Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase

Article abstract of DOI:10.1016/j.jinorgbio.2010.12.012

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn ²⁺ binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K_i = 14 μM) that is comparable to that of FBP (K_m = 2 μM) or its inert analog TBP (K_i = 1 μM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3 A?) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn²⁺. The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn²⁺ are not consistent with a strong interaction. To determine if Zn²⁺coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn²⁺ coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn²⁺ coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn ²⁺ coordination to binding of 8 to GlFBPA.

Full text of DOI:10.1016/j.jinorgbio.2010.12.012

Journal of Inorganic Biochemistry 105 (2011) 509–517
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Rational design, synthesis and evaluation of first generation inhibitors of the Giardia
☆,☆☆
lamblia fructose-1,6-biphosphate aldolase
Zhimin Li a, Zhengang Liu a, Dae Won Cho a, Jiwen Zou a, Maozhen Gong a, Robert M. Breece b,
Andrey Galkin c, Ling Li a, Hong Zhao a, Gabriel D. Maestas a, David L. Tierney b, Osnat Herzberg c,
a,
Debra Dunaway-Mariano a, , Patrick S. Mariano
⁎
⁎
a
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, United States
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, United States
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2010
Received in revised form 22 December 2010
Accepted 23 December 2010
Available online 30 December 2010
Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-
bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on
3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral
groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83,
and a Zn2+ binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl
phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The
3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (Ki =14 μM) that is comparable to
that of FBP (Km=2 μM) or its inert analog TBP (Ki=1 μM). The X-ray structure of the GlFBPA–inhibitor 8 complex
(2.3 Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of
coordination with Zn2+. The observed distances and orientation of the pyridone ring O=C–C–OH relative to Zn2+
are not consistent with a strong interaction. To determine if Zn2+coordination occurs in the GlFBPA–inhibitor
8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme
histidine ligands and an oxygen atom from the pyridone ring O=C–C–OH was indicated. Analysis of the Zn2+
coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn2+ coordination is
reserved for the enediolate-like transition state, accounting for minimal contribution of Zn2+ coordination to
binding of 8 to GlFBPA.
Keywords:
Fructose-1,6-biphosphate aldolase
Giardia lamblia
Zn2+ coordination
Hydroxypyridinone
EXAFS
Inhibitor design
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
bioterrorism category B organism. The drugs metronidazole and
tinidazole, which are currently used to treat giardiasis, produce
Giardia lamblia, a waterborne human parasite, inflicts billions of
people worldwide with a chronic infection (giardiasis) that leads to
malnutrition, growth retardation in children, and sometimes death [1–
3]. Because of its impact on impoverished countries, and the meager
effort expended to identify new therapies, giardiasis has been
designated as a World Health Organization neglected disease [4]. In
addition, the Center for Disease Control has classified Giardia as a
problematic side effects. Moreover, the rate of recurrence of infection
is high, and the existence of resistant strains has been documented [5–
8]. The cellular biology and biochemistry of G. lamblia are not well
defined nor are its host adaptation and survival tactics. However, the
G. lamblia genome sequence is now known [9] and, thus, understanding
and controlling G. lamblia pathogenicity are tractable objectives.
Recent studies in our laboratories have focused on the identification,
characterization and design of inhibitors of enzymes that constitute
potential G. lamblia drug targets. From the outset, the class II fructose
1,6-bisphosphate aldolase (FBPA) was viewed as a particularly
attractive target. FBPA catalyzes the reversible cleavage of D-fructose
1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and
D-glyceraldehyde 3-phosphate (G3P) (Fig. 1), a key step in the Embden–
Meyerhof–Parnas glycolytic pathway. Because G. lamblia lacks the
components of oxidative energy metabolism, the generation of ATP via
the glycolytic pathway is likely to be essential for trophozoite
colonization of the human gut [10–12]. This assumption gains support
from the finding that RNAi/antisense RNA FBPA gene silencing in
☆
Inhibitor synthesis was carried out by Z. L., D. W. C., J. Z., M. G. and G. D. M under
the direction of P. S. M.; protein preparations and kinetic studies were carried out by
Z. L., L. Li and H. Z. under the direction of D.D.-M.; EXAFS studies were carried out by
R. M. B. under the direction of D. L.T.; and the crystal structure determination was made by
A. G. under the direction of O. H. The manuscript was written by P.S.M. and D.D.-M.
☆☆
The atomic coordinates and structure factors have been deposited in the Protein
Data Bank.
Corresponding authors.
E-mail addresses: dd39@unm.edu (D. Dunaway-Mariano), mariano@unm.edu
⁎
(P.S. Mariano).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.12.012

510
Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
Fig. 1. The reaction pathways catalyzed by class I and class II fructose 1,6-bisphosphate aldolases.
Fig. 2. A. Surface representation of the substrate-binding site observed in the structure of the GlFBPA–TBP complex (PDB: 3GAY). The enzyme nitrogen atoms are colored, carbons
atoms white and oxygen atoms red. The Zn2+ cofactor is shown as a green sphere and the TBP ligand as yellow sticks. B. The stereoview of the GlFBPA–TBP complex (PDB: 3GAY)
substrate-binding site. Hydrogen bonds are represented with black dashed lines. The carbon atoms of the TBP are colored yellow, oxygen atoms red and phosphorus atoms orange.
