and mechanistic studies of MBIs, which led to the identification of a new cyclopentene-based analogue (6a), 25-times more efficient as an inactivator of GABA-AT compared to the parent compound (1R,3S,4S)-3-amino-4-fluorocyclopentane carboxylic acid (FCP, 4).

Full text of DOI:10.1021/acsmedchemlett.9b00672

pubs.acs.org/acsmedchemlett
Letter
Mechanism-Based Design of 3‑Amino-4-
Halocyclopentenecarboxylic Acids as Inactivators of GABA
Aminotransferase
Sida Shen, Peter F. Doubleday, Pathum M. Weerawarna, Wei Zhu, Neil L. Kelleher,
and Richard B. Silverman*
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ABSTRACT: Aminotransferases are pyridoxal 5′-phosphate-de-
pendent enzymes that catalyze reversible transamination reactions
between an amino acid and an α-keto acid, playing a critical role in
cellular nitrogen metabolism. It is evident that γ-aminobutyric acid
aminotransferase (GABA-AT), which balances the levels of
inhibitory and excitatory neurotransmitters, has emerged as a
promising therapeutic target for epilepsy and cocaine addiction
based on mechanism-based inactivators (MBIs). In this work, we
established an integrated approach using computational simulation,
organic synthesis, biochemical evaluation, and mass spectrometry
to facilitate our design and mechanistic studies of MBIs, which led
to the identification of a new cyclopentene-based analogue (6a),
25-times more efficient as an inactivator of GABA-AT compared to
the parent compound (1R,3S,4S)-3-amino-4-fluorocyclopentane
carboxylic acid (FCP, 4).
KEYWORDS: GABA aminotransferase, cyclopentene, deprotonation, rate constant, inactivation efficiency
minotransferases (ATs) are essential enzymes that
catalyze two coupled transamination reactions between
suggesting that modulation of the deficient level of GABA in
the CNS might produce an anticonvulsant effect. Among
various approaches to increase the brain concentrations of
GABA (e.g., GABA prodrugs and glutamic acid decarboxylase
(GAD) activators),4 mechanism-based inactivators (MBIs) of
GABA-AT are attractive because of their unique inactivation
mechanisms and their successful advancement into preclinical/
clinical stages.4 Unlike other irreversible inhibitors, MBIs are
unreactive prior to conversion into an active species in the
catalytic site of the target enzyme, thus minimizing unwanted
off-target effects.6
Vigabatrin (1, Sabril, Figure 1B), a MBI of GABA-AT,
exhibits anticonvulsant activity and was approved by the FDA
in 2009 as an adjunctive therapy for refractory partial seizures.7
Mechanistic studies revealed that it irreversibly inhibits GABA-
AT by covalent modification through two different mecha-
nisms, a Michael addition pathway (70%) and an enamine
pathway (30%), leading to its anticonvulsant effect.8,9 It was
A
an amino acid and an α-keto acid, thus playing an important
role in nitrogen metabolism in cells. All ATs require pyridoxal
5′-phosphate (PLP) as a cofactor, which is linked to a basic
lysine residue in the catalytic pocket through a Schiff base, to
convert an amino acid into the corresponding carbonyl
compound with concomitant conversion of PLP into pyridox-
amine 5′-phosphate (PMP) in the first half-reaction. In the
second half-reaction, ATs catalyze the reaction of PMP with an
acceptor α-keto acid to perform another transfer of an amino
group, thereby converting PMP back to PLP.1,2 Recent
findings have demonstrated that pharmacological inhibition
of certain ATs (e.g., γ-aminobutyric acid AT and ornithine
AT) is a therapeutic strategy aimed to treat neurological
disorders and cancers, respectively.3,4
γ-Aminobutyric acid aminotransferase (GABA-AT, E.C.
2.6.1.19) catalyzes the degradation of the prime inhibitory
neurotransmitter GABA to succinic semialdehyde (SSA) with
the generation of the major excitatory neurotransmitter L-
glutamate (L-Glu) from α-ketoglutarate (α-KG) (Figure 1A).4
Normal functioning of the central nervous system (CNS)
requires well-balanced levels of inhibitory and excitatory
neurotransmitters; a reduction in the level of GABA has
been implicated in the symptoms associated with epilepsy,5
Special Issue: Medicinal Chemistry: From Targets to
Therapies
Received: December 30, 2019
Accepted: February 13, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00672
© XXXX American Chemical Society
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A

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(Arg192 and Arg445).14,15 CPP-115 has been investigated in a
Phase I clinical trial,10 as a compassionate use medication and
as a treatment for infantile spasms,16 while OV329 suppressed
the release of brain dopamine at a dose of 0.1 mg/kg in a rat
model of cocaine addiction.14 Therefore, mechanism-based
inactivation of GABA-AT has served as an effective approach
to discover novel therapeutic treatments for different neuro-
logical disorders.
