Binding of α,α-disubstituted amino acids to arginase suggests new avenues for inhibitor design

Article abstract of DOI:10.1021/jm200443b

Arginase is a binuclear manganese metalloenzyme that hydrolyzes l-arginine to form l-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordin

Full text of DOI:10.1021/jm200443b

ARTICLE
pubs.acs.org/jmc
Binding of r,r-Disubstituted Amino Acids to Arginase Suggests
New Avenues for Inhibitor Design†
Monica Ilies,‡ Luigi Di Costanzo,§ Daniel P. Dowling,|| Katherine J. Thorn,^ and David W. Christianson*,#
‡Department of Chemistry, Drexel University, Philadelphia, Pennsylvania 19104-2875, United States
§RCSB Protein Data Bank, Department of Chemistry and Chemical Biology, Rutgers The State University of New Jersey, Piscataway,
New Jersey 08854-8087, United States
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, United States
^The Episcopal Academy, Newtown Square, Pennsylvania 19073, United States
#Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6323 United States
ABSTRACT: Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form
L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile
dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be
therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design
and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of R,
R-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-
methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I
and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the
additional R-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active
site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity
interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.
’ INTRODUCTION
cytokines such as interleukin-13,23ꢀ27 and arginase I and arginase
II single-nucleotide polymorphisms are identified in atopic
asthma.28,29 Arginase activity contributes to asthma pathology
through three metabolic functions:30ꢀ32 (1) arginase compro-
mises NO-dependent relaxation of airway smooth muscle, leading
to bronchoconstriction; (2) arginase enhances L-proline and
collagen biosynthesis, leading to the accumulation of fibrotic
tissue in the chronic asthmatic airway; (3) arginase enhances
polyamine biosynthesis, which stimulates cellular proliferation
and hyperplasia, e.g., of airway smooth muscle cells, in the
asthmatic airway. These disease manifestations can be blocked
by the use of arginase inhibitors in ex vivo and in vivo ex-
periments.15,21,22,33ꢀ35 Thus, the arginase isozymes are increas-
ingly considered as vital pharmaceutical targets for the treatment
of erectile dysfunction, asthma, and cardiovascular diseases linked
to aberrant arginase activity, such as atherosclerosis.36
The first high-affinity inhibitor of arginase to be reported was
the boronic acid analogue of L-arginine, 2-(S)-amino-6-borono-
hexanoic acid (ABH, Figure 1),37 which binds to human arginase
I (HAI) with Kd = 5 nM and human arginase II with Ki = 8.5
nM.38,39 ABH is also the most potent inhibitor known for
Plasmodium falciparum arginase (PFA), to which it binds more
weakly with Kd = 11 μM.40 The X-ray crystal structures of HAI
and rat arginase I complexed with ABH,15,38 as well as the
Arginase is a ubiquitous manganese metalloenzyme that cata-
lyzes the hydrolysis of L-arginine to form L-ornithine and urea. In
mammals, two isozymes have been identified with distinct tissue
distributions and subcellular localizations.1ꢀ5 Arginase I is a
cytosolic enzyme found predominantly in the liver, and arginase
II is a mitochondrial enzyme found at highest concentrations in
the kidney.6ꢀ9 However, both isozymes are constitutively ex-
pressed or induced in other tissues to regulate cationic amino acid
homeostasis in three critical metabolic pathways: (1) the regula-
tion of L-arginine levels for nitric oxide (NO) biosynthesis;10ꢀ15
(2) the regulation of L-ornithine levels for L-proline biosynthesis
to promote collagen production;16,17 (3) the regulation of
L-ornithine levels for polyamine biosynthesis to facilitate cellular
proliferation.2,18ꢀ20
Significantly, expression of arginase I and/or arginase II is up-
regulated in certain diseased tissues and cell types. For example,
given that arginase competes with NO synthase for their common
substrate L-arginine, aberrant arginase II activity in the penile
corpus cavernosum of the diabetic male attenuates NO biosynth-
esis and therefore compromises the NO-dependent relaxation of
cavernosal smooth muscle required for penile erection.21 More-
over, arginase I levels in the corpus cavernosum increase with age
and similarly compromise penile erection.22 Thus, erectile dys-
function can result from the up-regulation of either arginase I or
arginase II in different etiologies of the same disease. In another
example, arginase I is up-regulated in the asthmatic lung by Th2
Received: April 13, 2011
Published: July 05, 2011
r
2011 American Chemical Society
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avenues toward the development of R,R-disubstituted amino
acids as arginase inhibitors.
’ RESULTS
HAIꢀDFMO Complex. While DFMO is reported47 to be a
weak inhibitor of arginase activity in human colon carcinoma
cells with Ki = 3.9 mM, and while racemic DFMO was used in
crystal soaking experiments with HAI, the omit map in Figure 2a
clearly shows that the L-stereoisoimer of DFMO is bound
exclusively with full occupancy. This reflects the stereoselectivity
of the HAI active site for the binding of L-amino acids; e.g.,
L-arginine is a substrate for arginase, whereas D-arginine is not.48
Inhibitor binding does not cause any significant conformational
changes in the active site or elsewhere in the protein structure,
and the rms deviation is 0.26 Å for 314 CR atoms in comparison
with the unliganded enzyme. As for DFMO, the catalytic product
L-ornithine binds in the HAI active site without causing any
significant conformational changes. The rms deviation is 0.22 Å
for 314 CR atoms in comparison with the unliganded enzyme.
An omit map showing the binding of L-ornithine is found in
Figure 2b. Comparison of the two complexes reveals a generally
similar binding mode for DFMO and L-ornithine (Figure 2c).
Direct and water-mediated hydrogen bonds with the R-amino
and R-carboxylate groups are identical in each complex. These
interactions are crucial affinity determinants,43 and R,R-disub-
stitution clearly does not perturb them.
Chemistry. Since the active site of HAI readily accommodated
the additional R-substituent of DFMO without perturbing the
binding interactions or conformation observed for L-ornithine, we
synthesized the corresponding R,R-disubstituted derivatives of the
archetype HAI inhibitor, ABH (MABH and FABH; see Figure 1).
As shown in Scheme 1, the key intermediate in the syntheses of
MABH and FABH was ethyl 2-N-(diphenylmethyleneamino)hex-
5-enoate (2). While this compound was previously reported,49 we
developed an alternative synthetic pathway with a comparable
overall yield. We synthesized 2 from the commercially available
N-(diphenylmethylene)glycine ethyl ester (1), using the classic
mild deprotonation with potassium tert-butoxide (tBuOK),50,51
followed by alkylation with 4-bromo-1-butene. The use of benzo-
phenone imines of glycine alkyl esters as precursors of R-amino
acids is a well-known52 synthetic strategy to ensure selective
monoalkylation of the amino acid CR atom. We employed the
same mild base (tBuOK) to further deprotonate the CR in the
resulting derivative 2. Subsequent one-pot reaction with methyl
iodide and chlorodifluoromethane53 respectively yielded the cor-
responding methyl (3) and difluoromethyl (4) derivatives in good
yields as new compounds (Scheme 1). Hydroboration with
pinacolborane catalyzed by complexes of cyclooctadiene iridium
chloride dimer, [Ir(cod)Cl]2, with 1,1-bis(diphenylphosphino)-
methane (dppm)54 introduced the synthetic equivalent of the
boronic synthon in the terminal olefins 3 and 4, correspondingly
generating the intermediates 5 and 6. Complete deprotection of
derivatives 5 and 6 with 6 N HCl in tetrahydrofuran (THF) led to
the targeted compounds MABH (7) and FABH (8), respectively.