C. Cartoon representation of the inhibitor design.

Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
511
transfected G. lamblia trophozoites is lethal under standard culture
2.2. Crystallization and data collection
conditions [13].
FBPA function is also essential to the human host. Nevertheless,
through strategic design of mechanism-based inhibitors, it might be
possible to eliminate the activity of the G. lamblia FBPA (GlFBPA)
without interfering with the activity of the human FBPA. This proposal
is based on the fact that the GlFBPA and human FBPA evolved within
different protein fold families and thus possess different active site
structures and, most importantly, they employ different catalytic
mechanisms. GlFBPA is a class II aldolase [14], that uses a Zn2+
cofactor to activate FBP for retro-aldol cleavage [15–18] whereas the
human FBPA is a class I aldolase that utilizes an active site lysine to
activate cleavage of FBP via Schiff base formation [19–21] (Fig. 1).
Enzymes that employ Zn2+ in substrate activation are susceptible to
inhibition by small molecules that possess Zn2+-binding groups.
Examples of Zn2+-dependent enzymes which have been successfully
modulated in vivo with small molecule inhibitors equipped with a
“Zn2+-warhead” include carbonic anhydrase [22], matrix metallopro-
tease [23] and histone deacetylase [24].
The X-ray structures of GlFBPA bound with substrate and substrate
analogs provide the needed insight into the steric and electrostatic
features of the active site that govern ligand binding. Most helpful to
new inhibitor design is the 1.8 Å resolution structure of GlFBPA bound
with the inert substrate analog tagatose 1,6-bisphosphate (TBP) (a C(4)
epimer of FBP) [25]. This structure defines a solvent excluded pocket
that accommodates both the Zn2+ cofactor and the TBP C(1)–C(3) unit
(Fig. 2A) and shows that the TBP C(4)–C(6) unit extends along the
protein surface. The polar interactions observed between the TBP ligand
and the binding site residues are extensive (Fig. 2B). The Zn2+ cofactor is
bound to the enzyme through the coordination bonds formed with the
imidazole rings of His84, His178 and His210 (Fig. 2B).
The design of first generation of GlFBPA inhibitors described below
centers on a synthetically accessible scaffold that positions two highly
negatively charged, tetrahedral groups for interaction with the
numerous substrate C(1) and C(6) phosphate binding residues, a
hydrogen bond donor to the catalytic Asp83, and a Zn2+ binding
group (Fig. 2C). In the effort described, alkyl phosphonate/phosphate
substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were
synthesized and evaluated. The competitive inhibition constants for
these first generation inhibitors are reported and interpreted in the
context of X-ray crystallographic and solution X-ray absorption
(EXAFS) analyses of the inhibited enzyme. The major conclusions
are that the FBPA Zn2+ cofactor–substrate binding is weak at the
ground state but strong at the transition state and that effective
inhibitor design based on the use of a Zn2+ warhead must take into
account the steric and electrostatic properties of the Zn2+ ligand at
the transition state.
The crystals of the GlFBPA–inhibitor 8 complex were prepared by
soaking TBP-liganded crystals [25] with inhibitor 8. The TBP-liganded
crystals were grown at room temperature in hanging drops using the
vapor diffusion method. The protein solution containing 10 mM TBP
was mixed with an equal volume of mother liquor containing 22%
polyethylene glycol 3350 and 0.2 M NH4NO3. The crystals required a
period of 2–3 weeks to appear. Ligand exchange was accomplished by
4 rounds of transferring the crystals with a loop to a fresh solution
of mother liquor containing 20 mM inhibitor 8. The crystals diffracted
X-rays to a resolution of 2.3 Å. For data collection, the crystals were
transferred to solutions containing mother liquor, 20 mM inhibitor
8 and 20% glycerol, and flash-cooled in liquid nitrogen.
Diffraction data were acquired at the Southeast Regional —
Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced
Photon Source (APS, Argonne National Laboratory, Argonne, Illinois).
For data acquisition, the SER-CAT beamline was equipped with a
Quantex210 CCD detector. Data processing was carried out using
CrystalClear version 1.3.6 (Rigaku MSC Inc.). The statistics of data
collection are provided in Table 1.
2.3. Structure determination and refinement
The structure of the FBPA–inhibitor 8 complex was determined by
using Molecular Replacement techniques with the computer program
Phaser [26], employing the GlFBPA–phosphoglycolohydroxamate
structure (PDB code 2ISW) as the search model rather than the
GlFBPA–TBP structure in order to avoid phase bias. The Difference
Fourier maps indicated some alternative tracing. Structure refinement
was carried out using the CNS [27]. The final stages of refinement were
performed with REFMAC [28]. The two molecules in the asymmetric
unit were refined independently. The resulting models were
inspected and modified on a graphics workstation by the using
program ‘O’ [29]. Water molecules were added to the model based on
the Fo −Fc Difference Fourier electron density map (where Fo and Fc
are the observed and calculated structure factors, respectively), using
peaks with density ≥3σ as the acceptance criteria. Refinement
statistics are provided in Table 1. PROCHECK [30] was used for
analysis of geometry, QUANTA for molecular modeling and structural
Table 1
X-ray data collection and refinement statistics for the GlFBPA–inhibitor 8 complex.
