# Structure-Based Inhibitors Exhibit Differential Activities againstHelicobacter pylori and Escherichia coli Undecaprenyl Pyrophosphate Synthases

Authors: Chih-Jung Kuo; Rey-Ting Guo; I-Lin Lu; Hun-Ge Liu; Su-Ying Wu; Tzu-Ping Ko; Andrew H.-J. Wang; Po-Huang Liang
Journal: Journal of Biomedicine and Biotechnology
 (2008-01-03)

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## Abstract

Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase
(UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate  to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against
Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.


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## Body

## 1. Introduction

Undecaprenyl pyrophosphate
synthase (UPPS) catalyzes consecutive condensation reactions of farnesyl
pyrophosphate (FPP) with eight molecules of isopentenyl pyrophosphate (IPP) to
form C55 undecaprenyl pyrophosphate (UPP), which acts as a lipid
carrier to mediate bacterial peptidoglycan biosynthesis [1, 2].  This enzyme
belongs to a group of cis-prenyltransferases which catalyze cis-double
bonds during IPP condensation reactions [3, 4]. UPPS was first cloned from Micrococcus
luteus and Escherichia coli, and their amino acid sequences were
found conserved among the cis-prenyltransferases, but totally different
from those of the trans-prenyltransferases [5–7], implying different catalytic mechanism
[8, 9].Helicobacter
pylori is a pathogen which causes
chronic inflammation in the stomach [10]. The infection may evolve to peptic
ulcerations and gastric neoplasias. Due to its unusual ability to survive in
stomach under the low pH condition via proton pumps, H. pylori infection
becomes wide spreading and accounts for the increased cases of stomach
carcinogenesis [11]. Antibiotics, such as proton pump inhibitors (PPI), amoxicillin, and clarithromycin, are used to treat
the infected patients. When failed, empirical quadruple therapy
(PPI-bismuch-tetracyclin-metronidazole) is then used as the second-line therapy
[12]. Since UPPS is essential for bacterial survival, it could possibly serve
as a target for new antibiotics. Even though the complex structures of E. coli UPPS
with the FPP substrate or with its analogue (farnesyl thiopyrophosphate, FsPP)
and IPP have been obtained [9, 13], no UPPS structure-derived inhibitors have
been reported so far. As shown in this study, we solved the crystal structures
of H. pylori UPPS and performed structure-based
inhibitor discovery. Two hits were discovered through computer virtual
screening from 58,635 compounds, which exhibited different level of inhibition
against E. coli and H.
pylori UPPS.


## 2. Materials and Methods

### 2.1. Overexpression of H. pylori UPPS

The gene encoding UPPS from theH. pylori (ATCC43504) genomic DNA was amplified by using polymerase chain reaction (PCR).
The forward primer 5′-GGTATTGAGGGTCGCTTGGATAGCACTCTCAAA-3′ and reverse primer 5′-AGAGGAGAGTTAGAGCCCTAGCATTTTAATTCCCC-3′ were utilized in the PCR. The PCR product was purified from 0.8% agarose
gel electrophoresis. The DNA product was ligated with pET-32Xa/LIC vector
and transformed into E. coli BL21 (DE3) for protein
expression as previously described for expressing
E. coli UPPS [14].The C234A mutant was prepared by using QuikChange
Site-Directed Mutagenesis Kit in conjunction with the wild-type gene template
in the pET32Xa/Lic vector. The mutagenic
forward primer was5′-CGCAAATTCGGGGAATTAAAAGCCTAGTGAGGCTCTAACTCT-3′.
The procedure of mutagenesis utilized a supercoiled double-stranded DNA (dsDNA)
vector with an insert of interest and two synthetic forward
and backward primers containing the desired mutation. The mutation was
confirmed by sequencing the entire UPPS mutant gene of the plasmid obtained
from overnight culture. The correct construct was subsequently transformed
to E. coli BL21(DE3) for protein expression. The procedure
for protein purification followed our reported protocol [15]. Each purified mutant UPPS was
verified by mass spectroscopic analysis and its purity (>95%) was checked by
SDS-PAGE.