Binding Affinities of MABH and FABH. Dissociation con-
stants for the S-isomer of ABH and racemic MABH and FABH
against HAI determined by surface plasmon resonance (Figure 3)
and inhibition constants against PFA determined by kinetic assay
are recorded in Table 1. Curiously, both R,R-disubstituted
derivatives of ABH exhibit weaker affinity compared with ABH.
For HAI, MABH and FABH bind 49-fold and 1889-fold less
Figure 1. The amino acid substrate and product of the reaction
catalyzed by arginase, L-arginine and L-ornithine respectively, are com-
pared with DFMO and the boronic acid inhibitors BEC, ABH, MABH,
and FABH.
structure of the PFAꢀABH complex,40 reveal that the boronic
acid side chain undergoes nucleophilic attack by the metal-
bridging hydroxide ion observed in the unliganded enzyme41,42
to yield a tetrahedral boronate anion that mimics the tetrahedral
intermediate and its flanking transition states in catalysis. The
binding of the ABH analogue S-(2-boronoethyl)-L-cysteine
(BEC, Figure 1) to HAI and human arginase II occurs through
an identical mechanism.33,38
Structural comparisons of arginaseꢀABH and ꢀBEC com-
plexes reveal a conserved hydrogen bond network responsible for
the molecular recognition of the inhibitor R-amino and R-
carboxylate groups; these interactions contribute significantly
to enzymeꢀinhibitor affinity, as demonstrated in recent muta-
genesis studies with rat arginase I.43 Given that ABH analogues in
which the side chain is modified exhibit radically diminished
affinity,37,44,45 there is only one remaining possibility for the
derivatization of the ABH scaffold to yield new inhibitors: the
amino acid CR-H atom, which in the enzymeꢀinhibitor complex
is solvent exposed and oriented toward a region of the protein
surface that is currently “uncharted” with regard to inhibitor
binding. The design and synthesis of R,R-disubstituted amino
acids based on the high-affinity ABH scaffold (Figure 1) may thus
enable the generation and development of a new class of arginase
inhibitors to expedite the search for new arginase-directed
therapies. Notably, such therapies may target indications beyond
those outlined above. For instance, the inhibition of PFA has
been recently proposed as a potential adjuvant therapy for
accelerating the recovery of malaria patients.40,46
We now report the first study of the binding of R,R-disubsti-
tuted amino acids to arginase. We demonstrate initial proof-of-
principle with the X-ray crystal structures of HAI complexed with
2-(difluoromethyl)-L-ornithine (DFMO) and L-ornithine to
show that an additional R-substituent does not perturb the
intermolecular interactions of the amino acid product of the
arginase reaction. We then describe the design, synthesis, and
assay of two new R,R-disubstituted derivatives of ABH: 2-amino-
6-borono-2-methylhexanoic acid (MABH) and 2-amino-6-bor-
ono-2-(difluoromethyl)hexanoic acid (FABH) (Figure 1). X-ray
crystal structures of their complexes with HAI and PFA, together
with in vitro evaluations of inhibitory potency, illuminate new
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Figure 2. (a) Simulated annealing omit electron density map of the HAIꢀDFMO complex (cyan, contoured at 3.0σ), in which DFMO was omitted
from the structure factor calculation. Atoms are color-coded as follows: C = yellow, O = red, N = blue, F = black; Mn2+ ions and water molecules are
purple and red spheres, respectively. Metal coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively. (b)
Simulated annealing omit electron density map of the HAIꢀL-ornithine complex (cyan, contoured at 3.3σ). Atoms are color-coded as in (a). (c)
Superposition of the HAIꢀDFMO complex (red) and the HAIꢀL-ornithine complex (blue).
tightly, respectively; for PFA, MABH and FABH exhibit inhibi-
tory potencies based on Ki that are 26-fold and 200-fold weaker
than that observed for ABH. The structural basis for affinity loss is
not immediately evident for the binding of R,R-disubstituted
amino acids to HAI based on crystal structures of enzymeꢀinhibi-
tor complexes; however, that only the L-stereoisomer of each
inhibitor binds indicates that the affinity losses are only one-half
those measured for the racemic mixture (vide infra). Even so, it is
quite possible that affinity can be recaptured and enhanced with
longer R-substituents capable of making favorable interactions in
the active site. In contrast with HAIꢀinhibitor complexes, crystal
structures of PFAꢀinhibitor complexes reveal intersubunit inter-
actions with the R-substituents of MABH and FABH.
Crystal Structures of HAIꢀMABH and HAIꢀFABH Com-
plexes. The 1.60 Å resolution crystal structure of the
HAIꢀMABH complex reveals that inhibitor binding does not
cause any significant conformational changes in the active site or
elsewhere in the protein structure. The rms deviation is 0.24 Å for
313 CR atoms in comparison with the HAIꢀABH complex. The
electron density of MABH is well-defined and clearly indicates
that the L-stereoisomer of racemic MABH used in cocrystalliza-
tion experiments is exclusively selected for binding in the HAI
active site (Figure 4a). Superposition with the 1.29 Å resolution
crystal structure of human arginase IꢀABH complex shows
excellent overlap (Figure 4c), and enzymeꢀinhibitor hydrogen
bonds are identical in both complexes.
The 1.7 Å resolution electron density map of the HAIꢀFABH
complex similarly reveals the exclusive binding of the L-stereo-
isomer of racemic FABH used in cocrystallization experiments
(Figure 4b). Inhibitor binding does not cause any significant
overall conformational changes in the HAI structure, and the rms
deviation for 313 CR atoms is 0.29 Å when compared with the
structure of the HAIꢀABH complex. However, each fluorine
atom of the R-difluoromethyl group accepts hydrogen bonds
from solvent molecules, which in turn donate hydrogen bonds to
D183; additionally, it appears that T136 undergoes a 128ꢀ
conformational change about side chain torsion angle χ1 to
accommodate the binding of one of these solvent molecules
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Scheme 1. Synthesis of 2-Amino-6-borono-2-methylhexanoic Acid (MABH) and 2-Amino-6-borono-2-difluoromethylhexanoic
Acid (FABH)
Figure 3. Surface plasmon resonance sensorgrams for HAI showing the binding of (a) ABH, Kd = 18 ( 1 nM, (b) MABH, Kd = 0.88 ( 0.02 μM, and (c)
FABH, Kd = 34 ( 2 μM.