Data collection
Space group
P212121
58.58, 64.95, 171.45
20.0–2.3
2. Materials and methods
Cell dimension a, b, c (Å)
Resolution range (Å)
No. observations
2.1. Determination of GlFBPA inhibition constants of 1–10
108,611
No. unique reflections
23,705
86.5(89.1)
0.079(0.248)
Completeness (%)a
Initial velocities were measured for reactions initiated by the
addition of FBP (2–20 μM) to solutions containing GlFBPA (0.02 μM),
inhibitor (0–3Ki), 200 μM NADH, 5 units of triosephosphate isomerase,
and 2 units of glycerol-3-phosphate dehydrogenase in 50 mM K+HEPES
(pH 7.5; 25 °C). The progress of the reaction was continuously moni-
tored at 340 nM for conversion of NADH to NAD (Δε=6.2 mM−1 cm−1).
The initial velocity data were fitted to Eq. (1).
b
Rmerge
Refinement statistics
No. reflections
No. residues
22,497
635
339
0.199
No. water molecules
c
Rwork
Rfree
d
0.274
RMS deviation
Bonds (Å) 0.014; angles (°) 1.5
V0 = Vmax½Sꢀ= ½Kmð1 + ½Iꢀ= KiÞ + ½Sꢀꢀ:
ð1Þ
a
The values in parentheses are for the highest resolution shell.
Rmerge =Σhkl [(Σj | Ij −bIN |)/Σj | Ij |], for equivalent reflections.
Rwork =Σhkl | |Fo|−|Fc| |/Σhkl |Fo|, where Fo and Fc are the observed and calculated
b
c
In Eq. (1), [S]=substrate concentration, [I]=inhibitor concentration
V0=initial velocity, Vmax=maximum velocity and Km=Michaelis–
Menten constant, and Ki=the competitive inhibition constant.
structure factors included in the refinement, respectively.
d
Rfree is computed for 5% of reflections that were randomly selected and omitted
from the refinement.

512
Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
H
B
3. Results
N
N
N
N
N
3.1. Inhibitor design
N
The steric and electrostatic features of the GlFBPA substrate-
Ph
Ph
Ph
binding site, defined by the structure of the GlFBPA–TBP complex [25]
(Fig. 2A and B), guided the design of inhibitors (Fig. 2C). The multiple
hydrogen bonds formed with the TBP C(1) and C(6) phosphate groups
(Fig. 2B) suggested that the phosphate subsites supply a large fraction
of the binding energy in the ground state complex (Ki =1 μM [25];
ΔGbinding =−8 kcal/mol). Therefore, the inhibitor must include
comparable tetrahedral dianionic substituents in order to effectively
displace the substrate FBP, which also employs the phosphate groups
for tight binding [13]. Because the hydrogen bonds to TBP do not
involve the bridging oxygen atoms of the phosphate groups, a
phosphonate group, which provides greater chemical and metabolic
stability than the phosphate ester [35], might be a useful replacement
of the phosphate group.
Zn
O
H O
N
Fig. 3. Chemdraw representation of the 5-coordinate, trigonal bipyramidal complex tris
(pyrazoyl)borate–Zn2+–hydroxypyridinone complex adopted from reference [43].
alignment (Molecular Simulations Inc.), and PYMOL [31] for depiction
of the structures.
The selection of the Zn2+ binding group (“Zn2+ warhead”) was
influenced by the demonstrated success of the hydroxypyridinone-
based series of 6-membered ring Zn2+ chelating compounds developed
by Cohen and his co-workers for targeting the Zn2+ center of matrix
metalloproteases [36,37]. By first using the tris(pyrazoyl)borate-Zn2+
complexasa chemical model of thematrixmetalloprotease Zn2+ center,
these investigators showed that hydroxypyridinones displace the water
ligand and engage in strong bidentate coordination with the caged Zn2+
to generate a 5-coordinate, trigonal bipyramidal complex (Fig. 3) [36].
Subsequently, the potencies of the hydroxypyridinone Zn2+ binding
groups as inhibitors of the matrix metalloproteases were demonstrated,
as were their metabolic stabilities and lack of toxicity [38].
In silico modeling of the 3-hydroxy-2-pyridinone to the GlFBPA
active site [25] using the program AutoDock [39] indicated a good fit.
Importantly, the close proximities of the ring carbonyl and hydroxyl
functions with the Zn2+ and the Asp83, respectively, suggested the
potential for strong binding interactions. The model also indicated
that the attachment of the alkylphosphate/phosphonate substituents
to the 3-hydroxy-2-pyridinone ring nitrogen and the para-carbon
would place the anionic groups in the respective C(1) and C(6)
phosphate-binding subsites. The structures of the first generation
compounds 1–9, prepared to test this design strategy, are given in
Chart I. A close structural homolog, dihydroxypyridine 10, was also
prepared for evaluation.