### 2.2. Crystallization and Data Collection

H. pylori C234A UPPS mutant was crystallized using
the hanging drop method from Hampton Research (Laguna Niguel, Calif,
USA) by mixing 2 μL of the UPPS solution (10 mg/mL in 25 mM Tris,
150 mM NaCl, pH 8.0) with 2 μL of the mother
liquor (0.15 M KSCN, 15% PEG600, and 2% PEG5KMME),
and equilibrating with 500 μL of the mother liquor. Within 4 days,
crystals grew to dimensions of about 0.5 × 0.5 × 0.2 mm, and then the crystals were soaked with a cryoprotectant
solution of 0.2 M KSCN, 30% PEG600, and 5% PEG5KMME for 1 day. The structure of the C234A H. pylori UPPS in
complex with FsPP was obtained by soaking the crystals with cryoprotectant solution
of 2.5 mM MgCl2, 2.5 mM IPP,
2.5 mM FsPP, 0.15 M KSCN, 15% PEG600, and 2% PEG5KMME. However,
only the pyrophosphate of FsPP was found in the complex structure. The X-ray
diffraction datasets for the structures of the C234A UPPS mutant and the complex with FsPP were collected to 1.88 Å and 2.5 Å resolution, respectively.
Data for the C234A UPPS crystals were collected at beam line BL17B2 of the
National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan).
Data for the C234A UPPS complexed with FsPP were collected in
house using a Rigaku MicroMax002 X-ray generator equipped with an
R-Axis IV++ image plate detector. The diffraction data were processed using the
programs of HKL and HKL2000 [16]. Statistics for
the dataset are listed in Table 1. Prior to use in structural refinements, 5% randomly selected reflections were set aside for calculating Rfree as a monitor
[17].Table 1
Data collection and refinement statistics for the
orthorhombicH. pylori UPPS crystals
of the apoenyzme and the complex with thiopyrophosphate.
C234A mutation was included to prevent intramolecular
disulfide bond formation.
H. pylori UPPSH. pylori UPPS + PPiData collectionSpace groupP212121Resolution (Å)a25 to 1.88 (1.95 to 1.88)50 to 2.5 (2.59 to 2.5)Unit cell dimensionsa, b, c (Å)49.63, 58.91, 153.43No. of reflectionsObserved201171
(18692)137910
(12888)Unique35917
(3338)15618
(1432)Completeness
(%)95.4
(90.3)96.0
(91.2)Rmerge (%)5.5
(43.3)5.9
(15.9)I/σ(I)30.7
(4.1)42.3
(5.2)RefinementNo. of  reflectionsb34629
(3038)15084
(1330)Rwork (%)19.34
(22.91)21.44
(30.24)Rfree (%)24.00
(30.02)29.33
(37.43)Geometry
deviationsBond lengths (Å)0.01930.0061Bond angles (°)1.8171.157No. of
all non-H atoms34633449No. of
water molecules581134Mean B-values (Å2)39.5449.49Ramachandran plot (%)Most
favored92.192.3Additionally allowed7.97.7aValues in the parentheses are for the highest resolution shells.
bAll positive reflections are used in the refinements.


### 2.3. Structure Determination and Refinement

The crystal structure of C234A UPPS was determined by molecular replacement
method using theCrystallography & NMR System (CNS) program [18]. The orthorhombic crystal contained one UPPS dimer in an asymmetric unit. The models of PDB 1V7U (E. coli UPPS structure bound with FPP, chain A) [13] were used as search model to yield a good resolution for the H. pylori UPPS.
The space group was determined as P212121.
With all solvent and cofactor molecules removed, the model yielded an initial
R-value of 0.50 using all positive reflections at 1.88 Å resolution upon
rigid-body refinement.The 2Fo-Fc difference Fourier map showed clear electron densities for most
amino acid residues. The residues of catalytic loop of 58–67 in chain
A, 56–71 and 150–158 in chain B were disordered.
Subsequent refinement with incorporation of 581 water
molecules according to 1.0 σ  map level yieldedR and Rfree
values of 0.193 and 0.240, respectively, at 1.88 Å resolution.
By employing similar procedures, the C234A H. pylori
UPPS and the FsPP-complexed structures were refined with the
addition of cofactor and solvent molecules. All manual modifications of the
models were performed on an SGI Fuel computer using the program
O [19]. Computational refinements, which
included maximal likelihood and simulated-annealing protocols, were carried out
using CNS. The programs MolScript [20],
and Raster3D [21] were used in
producing figures.