(Figure 4c). Enzymeꢀinhibitor hydrogen bond interactions are
otherwise identical to those observed in the HAIꢀABH and
HAIꢀMABH complexes.
HAIꢀFABH complex (Figure 4). Attempted refinement with the
tetrahedral boronic acid form of each inhibitor resulted in
negative electron density peaks on both the boron atom and
the adjacent carbon atom. Such spurious peaks are not observed
when each inhibitor is refined with an intact boronic acid moiety.
Clear electron density for each inhibitor shows that a planar
boronic acid moiety binds to preclude the binding of the metal-
bridging solvent molecule typically observed in unliganded
arginase structures.41,42 Electron density for a water molecule
coordinated to MnA2+ is ∼2 Å from the boron atom of MABH
and FABH (e.g., see Figure 5b). Thus, MABH and FABH do not
bind as transition state analogues. Moreover, neither inhibitor
Crystal Structures of PFAꢀMABH and PFAꢀFABH Com-
plexes. The 1.9 Å resolution crystal structure of the PFAꢀ
MABH complex and the 2.0 Å resolution crystal structure of the
PFAꢀFABH complex (Figure 5) each reveal the exclusive
binding of the L-stereoisomer from the racemic inhibitor mixtures
used in cocrystallization experiments. Surprisingly, however, each
inhibitor binds to PFA with an intact boronic acid side chain; i.e.,
neither inhibitor binds in the tetrahedral boronate anion form as
observed in the PFAꢀABH complex40 or in the HAIꢀMABH or
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binds as a true substrate analogue, since the trigonal planar
boronic acid moiety of each inhibitor adopts a binding conforma-
tion with a CδꢀCεꢀBζꢀOη1 dihedral angle of ∼90ꢀ, which is
very different from the corresponding CδꢀNεꢀCζꢀNη1 dihe-
dral angle of 180ꢀ required by the planar guanidinium moiety of L-
arginine. This structural aberration may contribute to the weaker
affinity of MABH and FABH compared to ABH.
Inhibitor binding to PFA generally does not cause any signifi-
cant conformational changes in the protein structure. However,
active site solvent structure is slightly different in that the binding
of R,R-disubstituted amino acids displaces a water molecule that is
observed in the PFAꢀABH complex (Figure 5d). In comparison
with the PFAꢀABH complex, the rms deviations are 0.10 Å for
308 CR atoms and 0.12 Å for 308 CR atoms for the PFAꢀMABH
and PFAꢀFABH complexes, respectively. However, some local
structural changes accommodate the binding of R,R-disubstituted
amino acids. In particular, an alternative conformation with 50%
occupancy is observed for D272 in the PFAꢀMABH complex;
this residue hydrogen bonds with H381 in the PFAꢀABH
complex. The R-methyl group of MABH is 3.0 Å from the Nε2
atom of H381, and H381 rotates ∼5ꢀ about side chain torsion
angle χ1, so this van der Waals contact may destabilize the
H381ꢀD272 interaction. Higher thermal B factors are observed
for this portion of the inhibitor and the side chain of H381,
suggesting that there is some residual disorder in this region of the
enzymeꢀinhibitor complex.
Table 1. r,r-Disubstituted Amino Acids Synthesized as In-
hibitors of Human Arginase I (HAI) and Plasmodium falcip-
arum Arginase (PFA)
Inhibitor binding conformations are very similar in PFA
complexes with MABH and FABH (Figure 5d). Interestingly,
the R-difluoromethyl group of FABH does not trigger the
conformational change of D272 as observed in the PFAꢀMABH
complex. H381 undergoes a slightly greater conformational
change (∼12ꢀ) than that observed in the PFAꢀMABH complex,
perhaps due to a hydrogen bond interaction with one of the
inhibitor
HAI, Kd (μM)a
PFA, Ki (μM)b
ABH (S-isomer)
MABH (racemic)d
FABH (racemic)d
0.018 ( 0.001
0.88 ( 0.02
34 ( 2
10 ( 1c
260 ( 20
2000 ( 200
fluorine atoms of FABH (F N separation of 2.6 Å). However,
a Surface plasmon resonance. Errors are standard deviations of experi-
ments run in triplicate. b Kinetic colorimetric assay. Errors are standard
deviations of experiments run in triplicate. c From ref 40. d Since only the
L-stereoisomer is observed to bind in each crystal structure, the actual Kd
or Ki for the inhibitory stereoisomer is likely to be one-half that
measured for the racemic mixture.
3 3 3
as observed in the PFAꢀMABH complex, higher thermal B
factors characterize this portion of the inhibitor and the side
chain of H381, suggestive of some residual disorder in this region
of the enzymeꢀinhibitor complex. Such residual disorder may
compromise enzymeꢀinhibitor affinity.
Figure 4. (a) Simulated annealing omit electron density map of the HAIꢀMABH complex (cyan, contoured at 3.8σ), in which MABH was omitted
from the structure factor calculation. Atoms are color-coded as follows: C = yellow, O = red, N = blue, B = green; Mn2+ ions and water molecules are
purple and red spheres, respectively. Metal coordination and hydrogen bond interactions are indicated by red and green dashed lines, respectively. (b)
Simulated annealing omit electron density map of the HAIꢀFABH complex (cyan, contoured at 3.0σ), in which FABH was omitted from the structure
factor calculation. Atoms are color-coded as in (a), with F = black. (c) Superposition of the HAIꢀABH complex (blue), HAIꢀMABH complex (red),
and HAIꢀFABH complex (green).
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Figure 5. (a) Simulated annealing omit electron density map of the PFAꢀMABH complex (cyan, contoured at 3.5σ), in which MABH was omitted
from the structure factor calculation. Atoms are color-coded as follows: C = yellow (C = gray for H381 from the adjacent monomer), O = red, N = blue,
B = green; Mn2+ ions and water molecules are purple and red spheres, respectively. Red and green dashed lines indicate manganese coordination and
hydrogen bond interactions, respectively. (b) Simulated annealing omit electron density map of the PFAꢀMABH complex (cyan, contoured at 3.5σ), in
which MABH and the Mn2+A-bound water molecule were omitted from the structure factor calculation. The view is zoomed in on the binuclear
manganese cluster and oriented differently from that in (a) to clearly show that the electron density is most consistent with the binding of a trigonal
planar boronic acid moiety and a separate Mn2+A-bound water molecule. Similar electron density characterizes the PFAꢀFABH complex (data not
shown). Atoms are color-coded as in (a). (c) Simulated annealing omit electron density map of the PFAꢀFABH complex (cyan, contoured at 3.5σ), in
which FABH was omitted from the structure factor calculation. Atoms are color-coded as in (a), with F = black. (d) Superposition of the PFAꢀABH
complex (blue), PFAꢀMABH complex (red), and PFAꢀFABH complex (green).