In 2000, (1R,3S,4S)-3-amino-4-fluorocyclopentane carbox-
ylic acid (FCP, 4, Figure 1B) was reported as an inactivator of
GABA-AT.17 In 2004, crystallography with GABA-AT revealed
that FCP covalently modifies the Lys329-PLP linkage by
forming imine adduct M5 (Scheme 1) derived from an
enamine inactivation mechanism.18 The proposed inactivation
mechanism of FCP is initiated by FCP acting as a substrate to
form Schiff base M1 with PLP, followed by deprotonation
(M2) and subsequent elimination of fluoride ion to afford the
imine intermediate (M3). Subsequent Lys329 attack at the
imine moiety of M3 releases the enamine metabolite (M4) and
PLP is returned to Lys329. M4 covalently modifies the
Lys329−PLP complex to generate adduct M5 via an enamine
mechanism. However, except for the cocrystal structure, its
mechanism was not well supported. Therefore, at the
beginning of this work, we resynthesized FCP (Scheme S1)
and further elucidated its mechanisms of inactivation and
alternative turnover using mass spectrometry with the intent of
using this as a basis for new inactivator design.
Figure 1. Coupled transamination reactions of GABA-AT (A) and
structures of MBI representatives 1−5 (B).
In the present work, the kinetic constants for FCP against
GABA-AT (Table 1) indicate that FCP had a greater binding
also found to prevent cocaine addiction at a dose of 300 mg/kg
in a rat model.10 However, there are considerable concerns
regarding the permanent visual damage associated with long-
term vigabatrin administration, which results because of its low
inactivation efficiency and poor blood-brain barrier (BBB)
permeability, which demand high daily doses (1−3 g per day)
that eventually impair its clinical profile.11,12 Recent findings
identified cyclopentane-based analogue CPP-115 (2, Figure
1B) and cyclopentene-based analogue OV329 (3, Figure 1B),
which exhibit several hundred-fold improved inactivation
efficiency relative to vigabatrin.13,14 GABA-AT crystal
structures in complex with CPP-115/OV329 demonstrated
that their difluoromethylenyl groups are converted into a
carboxylate group in the binding site, and both compounds
inactivate the enzyme via tight electrostatic interactions
between the two carboxylate groups and two arginine residues
Table 1. Kinetic Constants of Analogues FCP and 6a−6c
a
with GABA-AT
k
inact/KI
cmpd
kinact (min−1
)
KI (mM)
(min−1mM−1
)
FCP (4)
6a
6b
6c
0.011 0.001
0.132 0.023
0.135 0.010
0.086 0.011
0.21 0.03
0.053 0.022
0.026 0.011
6.01 0.89
14.26 3.94
0.29 0.09
0.21
5.08
0.022
0.006
0.727
vigabatrin (1)
a
Data were collected from a time-dependent assay in duplicate
according to the experimental protocols in ref 14. kinact and KI values
were determined by the equation: kobs = kinact × [I]/(KI + [I]) and are
presented as means with standard errors.
a
Scheme 1. Inactivation and Turnover Mechanism of GABA-AT by FCP
a
Crystal structure (bottom right) of GABA-AT inactivated by FCP (green). Ligands and selected residues in GABA-AT are shown in stick
representation with atoms colored gray (carbon), red (oxygen), and blue (nitrogen). No PDB code was deposited for the GABA-AT crystal
structure, but we obtained coordinates from the authors.18
B
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a
Table 2. Intact Protein Mass Results of 6a−6c and FCP with GABA-AT
a
b
Data are presented as deconvoluted masses (in Daltons) with associated standard deviations around average protein masses. Mass shifts were
obtained by subtracting average native masses (no PLP attached) from average modified masses.