### 2.4. Computer Screening to Identify the Inhibitors

The X-ray structure ofH. pylori UPPS reported
here and the complex structure of E. coli UPPS (PDB
code 1V7U) were chosen as the templates in the virtual screening.
The program GOLD V2.1 was used to screen Maybridge
database, a commercially available compound database obtained from Maybridge
Chemical Company (Tintagel, Cornwall, England). The binding pocket for the docking study was defined as a 15 Å radius sphere centered on the active
site Asp13 of H. pylori UPPS or Asp26 of
E. coli UPPS. The scoring function,
GoldScore, implemented in GOLD was used to rank the docking positions of the compounds. 26 compounds with the highest score ranked by GoldScore were selected for inhibition assays.


### 2.5. IC50 Determination

The IC50 values of the two hits
were measured in a buffer of 100 mM Hepes (pH 7.5), 50 mM KCl,
0.5 mM MgCl2, and 0.1% Triton X-100,
containing 0.05 μM of
E. coli or H. pylori UPPS. The concentrations
of inhibitors used were ranged from 0 to 500 μM. To obtain the
IC50, the does-response curves were fitted with the equation,
A(I)=A(0)×{1−[I/(I+IC50)]},
where A(I) is the enzyme activity with inhibitor concentration I, A(0)
is enzyme activity without inhibitor, and I is the inhibitor concentration.


## 2.1. Overexpression of H. pylori UPPS

The gene encoding UPPS from theH. pylori (ATCC43504) genomic DNA was amplified by using polymerase chain reaction (PCR).
The forward primer 5′-GGTATTGAGGGTCGCTTGGATAGCACTCTCAAA-3′ and reverse primer 5′-AGAGGAGAGTTAGAGCCCTAGCATTTTAATTCCCC-3′ were utilized in the PCR. The PCR product was purified from 0.8% agarose
gel electrophoresis. The DNA product was ligated with pET-32Xa/LIC vector
and transformed into E. coli BL21 (DE3) for protein
expression as previously described for expressing
E. coli UPPS [14].The C234A mutant was prepared by using QuikChange
Site-Directed Mutagenesis Kit in conjunction with the wild-type gene template
in the pET32Xa/Lic vector. The mutagenic
forward primer was5′-CGCAAATTCGGGGAATTAAAAGCCTAGTGAGGCTCTAACTCT-3′.
The procedure of mutagenesis utilized a supercoiled double-stranded DNA (dsDNA)
vector with an insert of interest and two synthetic forward
and backward primers containing the desired mutation. The mutation was
confirmed by sequencing the entire UPPS mutant gene of the plasmid obtained
from overnight culture. The correct construct was subsequently transformed
to E. coli BL21(DE3) for protein expression. The procedure
for protein purification followed our reported protocol [15]. Each purified mutant UPPS was
verified by mass spectroscopic analysis and its purity (>95%) was checked by
SDS-PAGE.


## 2.2. Crystallization and Data Collection

H. pylori C234A UPPS mutant was crystallized using
the hanging drop method from Hampton Research (Laguna Niguel, Calif,
USA) by mixing 2 μL of the UPPS solution (10 mg/mL in 25 mM Tris,
150 mM NaCl, pH 8.0) with 2 μL of the mother
liquor (0.15 M KSCN, 15% PEG600, and 2% PEG5KMME),
and equilibrating with 500 μL of the mother liquor. Within 4 days,
crystals grew to dimensions of about 0.5 × 0.5 × 0.2 mm, and then the crystals were soaked with a cryoprotectant
solution of 0.2 M KSCN, 30% PEG600, and 5% PEG5KMME for 1 day. The structure of the C234A H. pylori UPPS in
complex with FsPP was obtained by soaking the crystals with cryoprotectant solution
of 2.5 mM MgCl2, 2.5 mM IPP,
2.5 mM FsPP, 0.15 M KSCN, 15% PEG600, and 2% PEG5KMME. However,
only the pyrophosphate of FsPP was found in the complex structure. The X-ray
diffraction datasets for the structures of the C234A UPPS mutant and the complex with FsPP were collected to 1.88 Å and 2.5 Å resolution, respectively.
Data for the C234A UPPS crystals were collected at beam line BL17B2 of the
National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan).
Data for the C234A UPPS complexed with FsPP were collected in
house using a Rigaku MicroMax002 X-ray generator equipped with an
R-Axis IV++ image plate detector. The diffraction data were processed using the
programs of HKL and HKL2000 [16]. Statistics for
the dataset are listed in Table 1. Prior to use in structural refinements, 5% randomly selected reflections were set aside for calculating Rfree as a monitor
[17].Table 1
Data collection and refinement statistics for the
orthorhombicH. pylori UPPS crystals
of the apoenyzme and the complex with thiopyrophosphate.
C234A mutation was included to prevent intramolecular
disulfide bond formation.
H. pylori UPPSH. pylori UPPS + PPiData collectionSpace groupP212121Resolution (Å)a25 to 1.88 (1.95 to 1.88)50 to 2.5 (2.59 to 2.5)Unit cell dimensionsa, b, c (Å)49.63, 58.91, 153.43No. of reflectionsObserved201171
(18692)137910
(12888)Unique35917
(3338)15618
(1432)Completeness
(%)95.4
(90.3)96.0
(91.2)Rmerge (%)5.5
(43.3)5.9
(15.9)I/σ(I)30.7
(4.1)42.3
(5.2)RefinementNo. of  reflectionsb34629
(3038)15084
(1330)Rwork (%)19.34
(22.91)21.44
(30.24)Rfree (%)24.00
(30.02)29.33
(37.43)Geometry
deviationsBond lengths (Å)0.01930.0061Bond angles (°)1.8171.157No. of
all non-H atoms34633449No. of
water molecules581134Mean B-values (Å2)39.5449.49Ramachandran plot (%)Most
favored92.192.3Additionally allowed7.97.7aValues in the parentheses are for the highest resolution shells.
bAll positive reflections are used in the refinements.