’ DISCUSSION
alkyl substituents, CH3 and CHF2, to initiate this study. As
discussed in detail in a recent review,55 the replacement of
hydrogen by fluorine is a commonly used strategy in medicinal
chemistry to increase the lipophilicity and metabolic resistance of
organic molecules. The introduction of fluorine can confer
greater pharmacokinetic and pharmacodynamic stability and
In the current study, we sought to determine the structural
characteristics and inhibitory properties of the most potent
arginase inhibitor known to date, ABH, when the amino acid
CR-H atom is substituted with alkyl groups. On the basis of the
structure of the HAIꢀDFMO complex, we selected the simplest
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can also influence binding affinity in that the CꢀF group of a
fluorinated inhibitor in an enzymeꢀinhibitor complex can be a
weak hydrogen bond acceptor.
the success of this particular strategy, with a variety of R-sub-
+
stituents such as ꢀ(CH2)4NH3 , ꢀ(CH2)3OPh, or ꢀ(CH2)3-
OPh-p-F yielding racemic ABH derivatives with Ki values ranging
from 10ꢀ8 to 10ꢀ10 M against both HAI and human arginase II.57
Future studies will continue the exploration of structureꢀaffinity
relationships for R,R-disubstituted amino acid inhibitors bearing
substituents capable of making favorable interactions in the T136
region, and such studies clearly promise to advance the devel-
opment of arginase inhibitors as possible therapeutic agents.
The structures of HAI and PFA complexed with the corre-
sponding inhibitors MABH and FABH reveal that the new R-
substituent of each inhibitor is directed out towarda new region of
the active site not previously identified to interact with bound
inhibitors. In HAI, this region of the active site is in the vicinity of
T136, so we now refer to this previously “uncharted” region of the
protein surface as the “T136 region”. Importantly, the current
work is the first to demonstrate that the arginase active site can
accommodate the binding of an R,R-disubstituted amino acid
without compromising the molecular recognition of the R-amino
and R-carboxylate groups, critical determinants of enzymeꢀ
inhibitor affinity.43 Both CꢀF groups of FABH accept hydrogen
bonds from active site solvent molecules in the HAIꢀFABH
complex, but these interactions presumably do not contribute to
enzymeꢀinhibitor binding affinity.
’ CONCLUSIONS
This work reports the design, multistep synthesis, assay, and
structural evaluation of two R,R-disubstituted amino acids,
MABH and FABH, in complex with HAI and PFA. This is the
first study describing the binding of R,R-disubstituted amino
acids to human and parasitic arginases, and X-ray crystal struc-
tures show that the additional R-substituent is readily accom-
modated in each enzyme active site. These structures highlight
new regions of the protein surfaces of HAI and PFA that can be
targeted for additional affinity interactions. Finally, this work
provides the first comparative structural insights on inhibitor
discrimination between a human arginase and a parasitic argi-
nase, which may facilitate the development of species-specific
inhibitors in the search for new arginase-directed drugs.
The residue corresponding to T136 of HAI in PFA is P228, so
the T136 region of HAI and the corresponding “P228 region” of
PFA differ in their three-dimensional contours and polarities.
These regions additionally differ because of the proximity of
H381 and its associated polypeptide chain in the adjacent
monomer, which constricts the P228 region of PFA relative to
the T136 region of HAI. If an R,R-disubstituted amino acid
inhibitor can ultimately be designed to interact with the P228
region of PFA, it may be possible to develop a species-specific
inhibitor of PFA that does not cross-react with HAI. Although a
CꢀF group of FABH accepts a hydrogen bond from H381 in the
PFAꢀFABH complex, this interaction does not enhance inhi-
bitor binding affinity.
It is surprising that while the R-amino and R-carboxylate
groups of MABH and FABH bind similarly within the active sites
of HAI and PFA, both exhibit decreased inhibitory potency in
comparison with ABH (Table 1). Moreover, it is particularly
surprising that while each inhibitor binds with a tetrahedral
boronate anion side chain to HAI, each inhibitor binds to PFA
with a trigonal planar boronic acid side chain. However, neither
MABH nor FABH bind to PFA as substrate analogues because of
a side chain conformation that would be unattainable for sub-
strate L-arginine. This structural feature and interactions between
the newly introduced R-substituent and H381 in an adjacent
subunit presumably contribute to the aberrant inhibitor binding
modes and lower affinities with PFA compared with HAI. While
previous studies56 suggest that PFA may have a significant role in
immune evasion and infection by the parasite P. falciparum, and
PFA inhibition is suggested46 as an adjuvant therapy in the
treatment of malaria patients, the current work demonstrates that
boronic acid analogues of ABH are not particularly effective
against PFA.
’ EXPERIMENTAL SECTION
Synthesis of r,r-Disubstituted Amino Acids. General
Procedures. All reagents were of at least 95% purity, purchased from
Sigma Aldrich Co. and Fisher Scientific, and used as received. All
solvents were of HPLC grade (Fisher Scientific or Sigma Aldrich Co).
For anhydrous conditions, solvents were freshly distilled under N2
(CH2Cl2 from P2O5, and THF from Na benzophenone). Reactions
were monitored by TLC with Sigma-Aldrich aluminum plates (silica gel
F254, 60 Å to 0.25 mm), visualized by quenching under UV light,
equilibrated in a glass chamber containing iodine, and/or stained with
ninhydrin solution. Flash column chromatography was performed using
Fisher Scientific silica gel 60 (230ꢀ400 mesh). High resolution mass
spectrometry (HRMS) was carried out using an instrument from LCT
Premier XE Micromass/Waters MS Technologies. Purities of all
synthesized and tested compounds were greater than 95% based on
1
HPLC analysis. H and 13C NMR spectra were recorded on Bruker
DMX 360 and DRX 500 spectrometers at 360 and 500 MHz for 1H, 90.6
and 125.6 MHz, respectively, for 13C, 282 MHz for 19F, and 128 MHz for
11B NMR. Assignments were made based on chemical shifts, signal
1
intensity, COSY, and HMQC sequences. H, 13C, 19F, and 11B NMR
chemical shifts (δ) are reported in ppm relative to the residual solvent
peaks. 1H NMR coupling constants (J) are reported in Hz, and multi-
plicities are denoted as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; bs, broad singlet. 11B NMR spectra are decoupled.