2.4. X-ray absorption spectra measurement
Buffered (50 mM K+HEPES, pH 7.5) GlFBPA (3 mM) samples were
prepared with 20% (v/v) glycerol, and loaded in Lucite cuvettes with
6 μm polypropylene windows, before rapid freezing in liquid nitrogen.
GlFBPA activity towards catalysis of FBP cleavage was assayed (vide
supra) in the presence of the 20% (v/v) glycerol and found to be
unchanged from that observed in buffer alone. The X-ray absorption
spectra were measured at the National Synchrotron Light Source
(Brookhaven National Lab, Upton, NY), beamline X3B, with a Si(111)
double crystal monochromator; harmonic rejection was accom-
plished using a Ni focusing mirror. Data collection and reduction
were accomplished according to published procedures [32]. The
reported spectra represent the average of the 6–8 scans per sample.
Both raw and Fourier filtered EXAFS data were fitted utilizing
theoretical amplitude and phase functions calculated with FEFF v. 8.00
[33]. The Zn–N scale factor and the threshold energy, ΔE0, were
calibrated to the experimental spectrum for tetrakis-1-methylimidazole
Zn(II) perchlorate, Zn(MeIm)4 and held fixed at 0.78 and −16 eV,
respectively, with E0 set to 9675 eV. First shell fits were then obtained
for all reasonable coordination numbers while allowing the absorber–
scatterer distance, Ras, and the Debye–Waller factor, σ2as, to vary. Fits to
unfiltered EXAFS, presented in the Appendix A (Fig. SI1), gave identical
results. In no case did inclusion of a mixed first shell, with distinct Zn–N
and Zn–O scattering contributions, result in either a significant
improvement in fit residual or resolvable Zn–N/Zn–O distances.
Multiple scattering contributions from coordinated histidine residues
were fitted using a set of combined multiple-scattering paths, according
to published procedures [34].
3.2. Synthesis of inhibitors 1–10
The synthetic sequences used for the preparation of the hydro-
xypyridinones 1–9 and the hydroxypyridine 10 are shown in
Schemes 1 and 2, respectively, and the complete experimental details
can be found in Appendix A. The preparation of N-phosphonomethyl-
3-hydroxy-2-pyridone 1 relied on initial N-alkylation of the benzyl-
R1
1 (R1 = CH2PO3H2 ; R2 = H)
6 (R1 = CH2PO3H2 ; R2 = CH2CH2PO3H2)
7 (R1 = CH2PO3H2 ; R2 = CH2OCH2PO3H2)
8 (R1 = CH2PO3H2 ; R2 = CH2CH2OPO3H2)
N
O
2 (R1 = H; R2 = CH2CH2PO3H2)
3 (R1 = H; R2 = CH2OCH2PO3H2)
OH
4 (R1 = H; R2 = CH2CH2OPO3H2)
9 (
R
) (R1 = CH2PO3H2 ; R2 = ( )-CHOHCH2OPO3H2)
R
R2
R
5 (R1 = H; R2 = ( )-CHOHCH2OPO3H2)
9 (
S
) (R1 = CH2PO3H2 ; R2 = ( )-CHOHCH2OPO3H2)
S
R1
OH
OH
10 (R1 = CH2PO3H2 ; R2 = CH2CH2OPO3H2)
N
R2
Chart I. The chemical structures of inhibitors 1–10.

Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
513
-2
CH2CH2PO3
CH2CH2PO(OiPr)2
OBn
O
OBn
O
OH
O
(iPrO)2POCH2Br
1. TMSBr
TMSBr
(96%)
OMOM
OH
(iPrO)2POCH2Br
K2CO3, MeCN
(80%)
2. H2, Pd/C
(84%)
N
N
H
N
17
-2
N
O
N
O
K2CO3, MeCN
(88%)
CH2PO(OiPr)2
CH2PO3
-2
CH2PO(OiPr)2
CH2PO3
12
11
1
26
6
CHO
OMOM
OMOM
OBn
nBuLi
BnOH
NaH
OMOM
OBn
-2
CH2OCH2PO(OiPr)2
OMOM
HCO2Me
Et2O
(70%
CH2OCH2PO3
OH
N
Cl
N
(iPrO)2POCH2Br
N
TMSBr
14
15
20
13
K2CO3, MeCN
(85%)
N
O
(90%)
N
O
CH=CHPO(OiPr)2
OMOM
CH2CH2PO(OiPr)2
OMOM
-2
CH2PO(OiPr)2
CH2PO3
(iPrO)2POCH2PO(OiPr)2
H2
27
7
NaH, THF
(85%)
Pd/C
(90%)
N
OBn