## 2.3. Structure Determination and Refinement

The crystal structure of C234A UPPS was determined by molecular replacement
method using theCrystallography & NMR System (CNS) program [18]. The orthorhombic crystal contained one UPPS dimer in an asymmetric unit. The models of PDB 1V7U (E. coli UPPS structure bound with FPP, chain A) [13] were used as search model to yield a good resolution for the H. pylori UPPS.
The space group was determined as P212121.
With all solvent and cofactor molecules removed, the model yielded an initial
R-value of 0.50 using all positive reflections at 1.88 Å resolution upon
rigid-body refinement.The 2Fo-Fc difference Fourier map showed clear electron densities for most
amino acid residues. The residues of catalytic loop of 58–67 in chain
A, 56–71 and 150–158 in chain B were disordered.
Subsequent refinement with incorporation of 581 water
molecules according to 1.0 σ  map level yieldedR and Rfree
values of 0.193 and 0.240, respectively, at 1.88 Å resolution.
By employing similar procedures, the C234A H. pylori
UPPS and the FsPP-complexed structures were refined with the
addition of cofactor and solvent molecules. All manual modifications of the
models were performed on an SGI Fuel computer using the program
O [19]. Computational refinements, which
included maximal likelihood and simulated-annealing protocols, were carried out
using CNS. The programs MolScript [20],
and Raster3D [21] were used in
producing figures.


## 2.4. Computer Screening to Identify the Inhibitors

The X-ray structure ofH. pylori UPPS reported
here and the complex structure of E. coli UPPS (PDB
code 1V7U) were chosen as the templates in the virtual screening.
The program GOLD V2.1 was used to screen Maybridge
database, a commercially available compound database obtained from Maybridge
Chemical Company (Tintagel, Cornwall, England). The binding pocket for the docking study was defined as a 15 Å radius sphere centered on the active
site Asp13 of H. pylori UPPS or Asp26 of
E. coli UPPS. The scoring function,
GoldScore, implemented in GOLD was used to rank the docking positions of the compounds. 26 compounds with the highest score ranked by GoldScore were selected for inhibition assays.


## 2.5. IC50 Determination

The IC50 values of the two hits
were measured in a buffer of 100 mM Hepes (pH 7.5), 50 mM KCl,
0.5 mM MgCl2, and 0.1% Triton X-100,
containing 0.05 μM of
E. coli or H. pylori UPPS. The concentrations
of inhibitors used were ranged from 0 to 500 μM. To obtain the
IC50, the does-response curves were fitted with the equation,
A(I)=A(0)×{1−[I/(I+IC50)]},
where A(I) is the enzyme activity with inhibitor concentration I, A(0)
is enzyme activity without inhibitor, and I is the inhibitor concentration.