affinity (KI = 0.053 mM) toward GABA-AT but a lower
maximum rate of inactivation (kinact) against GABA-AT (0.011
min−1) than vigabatrin, which eventually led to a modest
inactivation efficiency, defined by the kinact/KI ratio (0.20
min−1 mM−1).19 It was reported that FCP exhibited a partition
ratio of 147 with GABA-AT, meaning that 148 equiv of FCP
are turned over per active site for each equivalent of compound
leading to inactivation.17,18 Moreover, 148 equiv of fluoride ion
are released per inactivation event, detected with a fluoride
ion-selective electrode,15 indicating that all turnovers require a
fluoride ion elimination reaction.17,18 Tandem mass spectrom-
etry in the present study of FCP with GABA-AT identified two
main metabolites, whose masses fit enamine metabolite M4 or
its imine tautomer M6 ([M + H]+; theoretical, 128.0706;
observed, 128.0705); subsequent hydrolysis gave ketone
metabolite M7 ([M − H]−; theoretical, 127.0401; observed,
127.0391), all with supporting MS2 fragmentation (Scheme 1).
According to the literature, FCP shows a different turnover
mechanism when incubated with aspartate aminotransferase,
affording a ketone metabolite without releasing fluoride ion
(S11 in Scheme S2).20 In the current study with GABA-AT,
we did not detect this metabolite. Intact protein mass
spectrometry of GABA-AT inactivated by FCP produced a
mass shift caused by covalent modification, which corre-
sponded to PLP-bound ketone M8 (Scheme 1; theoretical,
357.06 Da; observed, 357.27 Da; shown in Table 2). Ketone
M8 is the product generated from imine adduct M5 via
hydrolysis under liquid chromatography and mass spectrom-
etry conditions, thereby validating the crystal structure.
Therefore, high-resolution intact protein mass spectrometry
is an important additional approach to facilitate protein adduct
structure determination. The inactivation and alternative
turnover mechanisms of FCP are summarized in Scheme 1.
After deprotonation to M2, exclusive fluoride ion elimination
affords M3, which is attacked by Lys329 at the iminium group,
releasing enamine metabolite M4. Most of enamine M4
tautomerizes to the corresponding imine (M6), which
undergoes hydrolysis to ketone M7, while only a very minor
portion of M4 (0.7% of FCP according to its partition ratio)
inactivates the enzyme by enamine addition to PLP, forming
M5.
good potency against GABA-AT (KI = 0.053 mM), similar to
that of CPP-115 (KI = 0.059 mM),14 a MBI that completed a
successful Phase I clinical trial. However, the low maximal rate
constant (kinact = 0.011 min−1) of FCP with GABA-AT limits
its inactivation efficiency. As illustrated in Scheme 1, there are
three steps leading to inactivation: deprotonation (M1 to M2),
fluoride elimination (M2 to M3), and enamine addition (M4
to M5). The deprotonation step was suggested as the rate-
determining step rather than the cleavage of the carbon−
fluorine bond in the original FCP article.17 Our recent findings
revealed that OV329 (Figure 1B) was about 10 times more
efficient than CPP-115 as an inactivator of GABA-AT.14
Computational simulations suggested that the incorporation of
an endocyclic double bond into the scaffold of CPP-115 is able
to bring the difluoromethylenyl group closer to the Lys329
residue, which is responsible for the enhanced binding affinity
of OV329 (KI = 0.010 mM).14 Furthermore, the added double
bond led to a 1.5-fold enhancement of the kinact value.
Additionally, it was also reported that the incorporation of a
double bond into 4-amino-5-fluoropentanoic acid (5, Figure
1B), the open-chain analogue of FCP, improved potency
against GABA-AT with a comparable rate constant.21
To explore the effect of the double bond on the scaffold of
FCP, we initially conducted molecular docking studies to
predict the binding poses of PLP-bound ligands in the binding
site of GABA-AT and quantum mechanical cluster calculations
to investigate the reaction profiles of the deprotonation steps.22
The docking pose comparison of M1 and M1′ (Figure 2A)
suggests that by incorporation of a double bond does not
change the salt bridge interactions between Arg192 and the
carboxylate group (Figure 2B) and retains the PLP-bound
ligand in a similar distance to Lys329, which abstracts the
adjacent proton (highlighted with red in Figure 2A). It should
be noted that the conformational change does bring the
fluorine atom closer to Lys329 (from 4.6 to 3.5 Å) (Figure
2C). Quantum mechanical cluster calculations of the
deprotonation step catalyzed by Lys329 (Figure 2D)
demonstrate that the ligand-PLP Schiff base M1′, containing
the added double bond, displays about a 3 kcal/mol lower
transition state (TS) energy in going to M2′ compared with
intermediate M1, generated from FCP, giving M2. This
indicates that deprotonation of M1′ is much easier than of M1.