N
H
O
16
17
-2
CH2CH2PO3
CH2CH2OH
CH2CH2OH
tBuLi;
H2
OH
OMOM
OMOM
14
TMSBr
(iPrO)2POCH2Br
ethylene
oxide
65%)
Pd/C
(90%)
(84%)
N
H
O
N
O
K2CO3, MeCN
(88%)
N
OBn
H
21
28
2
-2
CH2CH2OPO(OBn)2
CH2CH2OPO3
OH
CH2CH2OH
OMOM
1. (BnO)2PN(iPr)2
tetrazole
OMOM
1. H2, Pd/C
CH2OCH2PO(OiPr)2
OMOM
CH2OH
2. TMSBr
(65%)
2. MCPBA
(85%)
N
O
N
O
(iPrO)2POCH2Br
N
O
NaBH4
(94%)
OMOM
-2
15
CH2PO(OiPr)2
CH2PO3
CH2PO(OiPr)2
NaH, DMF
(65%)
N
OBn
30
8
N
OBn
29
19
18
AD-mix-
OH
CH=CH2
OMOM
-2
HO
(70%, 95%ee)
CH2OCH2PO3
OH
Ph3PCH3Br;
CH2OCH2PO(OiPr)2
OMOM
TBSCl
H2
TMSBr
(78%)
OMOM
15
AD-mix-
(70%, 97%ee)
tBuLi
(80%)
imadazole
(80%)
Pd/C
(90%)
N
OBn
N
OBn
N
O
N
H
O
31
32R (32S
)
OH
H
20
3
OMOM
OMOM
OMOM
OH
TBSO
TBSO
MOMCl
TBSO
H2
OMOM
OMOM
-2
DIEPA
(86%)
CH2CH2OPO3
OH
1. (BnO)2PN(iPr)2
tetrazole
CH2CH2OPO(OBn)2
OMOM
Pd/C
(90%)
)
1. H2, Pd/C
N
O
N
OBn
N
OBn
H
21
34R (34S
OMOM
2. EtSH
(90%)
33R (33S
)
OH
2. MCPBA
(70%)
35R (35S
)
OMOM
N
H
O
N
OBn
22
4
OMOM
OMOM
1. (BnO)2PN(iPr)2
tetrazole
TBSO
HO
(BnO)2POCH2OTf
TBAF
(90%)
OMOM
OMOM
K2CO3
(40%)
2. MCPBA
(86%)
OMOM
OMOM
OMOM
1. (BnO)2PN(iPr)2
tetrazole
HO
TBSO
N
O
N
O
TBAF
OMOM
O
CH2PO(OBn)2
36R (36S
CH2PO(OBn)2
(90%)
2. MCPBA
(68%)
37R (37S
)
OMOM
)
OMOM
N
OBn
N
H
OMOM
OMOM
OH
OH
24
35R
-2
(BnO)2OPO
O3PO
1. H2, Pd/C
OH
OMOM
-2
O3PO
(BnO)2OPO
1. H2, Pd/C
2. EtSH
(75%)
OH
O
OMOM
OBn
N
O
N
O
2. HCl
(65%)
-2
CH2PO(OBn)2
CH2PO3
N
H
5
N
38R (38S
)
OMOM
OH
9R (9S
)
25
Scheme 1. The reaction sequences used in the syntheses of inhibitors 1–9.
protected pyridone 11 with diisopropyl bromomethylphosphonate
followed by trimethylsilyl bromide promoted ester cleavage and
hydrogenolysis. For preparation of 3-hydroxy-4-pyridones 2 and 6,
methoxymethyl-protected 2-chloro-3-hydroxypyridine 13 was con-
verted to the benzyloxy analog 14. Sequential C-4 formylation and
olefination with tetraisopropyl methylene-bisphosphonate gave the
4-pyridyl vinylphosphonate diester 16. Reduction of the alkene
moiety in 16 was accompanied by hydrogenolytic cleavage of the
benzyl ether to give 17. Treatment of 17 with trimethylsilyl bromide
then furnished inhibitor 2. N-Alkylation of 17 with diisopropyl

514
Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
OTBDPS
OBn
OTBDPS
OBn
HO
N
HO
N
OTBDPS
O
LDA / THF
OH
OH
OH
OH
TBDPSCl
BnBr
H2O2 / KOH
H
N
N
N
imidazole / DMF
(70%)
OBn
OH
NaOH / MeOH
(48%)
N
N
N
H2O
(20%)
OBn
OH
CH3
42
CH3
CH3
CH3
40
41
43
39
OH
(35%)
OH
OTBDPS
OBn
PO(OH)2
OH
PO(OBn)2
OBn
1. (BnO)2PN(iPr)2
/ tetrazole / CH3CN
OBn
TBAF
H
2 / MeOH
(BnO)3P
N
N
THF
(65%)
N
N
Pd / C
(80%)
OBn
I2
(45%)
OBn
OH
OBn
2. mCPBA
(60%)
OPO(OBn)2
OPO(OBn)2
OPO(OH)2
OPO(OBn)2
44
45
46
10
Scheme 2. The reaction sequence used in the synthesis of inhibitor 10.
bromomethylphosphonate and ester deprotection led to inhibitor 6.
The pyridonyl ethylphosphonate 2 was prepared from intermediate
17 by treatment with trimethylsilyl bromide.
Reaction of the 4-pyridylcarboxaldehyde 15 with NaBH4 served as
the first step in the routes for preparation of 3-hydroxy-4-pyridones 3
and 7. The alcohol 14, formed in this manner, underwent O-alkylation
when treated with diisopropyl bromomethylphosphonate to produce
the corresponding ether 19, which following hydrogenolytic cleavage
formed 20. Treatment of 20 with trimethylsilyl bromide afforded
inhibitor 3.