Ethyl 2-N-(Diphenylmethyleneamino)hex-5-enoate (2). A
The decreased affinity of MABH and FABH in comparison
with the parent inhibitor ABH may be rationalized by analysis of
the crystal structures of their complexes with HAI. Here, all three
inhibitors clearly bind as analogues of the tetrahedral intermedi-
ate and its flanking transition state, and this feature significantly
contributes to high affinity. Given the presence of potential
hydrogen bonding groups in the T136 region of the HAI active
site, it appears that a more polar substituent would be more
favorably accommodated in this region of the active site. Ad-
ditionally, a longer R-substituent might be better suited for
capturing additional binding interactions in the protein land-
scape of T136 and beyond. Indeed, a recent report demonstrates
t
10% solution of BuOK (0.9 g, 8.25 mmol) in dry THF was added
dropwise to a 10% THF solution of N-(diphenylmethylene)glycine
ethyl ester (1) (2 g, 7.5 mmol) at ꢀ78 ꢀC, with stirring under N2. After
15 min, 4-bromobutene (2.4 mL, 22.5 mmol) in dry THF (5 mL) was
added at ꢀ78 ꢀC. After warming to room temperature, the reaction
mixture was stirred for 20 h and then quenched with NH4Cl (20 mL)
and water (5 mL). The resulting immiscible layers were separated, and
the aqueous layer was extracted with CH2Cl2 (4 ꢁ 20 mL). The
combined organic extracts were washed (brine), dried (Na2SO4), and
rotoevaporated. Purification by flash column chromatography (hexane/
ethyl acetate gradients) afforded 2 as a light-yellow oil (1.8 g, 75%). 1H
and 13C NMR (CDCl3) spectra confirmed literature data.49
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dx.doi.org/10.1021/jm200443b |J. Med. Chem. 2011, 54, 5432–5443

Journal of Medicinal Chemistry
ARTICLE
Ethyl2-N-(Diphenylmethyleneamino)-2-methylhex-5-enoate
(3). A solution of 2 (1.8 g, 5.6 mmol) in dry THF (20 mL) was treated
(molar ratio 2/tBuOK 1:1.1), under stirring and N2 purging. After
20 min, chlorodifluoromethane (4.8 g, 55.5 mmol) was added to the
reaction flask via needle. The resulting mixture was stirred at ꢀ78 ꢀC (10
min), then at room temperature (45 min), quenched with brine (5 mL),
and extracted with Et2O (4 ꢁ 15 mL). The combined organic extracts
were washed (brine), dried (Na2SO4), and rotoevaporated. The resulting
yellow oil was purified (flash column chromatography, hexane/ethyl
acetate gradients) to generate 4 as a colorless oil (1.4 g, 70%). 1H NMR
(CDCl3): δ 7.69ꢀ7.05 (m, 10H), 6.17 (t, J = 55, 1H, ꢀCHF2),
5.91ꢀ5.70 (m, 1H, ꢀHCdCH2), 5.04 (dq, J = 1.6, 17.1, 1H, HA,
ꢀHCdCHAHB), 4.95 (dd, J = 1.7, 10.2, 1H, HB, ꢀHCdCHAHB),
3.85ꢀ3.63 (m, 2H, ꢀOꢀCH2ꢀ), 2.45ꢀ2.20 (m, 2H, HA, HB, (CHF2)Cꢀ
CHAHBꢀ), 2.18ꢀ2.09 (m, 1H, HC, (CHF2)CꢀCH2ꢀCHCHD),
2.06ꢀ1.95 (m, 1H, HD, (CHF2)CꢀCH2ꢀCHCHD), 1.12 (t, J = 7.1,
3H, ꢀCH2ꢀCH3). 13C NMR (CDCl3): δ 176.6 (CO), 169.8 (CdN),
t
successively with 10% THF solutions of BuOK and methyl iodide,
respectively (molar ratio 2/tBuOK/CH3I 1:1.1:3). Subsequent workup
and flash column chromatography (hexane/ethyl acetate gradients)
1
yielded pure 3 as a colorless oil (1.5 g, 80%). H NMR (CDCl3): δ
7.63ꢀ7.54 (m, 2H, Ph), 7.39ꢀ7.22 (m, 6H, Ph), 7.20ꢀ7.05 (m, 2H,
Ph), 5.94ꢀ5.83 (m, 1H, ꢀHCdCH2), 5.04 (dq, J = 1.6, 17.1, 1H, HA,
ꢀHCdCHAHB), 4.98 (dd, J = 1.9, 10.2, 1H, HB, ꢀHCdCHAHB),
3.85ꢀ3.60 (m, 2H, ꢀOꢀCH2ꢀ), 2.40ꢀ2.25 (m, 1H, HA, (CH3)Cꢀ
CHAHBꢀCH2ꢀ), 2.22ꢀ1.94 (m, 3H, HB, HC, HD, (CH3)Cꢀ
CHAHBꢀCHCHDꢀ), 1.41 (s, 3H, (CH3)Cꢀ), 1.12 (t, J = 7.1, 3H,
ꢀCH2ꢀCH3). 13C NMR (CDCl3): δ 175 (CO), 166.8 (CdN), 141.5
0
(Cq, C1-Ph), 139 (ꢀCHdCH2), 137.7 (Cq, C1 -Ph), 130.3, 128.9, 128.8,
128.7, 128.2, 128.0 (all from Ph), 114.7 (ꢀCHdCH2), 66.3 (OꢀC),
60.6 (NꢀC(CH3)), 42.6 ((CH3)CꢀCH2ꢀ), 28.8 ((CH3)Cꢀ), 24.6
((CH3)CꢀCH2ꢀCH2ꢀ), 14.1 (ꢀCH2ꢀCH3). HRMS m/z 336.1960
(calcd for M + H, 336.1963).
0
140.3 (Cq, C1-Ph), 138.5 (ꢀCHdCH2), 136.7 (Cq, C1 -Ph), 130.9, 130.3,
129.2, 129.0, 128.5, 128.31, 128.27, 128.1 (all from Ph), 117.6 (t, 1JCF
=
250, CHF2), 114.9 (ꢀCHdCH2), 71.1 (bs, C(CHF2)ꢀN), 61.4
(OꢀC), 32.0 ((CHF2)CꢀCH2), 28.3 ((CHF2)CꢀCH2ꢀCH2ꢀ),
13.8 (ꢀCH2ꢀCH3). 19F NMR (CDCl3): δ ꢀ129.0 (ABX system
Ethyl 2-N-(Diphenylmethyleneamino)-2-methyl-6-(4,4,5,5-
tetramethyl[1,3,2]dioxaborolan-2-yl)hex-5-enoate (5). The
catalyst, a mixture of [Ir(cod)Cl]2 (150 mg, 0.2 mmol) and dppm (172 mg,
0.4 mmol), was weighed in the glovebox and dissolved with stirring in dry
CH2Cl2 (10 mL) under N2 at room temperature. Pinacolborane (2.6 mL,
18 mmol) and then a solution of 3 (1.5 g, 4.5 mmol) in dry CH2Cl2 (5 mL)
were added. 1H NMR monitoring showed the disappearance of signals for
the olefinic protons after 18 h, and the reaction mixture was then quenched
with water (5 mL). After separation, the aqueous layer was extracted with
Et2O (4 ꢁ 10 mL). The combined organic layers were washed with water
(5 mL), dried (Na2SO4), and rotoevaporated. The resulting crude orange
oil was further purified by flash column chromatography (hexane/ethyl
acetate gradients) and yielded 5 as a colorless oil (1.2 g, 60%). 1H NMR
(CDCl3): δ 7.60ꢀ7.51 (m, 2H, Ph), 7.40ꢀ7.22 (m, 6H, Ph), 7.18ꢀ7.08
(m, 2H, Ph), 3.78ꢀ3.55 (m, 2H, ꢀOꢀCH2ꢀ), 2.05ꢀ1.80 (m, 2H,
(CH3)CꢀCH2ꢀ), 1.52ꢀ1.45 (m, 3H, HB, HC, HD, (CH3)Cꢀ
CH2ꢀCHAHBꢀCHCHDꢀ), 1.42ꢀ1.35 (bs, 3H, (CH3)Cꢀ), 1.25ꢀ1.15
(m, 1H, HA, (CH3)CꢀCH2ꢀCHAHBꢀCHCHDꢀ), 1.23 (s, 12H,
4CH3 from pinacolboranyl), 1.1 (t, J = 7.1, 3H, CH2ꢀCH3), 0.81 (t,
J = 7.5, 2H, ꢀCH2B). 13C NMR (CDCl3): δ 174.4 (CO), 166.2 (CdN),
2
2
8 lines, JFF = 271, JHF = 56). HRMS m/z 372.1764 (calcd for M +
H, 372.1775).