The α,β-unsaturated carboxylate of M1′ leads to a lower pKa
With the mechanisms of FCP clarified, we directed our
attention to improving its inactivation efficiency. FCP exhibits
C
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a
Scheme 2. Synthetic Route to 6a
a
Reagents and conditions: (a) (i) p-anisyl alcohol, conc. HCl, rt; (ii)
NaH, TBAI, THF/DMF (10:1), 0 °C−rt; (b) m-CPBA, CHCl3,
reflux; (c) BF3·OEt2, AcOH, DCM, rt; (d) MOMCl, DIPEA, DCM,
rt; (e) K2CO3, MeOH/H2O, rt; (f) DAST, DCM, −78 °C−rt; (g) 6
N HCl, THF, 80 °C; (h) (i) TsCl, DIPEA, DMAP, CH3CN, rt, (ii)
ceric ammonium nitrate, CH3CN/H2O, rt; (i) (i) Boc2O, DIPEA,
DMAP, DCM, rt, (ii) K2CO3, MeOH, rt; (j) 4 N HCl, AcOH, 70 °C.
intermediate (10), containing a hydroxyl group on the
bridgehead, whose structure was confirmed by 2D NMR
spectrometry. Methoxymethyl (MOM) protection (11),
deacylation (12), and fluorination with DAST led to the
corresponding fluorinated bicycle (13) as a single diaster-
eomer. The full retention of relative configuration results from
the formation of a transient aziridinium intermediate.24
Subsequent MOM deprotection (14) and tosylation with
PMB deprotection afforded lactam 15, containing the tosylate
group on the bridgehead to act as a leaving group in the next
step. Introduction of a tert-butyloxycarbonyl (Boc) protecting
group on the lactam nitrogen of 15, followed by a one-pot
hydrolysis of the lactam and elimination of the tosylate with
K2CO3/MeOH afforded the desired cyclopentene key
intermediate (16). Removal of the Boc group and methyl
ester under acidic conditions afforded the final product (6a) as
a HCl salt, whose structure was confirmed by 1H, 13C, and 2D
NMR spectrometries.
To prepare two other cyclopentene analogues (6b and 6c),
bearing chlorine and bromine atoms, respectively, the synthetic
routes were initiated from tosylation (17) and PMB
deprotection of 10 to afford 18 (Scheme 3). After Boc
protection (19), a one-pot hydrolysis and elimination was
carried out with K2CO3/MeOH to obtain the desired
cyclopentene (20). Intermediate 20 was successfully converted
to the desired monochloro- (21a) or monobromo- (21b)
substituted compound using NCS or NBS, respectively; the
sole diastereomers formed may have been facilitated by
neighboring group participation in the halogenation step.17,26
Final cyclopentene products 6b and 6c were obtained as HCl
salts after deprotection.
Figure 2. Computational simulation results of PLP-bound ligands M1
and M1′. (A) The deprotonation reactions of PLP-bound ligand M1/
M1′ to M2/M2′. (B,C) Superimposed docking poses of M1 (purple)
and M1′ (cyan) with GABA-AT. Ligands and selected residues in
GABA-AT are shown in stick representation with atoms colored gray
(carbon), red (oxygen), orange (phosphorus), blue (nitrogen), and
light cyan (fluorine). (D) Reaction profile of the deprotonation
reactions of M1 to M2 (green) and M1′ to M2′ (blue) in aqueous
phase, calculating at a B3LYP/6-31+G(d,p) level of theory.
value for the adjacent proton, relative to that in M1, which
leads to a more efficient deprotonation step. As deprotonation
is considered the rate-determining step, we hypothesized that
the lower TS energy from M1′ to M2′ should improve the
overall inactivation rate, thereby enhancing the kinact value.
To assess the actual effect of this double bond, we
synthesized and evaluated several cyclopentene analogues
bearing different halogens. The synthetic route to prepare
cyclopentene compound 6a bearing a fluorine atom (Scheme
2) was initiated with (1R)-(−)-2-azabicyclo[2.2.1]hept-5-en-3-
one (7), which was protected with a p-methoxybenzyl (PMB)
group (8) and epoxidized with m-CPBA (9). Epoxide
rearrangement17,23−25 and selective acylation occurred under
BF3·OEt2/AcOH conditions to afford the bicyclic key
The kinetic constants shown in Table 1 indicate that (a)
Compared to FCP, new cyclopentene analogues 6a−6c
exhibited about a 10-fold enhancement of their rate constants
with GABA-AT. (b) Chloro- (6b) and bromo-substituted (6c)
analogues showed much lower binding affinities to GABA-AT,
D
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a
Scheme 3. Synthetic Route to 6b and 6c
Scheme 4. Inactivation and Turnover Mechanism of GABA-
AT by 6a
a
Reagents and conditions: (a) TsCl, DIPEA, DMAP, CH3CN, rt; (b)
ceric ammonium nitrate, CH3CN/H2O, rt; (c) Boc2O, DIPEA, DCM,
rt; (d) K2CO3, MeOH, rt; (e) NBS or NCS, Ph3P, DMF, rt; (f) 4 N
HCl, AcOH, 70 °C.