The synthetic sequence employed to prepare the N-phosphono-
methyl phosphates 4 and 8 began with the protected dihydroxypyridine
14 (Scheme 1). Introduction of the C-4 hydroxyethyl group was
performed by reaction of the C-4 anion of 18 with ethylene oxide.
Hydrogenolytic removal of the benzyl group followed by N-phospho-
nomethylation gave the alcohol 29. Transformation of 29 to the dibenzyl
phosphate diester 30 was achieved by reaction with dibenzyl N,N-
diisopropylphosphite followed meta-chloroperoxybenzoic acid oxida-
tion. Finally, a two-step deprotection sequence was used to convert 30
to inhibitor 8. The phosphate analog 4 was produced from intermediate
21 by phosphorylation of the alcohol moiety and deprotection.
The route to 9R began with Wittig olefination of aldehyde 15 to
produce alkene 31. Sharpless dihydroxylation [40] with AD-mix-β
provided diol 32R with a 95% ee. Differential protection of the
hydroxyl groups in 32R followed by hydrogenolysis generated
pyridone 35R, which was N-alkylated using dibenzyl phosphono-
methyltriflate to form 36R. Selective liberation of the terminal
hydroxyl group provided alcohol 37R, which was then converted to
inhibitor 9R by using a phosphorylation–deprotection sequence.
Inhibitor 9S was prepared by using the same route employed for 9R
except that the Sharpless AD-mix-α methodology was used to carry
out dihydroxylation of 31. The phosphate 5 was formed from
intermediate 35R by tert-butyldimethyl silyl deprotection, alcohol
phosphorylation, and benzyl and methoxymethyl ether removal.
Finally, the route used for preparation of the dihydroxypyridine
derivative 10 was initiated by conversion of pyridoxal (39) to the
protected pyridine 42. Formylation of 42 followed by sequential
phosphate and phosphonate introduction gave 46, which undergoes
hydrogenolysis to form inhibitor 10.
3.3. Kinetic evaluation of compounds 1–10 as GlFBPA inhibitors
Steady-state kinetic methods were used to access the binding
affinities of compounds 1–10 as inhibitors of the GlFBPA catalyzed
conversion of FBP to DHAP and G3P at pH 7.5 and 25 °C. The initial
velocity data, measured at varying FBP concentrations and changing
fixed inhibitor concentrations, demonstrated that competitive inhibi-
tion was taking place in each case and yielded the inhibition constants
listed in Table 2. Compounds 1–5, which possess only one of the
phosphoryl groups, were observed to bind significantly less tightly than
inhibitors 7–10, which possess two properly positioned phosphoryl
groups. This finding shows that both alkylphosphate/phosphonate
ring substituents (R1 and R2) contribute to inhibitor binding affinity.
Comparisons of the inhibition constants of inhibitors 7–9 to that of
inhibitor 6, indicate that the longer R2 linker generates more binding
energy. Elaboration of inhibitor 8 by incorporation of a (S)-hydroxyl
substituent on the R2 linker was found to have little effect on binding as
shown by the inhibition constant of inhibitor 9(S). However, incorpo-
ration of a (R)-hydroxyl substituent caused a slight impairment in
binding as noted for inhibitor 9 (Table 2). Replacement of the Zn2+
binding group O=C–C–OH in inhibitor 8 with the HO–C–C–OH moiety
in inhibitor 10 did not alter the value of the inhibition constant.
3.4. X-ray crystallographic structure determination of GlFBPA bound
with compound 8
In order gain further insight into the structural determinants of
inhibitor binding, the X-ray structure of GlFBPA, complexed with
inhibitor 8, was determined. The 2.3Å resolution structure was
obtained by first soaking GlFBPA–TBP crystals [25] with inhibitor
8 to replace the TBP ligand. The crystallization and refinement
statistics are listed in Table 1, and the stereo-picture depicting of the
ligand electron density is shown in Fig. 4A. As predicted by analysis of
the docking model, the CH2PO3 group of inhibitor 8 fits nicely in the
desolvated pocket where the TBP (C1)phosphate binds [25]. Likewise,
the CH2CH2OPO3 substituent of inhibitor 8 is located in the depression
at the protein surface with its phosphate group bound by the residues
that form the TBP C(6)phosphate binding site (Fig. 4B). Comparison of
the GlFBPA active site binding interactions observed with inhibitor
8 (Fig. 4C) to those occurring with TBP (Fig. 2B) leads to the
Table 2
GlFBPA inhibition constants (Ki) measured for compounds 1–10 vs FBP at pH 7.5 and
25 °C (see the experimental procedures in the Appendix for details).