Ethyl 2-N-(Diphenylmethyleneamino)-2-difluoromethyl-
6-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)hex-5-enoate
(6). A solution of 4 (1.4 g, 4.2 mmol) in dry CH2Cl2 (5 mL) was reacted
with a mixture of [Ir(cod)Cl]2/dppm and pinacolborane following the
same experimental procedure as described for the synthesis of 5. Flash
column chromatography (pentane/Et2O gradients) generated 6 as a
colorless oil (0.95 g, 45%). 1H NMR (CDCl3): δ 7.65ꢀ7.11 (m, 10H),
6.16 (t, J = 56, 1H, ꢀCHF2), 3.81ꢀ3.65 (m, 2H, ꢀOꢀCH2ꢀ),
2.1ꢀ1.80 (m, 2H, HA, HB, (CHF2)CꢀCHAHBꢀ), 1.60ꢀ1.38 (m,
4H, HC, HD, HE, HF, (CHF2)CꢀCH2ꢀCHCHDꢀCHEHFꢀ), 1.18 (s,
6H, 2CH3 from pinacolboranyl), 1.16 (s, 6H, 2CH3 from
pinacolboranyl), 1.1 (t, J = 7.1, 3H, CH2ꢀCH3), 0.85ꢀ0.70 (m, 2H,
ꢀCH2B). 13C NMR (CDCl3): δ 170.0 (CO), 169.5 (CdN), 140.3 (Cq,
0
C1-Ph), 136.8 (Cq, C1 -Ph), 130.8, 129.1, 129.0, 128.4, 128.2, 128.1 (all
3
from Ph), 83.1 (2C, Cq from pinacolboranyl), 71.1 (t, JCF = 21,
(CHF2)Cꢀ), 61.3 (OꢀC), 32.9 ((CHF2)CꢀCH2), 26.4 ((CHF2)Cꢀ
CH2ꢀCH2ꢀ), 25.1 ((CHF2)CꢀCH2ꢀCH2ꢀCH2ꢀ), 25.0 (2CH3
from pinacolboranyl), 24.9 (2CH3 from pinacolboranyl), 13.9
(ꢀCH2ꢀCH3), 11.3 (CH2ꢀB). 11B NMR (CDCl3): δ 32.5. 19F
0
141.6 (Cq, C1-Ph), 137.5 (Cq, C1 -Ph), 130.2, 129.3, 129.0, 128.9, 128.3,
128.0 (all from Ph), 83.2 (2C, Cq from pinacolboranyl), 66.8 (OꢀC),
60.5 (NꢀC(CH3)), 43.2 ((CH3)CꢀCH2), 27 ((CH3)Cꢀ), 25.2 (4C,
4CH3 from pinacolboranyl), 24.9, 24.6 (C(CH3)ꢀCH2ꢀCH2ꢀ
CH2ꢀ), 14.2 (ꢀCH2ꢀCH3), 11.5 (bs, CH2ꢀB). 11B NMR (CDCl3):
δ 38.2. HRMS m/z 464.2979 (calcd for M + H, 464.2972).
NMR (CDCl3): δ ꢀ129.3 (ABX system, eight lines, 2JFF = 274, 2JHF
56.5). HRMS m/z 500.2772 (calcd for M + H, 500.2783).
=
2-Amino-6-borono-2-(difluoromethyl)hexanoic Acid (FABH,
8). Intermediate 6 (0.9 g, 1.8 mmol) was deprotected by the same
experimental protocol utilized for the deprotection of 5. Flash column
chromatography (CHCl3/MeOH/NH4OH/iPrOH gradients) afforded
8 as a white precipitate (0.37 g, 92%). 1H NMR (D2O): δ 6.19 (t, J = 54,
1H, ꢀCHF2), 1.96ꢀ1.88 (m, 1H, HA, (CHF2)CꢀCHAHB), 1.78ꢀ1.68 (m,
1H, HB, (CHF2)CꢀCHAHB), 1.53ꢀ1.30 (m, 3H, HB, HC, HD, (CHF2)Cꢀ
CH2ꢀCHAHBꢀCHCHDꢀ), 1.30ꢀ1.08 (m, 1H, (CHF2)CꢀCH2ꢀ
CHAHBꢀCHCHDꢀ), 0.76 (t, J = 7.5, 2H, ꢀCH2B). 13C NMR (D2O):
2-Amino-6-borono-2-methylhexanoic Acid (MABH, 7). The
protected derivative 5 (1.2 g, 2.6 mmol) was stirred with 6 N HCl/THF
2:1 (4 h, 70 ꢀC), cooled to room temperature, and extracted with Et2O
(4 ꢁ 25 mL) to remove the benzophenone. The aqueous layer was
rotoevaporated to dryness, retaken in 6 N HCl (50 mL), and stirred at
80ꢀ90 ꢀC until TLC monitoring (MeOH/CHCl3/NH4OH 8:2:0.5)
revealed complete deprotection (after∼24 h). Rotoevaporationat 40 ꢀC,
followed by flash column chromatography (CHCl3/MeOH/NH4OH
gradients) afforded 7 as a white powder (0.46 g, 93%). 1H NMR (D2O):
δ 1.94ꢀ1.87 (m, 1H, HA, (CH3)CꢀCHAHBꢀ), 1.80ꢀ1.65 (m, 1H, HB,
(CH3)CꢀCHAHBꢀ), 1.47 (s, 3H, (CH3)Cꢀ), 1.48ꢀ1.32 (m, 3H, HB,
HC, HD, (CH3)CꢀCH2ꢀCHAHBꢀCHCHDꢀ), 1.29ꢀ1.13 (m, 1H,
(CH3)CꢀCH2ꢀCHAHBꢀCHCHDꢀ), 0.79 (t, J = 7.5, 2H, ꢀCH2B).
13C NMR (D2O): δ 177 (CO), 61.6 (NꢀC(CH3)), 36.9 ((CH3)Cꢀ
CH2), 25.9 ((CH3)Cꢀ), 23.5, 22.5 ((CH3)CꢀCH2ꢀCH2ꢀCH2ꢀ),
13.9 (bs, CH2ꢀB). 11B NMR (D2O): δ 36.25. HRMS m/z 190.1258
(calcd for M + H, 190.1250).