corresponding to the larger sizes of the halogens, while the
fluoro-substituted analogue (6a) showed a 2-fold enhancement
of binding affinity compared to FCP, thereby establishing that
6a is about 25-times more efficient than FCP as an inactivator
of GABA-AT. The correspondence of the inactivation rate
constants for 6a−6c supports the notion that the rate-
determining step is deprotonation rather than carbon−halogen
bond cleavage.
The irreversibility of the inhibition of GABA-AT by 6a was
assessed by dialysis experiment. After 4 days of dialysis against
buffer containing excess PLP and α-KG, there was no
regeneration of enzyme activity, confirming that 6a is an
inactivator of GABA-AT (Figure S1A). Consequently, we
investigated the potential adduct structures generated from
6a−6c using intact protein mass spectrometry. The results
shown in Table 2 demonstrate that 6a−6c, bearing different
halogens, resulted in identical molecular mass shifts after
inactivation of GABA-AT, which match the ketone−PLP
adduct structure (M6′ in Scheme 4; calculated, 355.05 Da;
found, 354.02−355.42 Da) generated from an enamine
addition pathway. Hydrolysis of the imine in M5′ to M6′
might have occurred during chromatography and mass
spectrometry. This suggests that they all covalently bind to
GABA-AT through the same mechanism as FCP (Scheme 1)
after forming M3′ via halide ion elimination (Scheme 4).
Superimposed docking poses in Figure 3A of 6a−6c exhibit
similar binding poses as FCP after forming PLP-bound
intermediates, such as M1′. Docking studies of FCP and
6a−6c in the binding site of GABA-AT show that the amino
groups of FCP and 6a display similar orientations toward the
Lys329-PLP linkage (Figure 3B), which is beneficial for the
reaction with the PLP ligand in the initial binding step, while
the amino groups of 6b and 6c orient away from the Lys329-
bound PLP (Figure 3C). These modeling results suggest that
the initial binding step with Lys329-PLP may play the most
significant role in influencing their binding affinity, leading to
the distinct KI values.
H]−; calculated, 125.0244; found, 125.0234), generated by
fluoride ion elimination and enamine hydrolysis, as the major
metabolite with its confirmatory MS2 fragmentation pattern.
No ketone metabolite containing a fluorine atom was detected,
indicating that simple hydrolysis of M2′ does not occur.
The inactivation and turnover mechanism for 6a are
proposed in Scheme 4. After capturing the PLP ligand from
Lys329, PLP-bound M1′ undergoes deprotonation and
fluoride ion elimination to form the imine intermediate
(M3′). According to the enamine pathway, Lys329 attacks
the imine moiety of M3′, which leads to the release of enamine
metabolite M4′, mostly hydrolyzing to M7′. A small amount of
M4′ (2.2% of 6a according to its partition ratio) attaches to
the Lys329-PLP linkage, forming M5′, and inactivates the
enzyme in a manner similar to that by FCP. Compared to
original inactivator FCP, incorporation of the extra double
bond decreases the partition ratio (147 for FCP18 vs 44 for 6a)
and leads to a 2-fold greater binding affinity with reduced
transition state energy for the rate-determining deprotonation
step, which is responsible for a 12-fold enhancement in the rate
constant, making 6a 25-fold more efficient as an inactivator of
GABA-AT than FCP.
We also assessed the selectivity of 6a over other ATs,
including ornithine aminotransferase (Orn-AT), aspartate
aminotransferase (Asp-AT), and alanine aminotransferase
(Ala-AT). In a concentration-dependent assay,14 6a showed
no inhibitory effect on Asp-AT or Ala-AT up to a
concentration of 10 mM. In a time-dependent assay against
The partition ratio for 6a was determined to be 44, and 44
equiv of fluoride ions were release per inactivation event
(Figure S1B), indicating that 97.8% of 6a underwent fluoride
ion elimination per inactivation event. Tandem mass spectra of
6a with GABA-AT identified ketone M7′ (Scheme 4) ([M −
Orn-AT,27 6a displayed a comparable rate constant (kinact
=
0.143 0.014 min−1) but 10-fold weaker binding affinity (KI =
0.25 0.06 mM) relative to its effect with GABA-AT.