## 3. Results

### 3.1. 3D Structures of H. pylori UPPS

To develop structure-based inhibitors, the crystal structures ofH. pylori UPPS were solved in this study. One is the structure of H. pylori UPPS containing C234A mutation to prevent intra-molecular disulfide
bond formed during the long period of crystallization
process (Figure 1(a)), and
the other is the structure of C234A complexed with FsPP, but only the pyrophosphate portion is visible (Figure 1(b)). The
C234A mutant has unchanged kinetic property compared with the wild type
(kcat, FPP
Km and IPP Km
of C234A were 0.20±0.08 s-1, 0.15±0.04 μM
and 9.6±0.2 μM,
almost equal to
0.22±0.05
s-1,
0.11±0.02 μM and
9.2±0.1 μM for the wild
type, resp.). The overall structure of H. pylori
UPPS was similar to that of E. coli UPPS
[22]. The protein is a dimer and each
subunit contains a catalytic domain and a pairing domain. Two subunits are
tightly associated through the central β-sheet and a pair of long α-helices (α5
and α6). However, H. pylori UPPS has a 1.5-turn shorter α5
helix in the dimer interface. This may weaken the dimer formation for
H. pylori UPPS. The
catalytic domain is composed of six β-strands and four β-helices and the
central tunnel-shaped active site is surrounded by 2 α-helices (α2 and α3) and
4 β-strands (βA-βB-βD-βC) (Figure 1(a)).Crystal structures of H. pylori UPPS. (a) Two subunits of the apoenzyme are superimposed. The most obvious disposition occurs in α3
helix which adopts an open form and a closed form in subunit A and B,
respectively. At the top of the tunnel-shaped crevice surrounded by 2α-helices
and 4β-strands is the substrate-binding site. Phe124 located at the bottom of the
H. pylori UPPS tunnel adopts a similar position to that of Leu137
in E. coli UPPS, essential for determining product chain length.
(b) Superimposition of active site structures of H. pylori UPPS with FsPP and E. coli UPPS with FsPP, Mg2+,
and IPP [9]. The active site residues in
H. pylori UPPS are shown in pink and those in
E. coli UPPS in white for carbon-carbon bonds in
ball-and-stick model. The thiopyrophosphate (visible in crystal structure) is shown in black, the nitrogen atoms and Mg2+ ion are shown in blue, and oxygen atoms are shown in red. Asp13 in H. pylori UPPS occupies a similar
position to that of Asp26 in E. coli UPPS to coordinate
with an Mg2+ for binding with the pyrophosphate leaving group of FPP.
(a)(b)At the bottom of the tunnel, a large amino acid F124 occupies a similar position to that of L137 at the bottom ofE. coli UPPS tunnel, which is a key residue to shield the final product and determine its chain length
[22]. At the top of this tunnel, several amino acids including D13, R17, R26, H30, F57, S58, R180, and E184 are located in the substrate binding site (Figure 1(b)).
The position of the pyrophosphate (shown in black sticks in Figure 1(b)) of FsPP in the complex is almost identical to that of the FPP pyrophosphate in the E. coli UPPS active site
[13].The positions of the α3 helix in the two subunits ofH. pylori UPPS are slightly different
(Figure 1(a)),
resembling the open and closed forms of E. coli
UPPS [22]. H. pylori
UPPS A-chain strongly resembles the Triton-bound open form of
E. coli UPPS [23],
with root mean square deviation (r.m.s.d) of 0.78 Å for
200 match pairs of α-carbon atoms. Compare to the closed-form structure of
E. coli UPPS with FsPP and IPP bound
[9], the H. pylori
UPPS B-chain is with the r.m.s.d. of 1.08 Å for 191 match pairs of α-carbon atoms. This suggests
a conformational change in the H. pylori UPPS reaction.


### 3.2. Virtual Screening of the H. pylori UPPS Inhibitors

Based on the structures, computer virtual screening was carried out to search for
selective inhibitors ofE. coli and H. pylori UPPS. The screening
procedure is summarized in Figure 2. The crystal structure of E. coli UPPS
bound with FPP (1V7U) was used as a template first for the virtual screening
since the electron density of a small loop responsible for conformational
change near the active site is not visible in H. pylori UPPS, which
might confound the virtual screening result. A compound database containing
58,635 compounds available from Maybridge Chemical Company were screened using
the program GOLD V2.1. Each compound in the database was docked into the active
site of E. coli UPPS, defined as 15 Å
radius sphere around Asp26, an essential residue responsible to coordinate with
the catalytic Mg2+. The docked molecules were then ranked by the
GoldScore fitness function, according to the sum of H-bond energy, van der
Waals energy, internal ligand van der Waals and internal torsional strain
energy. The top 26 compounds ranked by GoldScore were then purchased and
experimentally evaluated for their ability to inhibit H. pylori and E. coli UPPS.Figure 2
The flow chart for computer screening of H. pylori UPPS inhibitors. The active zone for screening was focused on Asp13, an important amino acid residue for coordinating with catalytic Mg2+.
In parentheses are the numbers of compounds. BTB06061 and HTS04781 are
the final hits.