δ 171.5 (d, 4JCF = 6.1, CO), 116.0 (t, 1JCF = 246, CHF2), 65.5 (dd, 3JCF
=
4
16.5, 20, (CHF2)Cꢀ), 31.2 (d, JCF = 3.2, (CHF2)CꢀCH2), 25.1
((CHF2)CꢀCH2ꢀCH2ꢀ), 23.4 ((CHF2)CꢀCH2ꢀCH2ꢀCH2ꢀ),
13.7 (bs, CH2ꢀB). 11B NMR (D2O): δ 21.6. 19F NMR (D2O): δ
2
2
ꢀ130.0 (ABX system, eight lines, JFF = 278, JHF = 54). HRMS m/z
226.1057 (calcd for M + H, 226.1062).
Surface Plasmon Resonance (SPR). The binding affinities of
MABH and FABH to HAI were determined by SPR on a Biacore 3000
instrument according to a previously reported procedure58 except that
all measurements were made at pH 8.5 and inhibitor concentrations
were 0ꢀ200 nm for ABH, 0ꢀ25 μM for MABH, and 0ꢀ500 μM for
FABH. In this measurement, HAI is immobilized on carboxymethylated
Ethyl 2-N-(Diphenylmethyleneamino)-2-(difluoromethyl)-
hex-5-enoate (4). A solution of 2 (1.8 g, 5.6 mmol) in dry THF
t
(10 mL) was cooled to ꢀ78 ꢀC and treated with 20% BuOK/THF
5439
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Journal of Medicinal Chemistry
ARTICLE
Table 2. X-ray Crystallographic Data Collection and Refinement Statistics
complex
parameter
resolution limit, Å
HAIꢀDFMO
HAIꢀL-Orn
HAIꢀMABH
HAIꢀFABH
PFAꢀMABH
PFAꢀFABH
50ꢀ1.70
50ꢀ1.43
50ꢀ1.60
50ꢀ1.70
128641/68384
99.0/98.2
50ꢀ1.90
404894/44684
99.9/99.2
50ꢀ2.0
total/unique reflections
129926/67814
99.9/100.0
0.101/0.412
11.0/2.7
233126/116771
99.8/100.0
0.067/0.640
35.6/2.0
82598/8046
98.4/95.6
0.050/0.298
25.1/2.7
412761/38494
99.9/98.7
0.090/0.587
23.6/2.7
completeness (%) (overall/outer shell)
Rmerge (overall/outer shell)a
I/σ(I) (overall/outer shell)
0.148/0.577
11.6/2.3
0.081/0.444
23.0/3.4
Refinement
R/Rfree
0.161/0.202b
0.147/0.185b
0.129/0.163b
0.136/0.178b
0.163/0.182c
0.167/0.183c
protein atomsd
manganese ionsd
ligand atomsd
4782
4
4782
4
4764
4
4778
4
2416
2
2416
2
24
18
28
32
13
15
water moleculesd
345
393
765
760
212
203
rms deviation
bond length, Å
bond angle, deg
dihedral angle, deg
PDB accession code
0.005
1.3
0.006
1.4
0.006
1.0
0.006
1.0
0.007
1.1
0.006
0.9
23
23
17
18
13
13
3GN0
3GMZ
3SJT
3SKK
3SL1
3SL0
a Rmerge = ∑|I ꢀ ÆIæ|/∑I, where I is the observed intensity and ÆIæ is the average intensity calculated for replicate data. b Rtwin = ∑|[|Fcalc/A|2 + |F
| ]
2 1/2
calc/B
ꢀ |Fobs||/∑|Fobs| for reflections contained in the working set. |Fobs| is the observed structural factor amplitude, and |Fcalc/A| and |Fcalc/B|are the structure
factor amplitudes calculated for twin domains A and B, respectively. Rtwin underestimates the residual error in the model over the two twin-related
reflections by a factor of approximately 0.7. The same expression describes Rtwin/free, calculated for test set reflections excluded from refinement. c R =
∑||Fobs| ꢀ |Fcalc||/∑|Fobs|, where |Fobs| and |Fcalc| are the observed and calculated structure factor amplitudes, respectively. The same expression
describes Rfree, calculated for test set reflections excluded from refinement. d Per asymmetric unit.
dextran over the gold surface of sensor chip CM5 and the inhibitor is
added to a buffer solution that continuously flows over the sensor
surface. Inhibitor binding to the immobilized enzyme causes a change in
the angle of reflection of polarized light used to interrogate the glass
sensor support. Angular changes are recorded in real time as “response
units” in the sensorgrams shown in Figure 3; these changes are
proportional to the concentration of enzyme-bound inhibitor and thus
allow for real time monitoring of the enzymeꢀinhibitor interaction as a
function of inhibitor concentration. Dissociation constants are calcu-
lated using a 1:1 Langmuir interaction model.59
Enzyme Inhibition Assay. The inhibition of PFA by MABH and
FABH was analyzed using a colorimetric method,60 as previously
described.40 Briefly, the reaction of urea with R-isonitrosopropiophe-
none was measured at a wavelength of 550 nm using the Envision plate
reader (courtesy of Dr. Scott Diamond, Institute of Medicine and
Engineering, University of Pennsylvania). The arginase reaction was
performed in 50 mM Tris (pH 8.0), 0.5 mM TCEP, 1 mM MnCl2, 0.2
μM protein, 0ꢀ100 mM inhibitor, and 20 mM L-arginine for 5ꢀ16 min
at 37 ꢀC. Assay mixture and protein were preincubated at 37 ꢀC for 1 min
before initiating the reaction. The assay mixture (20 μL) was stopped
with a sulfuricꢀphosphoric acid/R-isonitrosopropiophenone mixture
(140 μL). Reaction points were developed in a thermocycler (90 ꢀC, 1
h), followed by incubation at 21 ꢀC (15 min). The Ki values for the
racemic mixtures of MABH and FABH were calculated using the
ChengꢀPrusoff equation61 with the software Graphpad Prism (2008).
Crystallography: HAI Complexes. Crystals of the HAIꢀDFMO
and HAIꢀL-ornithine complexes were prepared by soaking crystals of
unliganded HAI prepared as described42 in 0.1 M bis-Tris (pH 6.5), 20%
PEG monomethyl ether, and 20 mM DFMO or 20 mM L-ornithine for 2
days. Crystals of the HAIꢀMABH and HAIꢀFABH complexes were
prepared by cocrystallization in hanging drops at 21 ꢀC. Drops contain-
ing 3 μL of protein solution [3.5 mg/mL HAI, 50 mM bicine (pH 8.5),
2 mM MABH, 100 μM MnCl2] and 3 μL of precipitant solution [0.1 M
HEPES (pH 7.0), 22ꢀ28% Jeffamine] were equilibrated against a 1 mL
reservoir of precipitant solution. Crystals appeared overnight and grew
with typical dimensions of 0.5 mm ꢁ 0.2 mm ꢁ 0.2 mm. All crystals were
cryoprotected in a precipitant solution containing 32% Jeffamine prior
to flash cooling in liquid nitrogen.