Compound
Ki (μM)
Compound
Ki (μM)
1
2
3
4
5
6
1200 200
810 50
970 50
900 200
2300 500
800 100
7
8
9R
9S
10
TBP
110 10
14
90
10
15
1
4
6
2
2

Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
515
Fig. 4. A. Stereoviews of the GlFBPA–inhibitor 8 complex electron density map showing the electron density of inhibitor 8 and the Zn2+ cofactor. B. Superposition of the structures of
the GlFBPA–TBP and GlFBPA–inhibitor 8 complexes. The FBP is colored yellow and the inhibitor 8 is colored cyan (carbon), red (oxygen) and orange (phosphorus). The surface of the
GlFBPA–inhibitor 8 complex is shown in gray. C. Chemdraw representation of the nonbonding interactions observed for inhibitor 8 in the GlFBPA–inhibitor 8 complex. The distances
are provided in Ås.
conclusion that inhibitor scaffold design is successful. Specifically,
each of the targeted hydrogen bond donors or acceptors observed in
the GlFBPA–TBP complex along with the Asp83, which forms a
hydrogen bond to the pyridone ring OH, are observed to favorably
interact with the ligand in the GlFBPA–inhibitor 8 complex, although
no additional binding energy is gained through incorporation of the
hydroxyl substituent on the first carbon atom of the CH2CH2OPO3
group (compounds 9S and 9R) (Table 2).
The binding of inhibitor 8 to GlFBPA, in analogy to substrate
binding, induced the crucial conformational change that locks down
the active site and places the Zn2+ in the proximity of the reaction
center.1 This change in Zn2+ coordination is depicted in Fig. 5. In order
to gain binding energy through interaction of the Zn2+cofactor with
the 3-hydroxy-2-pyridinone unit, the Zn2+ must form short coordi-
nation bonds (2.1 Å) with the O=C–C–OH oxygen atoms. The high B
factor observed for the Zn2+, coupled with elongated bond distances
between Zn2+ and the three histidine ligands (2.4–2.8 Å) and the
E135
E135
O
O
O
O
H210
Zn
N
NH
H210
H178
N
NH
H178
NH
H84
N
N
HN
NH
H84
N
Zn
N
HN
O
O
1
The change in the Zn2+ coordination depicted in Fig. 5 is based on the comparison
D83
H
O
of the structures of H. pylori apo FBPA (determined at 1.8 Å resolution; PDB accession
code 3C4U) and the corresponding liganded structures (determined at 2.3 Å
resolution; PDB accession codes 3C56 and 3C52) as well as on the comparison of
the structure of G. intestinalis apo FBPA (determined at 2.9 Å resolution; PDB accession
code 3GAK) and the TBP liganded structure (determined at 1.8 Å resolution; PDB
accession code 2ISW).
O
D83
R
N
R
O
O
Fig. 5. Chemdraw representation of the anticipated change in the GlFBPA Zn+2
coordination state upon inhibitor 8 association.

516
Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
TBP are nearly indistinguishable from the unliganded enzyme. The
XANES and EXAFS for GlFBPA–PGH show some minor perturbations
relative to theunligandedenzyme. However, the slight(0.04 Å) increase
in first shell bond length is insufficient to suggest an increase in
coordination number, although it may be indicative of slight geometric
differences in the TBP and PGH complexes. The data for the GlFBPA–
inhibitor 8 complex show minimal perturbation of the XANES and only
0.007 Å difference in average first shell bond length, which we take to
indicate that Zn2+ in the GlFBPA–8 complex coordinates to one oxygen
atom of the pyridinone O=C–C–OH unit of inhibitor 8.
4. Discussion
The EXAFS data measured for the GlFBPA–inhibitor 8 complex
suggests that the Zn2+ cofactor does engage one of the pyridinone
ring oxygen atoms in coordination. However, based on inspection of
recent X-ray structures of liganded bacterial class II FBPAs we surmise
that the coordination bond will be weak. Specifically, it was reported
by Fonvielle et al. [41] that N-(3-hydroxypropyl)-glycolohydroxamic
acid bisphosphate (PGH-PrP), a hydroxamic acid analog of FBP, binds
to the H. pylori FPBA with high affinity (Ki =0.01 μM) yet without the
anticipated short-bond, in-plane, bidentate coordination geometry
between the hydroxamic group and the Zn2+ cofactor. The long-range
(C=O at 2.9 Å and N–OH at 2.5 Å), “out-of-plane” interaction between
Zn2+ and the hydroxamic acid group indicates that the binding energy
is primarily derived from hydrogen bonding interactions between
active site residues and the phosphonate and hydroxamic acid groups.
PGH-PrP presents both a flexible, substrate-like scaffold and a
powerful Zn2+ binding group. The absence of tight, bidentate Zn2+
coordination in this complex is striking, but can be rationalized in
light of the respective structures of the Mycobacterium tuberculosis
FBPA bound with FBP or the charged enediolate form of DHAP
reported by Mesecar et al. [42]. Whereas the Zn2+ is observed to be
centered above the plane of FBP O=C(2)–C(3)(OH)–C(4)OH moiety
and thus not engaged in strong coordination to any one of the three
potential oxygen ligands, the DHAP enediolate participates in strong,
in-plane bidentate coordination of Zn2+ (C(1)O at 2.1 Å and C(2)O at
2.2 Å). The DHAP enediolate is the reaction intermediate formed by
the C(3)–C(4) cleavage step of FBPA catalysis (Fig. 1).