### 3.3. Inhibition Against E. coli and H. pylori UPPS

Of these 26 compounds, 2 compounds
numbered BTB06061 and HTS04781, were found inhibitory toH. pylori UPPS almost equally with IC50 values of 350 μM and 362 μM, respectively (Figure 3). The IC50 values of these two compounds against the C234A and wild-type
enzyme were almost equal. As revealed by the predicted models shown in
Figures 3(a) and 3(b),
two inhibitors are likely bound to H. pylori UPPS with
a similar orientation to that of the substrate FPP. The sulfur atom in the thiazole ring of BTB06061 may form H-bonds with Asn15 and His30 while the SO2 group is hydrogen bound with Met12. In addition, the aromatic rings of BTB06061
form hydrophobic interactions with the surrounding hydrophobic residues, including Val34, Leu37, Ala56 and Tyr79. As shown in the predicted model of HTS04781 with
H. pylori UPPS, the sulfonamide group forms H-bonds with Gly16 and Arg26 and the N atom in the tetracyclic ring is hydrogen bound to the main chain
of Met12. Extensive hydrophobic interactions were found between the tetracyclic ring with the surrounding residues including Met12, His30, Gly33 and Val34.Computer virtual screening of the H. pylori UPPS inhibitors. Two compounds, BTB06061 shown in (a) and HTS04781 in (b), were identified from the computer fitting of the Maybridge compounds into the active site of E. coli and H. pylori UPPS. The data of enzyme activities in the presence of different concentrations of the inhibitors were used to determine the
IC50 values of the inhibitors. The compounds displayed IC50 of 350 and 363 μM, respectively, in inhibiting
H. pylori UPPS activity. However, the IC50 of BTB06061 became 71 μM in inhibiting E. coli UPPS and HTS04781 was
almost inactive against the enzyme. The modeled structures of the inhibitor bound in
the active site of H. pylori UPPS are shown at the bottom.
(a)(b)Surprisingly, BTB06061 showed 5-fold smaller IC50 (71 μM)
against E. coli UPPS and HTS04781 almost did not inhibit
E. coli UPPS, although two compounds inhibited
H. pylori equally. From the modeling (not
shown), the smaller entrance in E. coli UPPS compared to
H. pylori UPPS at the top of the tunnel due to the partial blockage by the amino acids such as Trp75 from the flexible loop might restrict, or at least partially limit the access of bulky compound HTS04781 that contains four rigid aromatic rings to the active site, thereby leading to the loss of inhibitory activity when
competing with the substrate for binding.