X-ray diffraction data from all crystals were collected at GM/CA-
CAT beamline 23-ID-D or NE-CAT beamline 24-ID-C at the Ad-
vanced Photon Source (APS, Argonne, IL). Diffraction intensities
measured from crystals of HAI complexes exhibited symmetry con-
sistent with apparent space group P6 (unit cell parameters a = b = 90.3 Å,
c = 69.3 Å). Intensity data integration and reduction were performed
using the HKL2000 suite of programs.62 Data reduction statistics are
recorded in Table 2. As with crystals of other HAI complexes,38,42
deviations from ideal Wilson statistics were observed with ÆI2æ/ÆIæ2 = 1.5,
indicating perfect hemihedral twinning.63 The structure of each enzy-
meꢀligand complex was solved by molecular replacement using the
program Phaser64 with chain A of unliganded HAI (PDB accession code
2ZAV, less water molecules)42 used as a search probe against twinned
data. In order to calculate electron density maps, structure factor
amplitudes (|Fobs|) derived from twinned data (|Iobs|) were deconvo-
luted into structure factor amplitudes corresponding to twin domains A
and B (|Fobs/A| and |Fobs/B|, respectively) using the structure-based
algorithm of Redinbo and Yeates63 implemented in CNS.65 Electron
density maps were visualized with the graphics software COOT.66 After
initial rigid body refinement with CNS,65 refinement of each complex
was performed against twinned data using PHENIX.67 After water
molecules were located and fit into the map, gradient omit maps revealed
the bound ligand in the active site of each monomer in the asymmetric
unit. Ligand atoms were refined with full occupancy and exhibited
atomic B factors consistent with the average B-factor calculated for the
entire protein. The quality of each final model was assessed using
PROCHECK.68 Final refinement statistics are recorded in Table 2.
5440
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Journal of Medicinal Chemistry
ARTICLE
Crystallography: Plasmodium falciparum Arginase Com-
plexes. Recombinant PFA was prepared and purified as described, and
crystals of complexes with MABH and FABH were prepared using
conditions similar to those employed in the structure determination of
the PFAꢀABH complex.40 Briefly, a 2 μL sitting drop of enzy-
meꢀinhibitor complex [5 mg/mL PFA, 50 mM Tris (pH 8.0),
200 mM NaCl, 1 mM MnCl2, 1 mM tris(2-carboxyethyl)phosphine
(TCEP), 5 mM inhibitor] was mixed with a 2 μL drop of precipitant
solution [1.2 M sodium/potassium phosphate (pH 8.0)] and equili-
brated against 500 μL of precipitant solution in the reservoir at 21 ꢀC.
Crystals appeared within 1ꢀ2 days and were harvested, cryoprotected
in 30% Jeffamine ED-2001, 0.1 M HEPES (pH 7.0), 50 μM inhibitor,
and flash-cooled in liquid nitrogen. Diffraction data were measured on
NE-CAT beamline 24-ID-E at APS. Data collection statistics are
recorded in Table 2.
’ ABBREVIATIONS USED
ABH, 2-(S)-amino-6-boronohexanoic acid; APS, Advanced Photon
Source; BEC, S-(2-boronoethyl)-L-cysteine; DFMO, 2-(difluoro-
methyl)-L-ornithine; FABH, 2-amino-6-borono-2-(difluoromethyl)-
hexanoic acid; HAI, human arginase I; HRMS, high resolution
mass spectrometry; MABH, 2-amino-6-borono-2-methylhexanoic
acid; NO, nitric oxide; PFA, Plasmodium falciparum arginase;
SPR, surface plasmon resonance
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properties of arginases. Comp. Biochem. Physiol., Part B: Biochem. Mol.
Biol. 1996, 114, 107–132.
(3) Wu, G.; Morris, S. M. Arginine metabolism: nitric oxide and
beyond. Biochem. J. 1998, 336 (Part 1), 1–17.
(4) Morris, S. M., Jr. Regulation of enzymes of the urea cycle and
arginine metabolism. Annu. Rev. Nutr. 2002, 22, 87–105.
(5) Morris, S. M., Jr. Recent advances in arginine metabolism: roles
and regulation of the arginases. Br. J. Pharmacol. 2009, 157, 922–930.
(6) Borcic, O.; Straus, B. Separation of arginase isoenzymes from
human tissues by agar gel electrophoresis. J. Clin. Chem. Clin. Biochem.
1976, 14, 533–535.
Data were indexed and merged using HKL2000.62 Molecular re-
placement calculations were performed with PHASER64 using the atomic
coordinates of PFA less inhibitor and solvent atoms (PDB accession code
3MMR)40 as a search probe for rotation and translation function
calculations. Iterative cycles of refinement and model building were
performed using PHENIX67 and COOT,66 respectively, in order to
improve each structure as guided by Rfree. Refinement included six
defined TLS groups identified by the TLS motion determination
server.69 As also observed in the PFAꢀABH complex,40 the N-terminus
was disordered (continuous main chain electron density starts at K22)
and residues G72ꢀN153 in the L2 loop were disordered. The quality of
each final model was assessed using PROCHECK.68 Refinement statis-
tics are recorded in Table 2.
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Accession Codes
†Atomic coordinates of human arginase I complexes with
DFMO, L-ornithine, MABH, and FABH have been deposited
in the Protein Data Bank (www.rcsb.org) with accession codes
3GN0, 3GMZ, 3SJT, and 3SKK, respectively; coordinates of
P. falciparum arginase complexes with MABH and FABH have
been deposited with accession codes 3SL1 and 3SL0, respectively.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 215-898-5714. Fax: 215-573-2201. E-mail: chris@
sas.upenn.edu.
’ ACKNOWLEDGMENT
These studies were supported by NIH Grant GM49758. We
thank Dr. Richard Pottorf of Provid Pharmaceuticals, Inc., for
helpful discussions; Dr. Steven Seeholzer, Dr. Hua Ding, and the
Protein Core Facility at Children’s Hospital of Philadelphia, PA,
for assistance with surface plasmon resonance measurements;
Dr. Scott Diamond at the Institute of Medicine and Engineering,
University of Pennsylvania, for assistance with kinetic colori-
metric assays; and Dr. Rakesh Kohli for recording the high
resolution mass spectra. We also thank the Northeastern Colla-
borative Access Team (NE-CAT) and the National Institute of
General Medical Sciences and National Cancer Institute Colla-
borative Access Team (GM/CA-CAT) for beamlines at the APS
and for access to X-ray crystallographic data collection facilities.
Use of the Advanced Photon Source is supported by Award RR-
15301 from the National Center for Research Resources at the
National Institutes of Health and by the U.S. Department of
Energy, Office of Basic Energy Sciences, under Contract No. DE-
AC02-06CH11357.
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