Taken together, the structures of FBPA bound with substrate (FBP)
or substrate mimics (TBA, PGH-PrP and inhibitor 8) provide solid
evidence that the Zn2+ cofactor does not engage the substrate in
strong coordination bonding and, thus, it does not significantly
contribute to the substrate binding energy. The structure of FBPA
bound with the DHAP enediolate is definitive proof that the Zn2+
cofactor engages in strong coordination bonding as the substrate
changes to product along the reaction coordinate. The ability of a Zn2+
cofactor to modify its coordination with the reactant as it is transformed
along the enzyme catalyzed reaction coordinate is well documented
[43].
Fig. 6. Fourier transformed Zn K-edge EXAFS (solid lines) and corresponding best fits
(open symbols, see Table 3) measured for unliganded GlFBPA (labeled “wild-type”) and
GlFBPA complexes of inhibitor 8, PGH and TBP. See the experimental procedures in the
Appendix for details.
O=C–C–OH group of compound 8 (2.7–2.9 Å), indicated that the Zn2+
site is not well ordered in the structure. Therefore, we interrogated
the Zn2+ coordination state in the GlFBPA–inhibitor 8 complex in
solution (see below).
3.5. X-ray absorption spectral determination of GlFBPA complexes
Solution X-ray absorption spectroscopy, which offers an alternative
method for examining Zn2+ coordination in protein complexes, was
usedasanindependentmethodtodetermineifcompound8coordinates
to the GlFBPA Zn2+. X-ray absorption spectra were obtained for the
unliganded enzyme and for GlFBPA bound with inhibitor 8, as well as for
GlFBPA bound with the inhibitors phosphoglycolohydroxamate (PGH)
[13] and TBP [25]. The Fourier transformed Zn K-edge EXAFS spectra are
shown in Fig. 6 and curve fittingresults are summarized inTable 3 and in
Appendix A (Table SI1); XANES spectra and fits to raw EXAFS data are
presented in Fig. SI1 and Table SI2 (Appendix A), respectively. Although
high-resolution X-ray data are not available for the unliganded GlFBPA,
it is reasonable to assume that the 4-coordinate geometry observed for
the unliganded H. pyloriFBPA (three His ligandsplus the Glu ligand) [25]
is representative of that of the unliganded GlFBPA (Fig. 5). The XANES
and EXAFS data for unliganded GlFBPA (Figs. 6 and SI1 (Appendix A),
Table 3) support this expectation. The best fit average bond length of
2.03 Å is wholly consistent with those determined crystallograhically
for four-coordinate Zn(II) complexes containing all low-Z (N/O) donors.
High-resolution (1.8 Å) X-ray structures are available for the GlFBPA–
PGH [13] and GlFBPA–TBP [25] complexes, and these structures clearly
define a 4-coordinate Zn2+, consisting of three histidine ligands (H210,
H178, H84) and an oxygen atom from the inhibitor ligand. The XANES
spectrum, and the EXAFS-determined bond length of 2.03 Å for GlFBPA–
Table 3
Best fits to EXAFS data measured for apo GlFBPA and GlFBPA–ligand complexes.a
Zn–Hisb
Rfc
Ru
c
Model
Zn–N/O
apo GlFBPA
GlFBPA–8
GlFBPA–PGH
GlFBPA–TBP
4 at 2.030 (6.7)
4 at 2.023 (6.4)
4 at 2.068 (4.6)
4 at 2.031 (8.5)
2.91 (3.4)
2.92 (2.6)
2.92 (5.4)
2.95 (10)
3.17 (3.8)
3.19 (1.7)
3.20 (2.1)
3.17 (2.9)
4.22 (14)
4.20 (24)
4.18 (8.3)
4.20 (11)
4.41 (16)
4.42 (19)
4.40 (18)
4.40 (16)
106
77
78
448
357
366
467
102
a
Distances (Å) and disorder parameters (in parentheses, σ2 (10−3 Å2)) shown derive from integer coordination number fits to filtered EXAFS data [Δk=1–11 Å−1; ΔR=0.3–
4.0 Å].
b
c
Multiple scattering paths represent combined paths, as described previously (see Materials and methods).
È
É
É
N
2
2
∑
½Reðχi calc Þꢀ
+
Im
χ
½
ð
Þꢀ
Þꢀ
i
calc
Goodness of fit (Rf for fits to filtered data; Ru for fits to raw data) defined as 1000⁎ i = 1
, where N is the number of data points.
È
N
2
2
∑
Re χ
ð
+ Im χ
i
obs
½
Þꢀ
½
ð
i
obs
i = 1

Z. Li et al. / Journal of Inorganic Biochemistry 105 (2011) 509–517
517
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Abbreviations
FBP
Fructose 1,6 bisphosphate
FBPA
fructose 1,6 bisphosphate aldolase
GlFBPA Giardia lamblia fructose 1,6 bisphosphate aldolase
TBP
tagatose 1,6 bisphosphate
G3P
glyceraldehyde 3-phosphate
DHAP
HEPES
EXAFS
HEPES
EXAFS
dihydroxyacetone phosphate
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
extended X-ray absorption fine structure
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
extended x-ray absorption spectroscopy
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2002.
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