E
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Letter
Authors
Sida Shen − Department of Chemistry, Chemistry of Life
Processes Institute, 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
Peter F. Doubleday − Department of Molecular Biosciences,
Northwestern University, Evanston, Illinois 60208, United
States
Pathum M. Weerawarna − Department of Chemistry,
Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, and Center for Developmental
Therapeutics, Northwestern University, Evanston, Illinois
60208, United States
Wei Zhu − Department of Chemistry, Chemistry of Life Processes
Institute, Center for Molecular Innovation and Drug Discovery,
and Center for Developmental Therapeutics, Northwestern
University, Evanston, Illinois 60208, United States;
orcid.org/0000-0003-0197-9911
Neil L. Kelleher − Department of Chemistry, Chemistry of Life
Processes Institute, 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
Figure 3. Superimposed docking poses of FCP (blue), 6a (cyan), 6b
(yellow), and 6c (pink) in Schiff bases with PLP from GABA-AT (A).
Docking study results for FCP (blue) and 6a (cyan) prior to reaction
with Lys329-PLP (B). Docking study results for 6b (yellow) and 6c
(pink) prior to reaction with Lys329-PLP (C). Ligands and selected
residues in GABA-AT are shown in stick representation with atoms
colored gray (carbon), red (oxygen), orange (phosphorus), blue
(nitrogen), light cyan (fluorine), green (chlorine), and brown
(bromine).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00672
Author Contributions
S.S. designed the molecules, carried out most of the
experiments, and wrote the initial draft of the manuscript;
P.F.D. performed all of the mass spectral experiments and
interpreted the data; P.M.W. carried out quantum chemical
cluster calculations; W.Z. conducted molecular docking
studies; N.L.K. directed the mass spectrometry experiments;
and R.B.S. directed the medicinal chemistry work and edited
the drafts of the manuscript. All authors have read and edited
the manuscript and have given their approval of the final
version.
In summary, we have established a GABA-AT MBI
discovery strategy using computational simulation, organic
synthesis, mechanistic enzymology, and intact protein- and
small molecule mass spectrometry. These approaches allowed
us to better understand inactivation and alternative turnover
mechanisms of a known inactivator, FCP. This strategy
facilitated the design of new MBIs, leading to the identification
of compound 6a as a more efficient inactivator of GABA-AT
than FCP.
Funding
This research was supported by NIH grants (Grant R01
DA030604 to R.B.S. and Grant P30 DA018310 to N.L.K.) and
NSF grant (Grant 2015210477 to P.F.D.). This work made use
of the IMSERC at Northwestern University, which has
received support from the Soft and Hybrid Nanotechnology
Experimental (SHyNE) Resource (NSF Grant NNCI-
1542205), the State of Illinois, and the International Institute
for Nanotechnology (IIN).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00672.
Notes
Supplementary figures and schemes and details of
methods, syntheses, and spectra (PDF)
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
AUTHOR INFORMATION
Corresponding Author
We thank our collaborators Dr. Dali Liu and Arseniy Butrin at
Loyola University Chicago for providing purified ornithine
aminotransferase.
■
Richard B. Silverman − Department of Chemistry, Chemistry of
Life Processes Institute, Center for Molecular Innovation and
Drug Discovery, and Center for Developmental Therapeutics,
Department of Molecular Biosciences, and Department of
Pharmacology, Northwestern University, Evanston, Illinois
60208, United States; orcid.org/0000-0001-9034-1084;
Phone: +1-847-491-5653; Email: r-silverman@
northwestern.edu
ABBREVIATIONS
■
MS, mass spectrometry; MS2, tandem mass spectrometry; m-
CPBA, meta-chloroperoxybenzoic acid; DAST, diethylamino-
sulfur trifluoride; TBAI, tetra-n-butylammonium iodide; THF,
tetrahydrofuran; DMF, N,N-dimethylformamide; DIPEA, N,N-
diisopropylethylamine; DCM, dichloromethane; Ts, tosyl;
F
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ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
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DMAP, 4-dimethylaminopyridine; NCS, N-chlorosuccinimide;
NBS, N-bromosuccinimide.
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