## 3.1. 3D Structures of H. pylori UPPS

To develop structure-based inhibitors, the crystal structures ofH. pylori UPPS were solved in this study. One is the structure of H. pylori UPPS containing C234A mutation to prevent intra-molecular disulfide
bond formed during the long period of crystallization
process (Figure 1(a)), and
the other is the structure of C234A complexed with FsPP, but only the pyrophosphate portion is visible (Figure 1(b)). The
C234A mutant has unchanged kinetic property compared with the wild type
(kcat, FPP
Km and IPP Km
of C234A were 0.20±0.08 s-1, 0.15±0.04 μM
and 9.6±0.2 μM,
almost equal to
0.22±0.05
s-1,
0.11±0.02 μM and
9.2±0.1 μM for the wild
type, resp.). The overall structure of H. pylori
UPPS was similar to that of E. coli UPPS
[22]. The protein is a dimer and each
subunit contains a catalytic domain and a pairing domain. Two subunits are
tightly associated through the central β-sheet and a pair of long α-helices (α5
and α6). However, H. pylori UPPS has a 1.5-turn shorter α5
helix in the dimer interface. This may weaken the dimer formation for
H. pylori UPPS. The
catalytic domain is composed of six β-strands and four β-helices and the
central tunnel-shaped active site is surrounded by 2 α-helices (α2 and α3) and
4 β-strands (βA-βB-βD-βC) (Figure 1(a)).Crystal structures of H. pylori UPPS. (a) Two subunits of the apoenzyme are superimposed. The most obvious disposition occurs in α3
helix which adopts an open form and a closed form in subunit A and B,
respectively. At the top of the tunnel-shaped crevice surrounded by 2α-helices
and 4β-strands is the substrate-binding site. Phe124 located at the bottom of the
H. pylori UPPS tunnel adopts a similar position to that of Leu137
in E. coli UPPS, essential for determining product chain length.
(b) Superimposition of active site structures of H. pylori UPPS with FsPP and E. coli UPPS with FsPP, Mg2+,
and IPP [9]. The active site residues in
H. pylori UPPS are shown in pink and those in
E. coli UPPS in white for carbon-carbon bonds in
ball-and-stick model. The thiopyrophosphate (visible in crystal structure) is shown in black, the nitrogen atoms and Mg2+ ion are shown in blue, and oxygen atoms are shown in red. Asp13 in H. pylori UPPS occupies a similar
position to that of Asp26 in E. coli UPPS to coordinate
with an Mg2+ for binding with the pyrophosphate leaving group of FPP.
(a)(b)At the bottom of the tunnel, a large amino acid F124 occupies a similar position to that of L137 at the bottom ofE. coli UPPS tunnel, which is a key residue to shield the final product and determine its chain length
[22]. At the top of this tunnel, several amino acids including D13, R17, R26, H30, F57, S58, R180, and E184 are located in the substrate binding site (Figure 1(b)).
The position of the pyrophosphate (shown in black sticks in Figure 1(b)) of FsPP in the complex is almost identical to that of the FPP pyrophosphate in the E. coli UPPS active site
[13].The positions of the α3 helix in the two subunits ofH. pylori UPPS are slightly different
(Figure 1(a)),
resembling the open and closed forms of E. coli
UPPS [22]. H. pylori
UPPS A-chain strongly resembles the Triton-bound open form of
E. coli UPPS [23],
with root mean square deviation (r.m.s.d) of 0.78 Å for
200 match pairs of α-carbon atoms. Compare to the closed-form structure of
E. coli UPPS with FsPP and IPP bound
[9], the H. pylori
UPPS B-chain is with the r.m.s.d. of 1.08 Å for 191 match pairs of α-carbon atoms. This suggests
a conformational change in the H. pylori UPPS reaction.


## 3.2. Virtual Screening of the H. pylori UPPS Inhibitors

Based on the structures, computer virtual screening was carried out to search for
selective inhibitors ofE. coli and H. pylori UPPS. The screening
procedure is summarized in Figure 2. The crystal structure of E. coli UPPS
bound with FPP (1V7U) was used as a template first for the virtual screening
since the electron density of a small loop responsible for conformational
change near the active site is not visible in H. pylori UPPS, which
might confound the virtual screening result. A compound database containing
58,635 compounds available from Maybridge Chemical Company were screened using
the program GOLD V2.1. Each compound in the database was docked into the active
site of E. coli UPPS, defined as 15 Å
radius sphere around Asp26, an essential residue responsible to coordinate with
the catalytic Mg2+. The docked molecules were then ranked by the
GoldScore fitness function, according to the sum of H-bond energy, van der
Waals energy, internal ligand van der Waals and internal torsional strain
energy. The top 26 compounds ranked by GoldScore were then purchased and
experimentally evaluated for their ability to inhibit H. pylori and E. coli UPPS.Figure 2
The flow chart for computer screening of H. pylori UPPS inhibitors. The active zone for screening was focused on Asp13, an important amino acid residue for coordinating with catalytic Mg2+.
In parentheses are the numbers of compounds. BTB06061 and HTS04781 are
the final hits.


## 3.3. Inhibition Against E. coli and H. pylori UPPS

Of these 26 compounds, 2 compounds
numbered BTB06061 and HTS04781, were found inhibitory toH. pylori UPPS almost equally with IC50 values of 350 μM and 362 μM, respectively (Figure 3). The IC50 values of these two compounds against the C234A and wild-type
enzyme were almost equal. As revealed by the predicted models shown in
Figures 3(a) and 3(b),
two inhibitors are likely bound to H. pylori UPPS with
a similar orientation to that of the substrate FPP. The sulfur atom in the thiazole ring of BTB06061 may form H-bonds with Asn15 and His30 while the SO2 group is hydrogen bound with Met12. In addition, the aromatic rings of BTB06061
form hydrophobic interactions with the surrounding hydrophobic residues, including Val34, Leu37, Ala56 and Tyr79. As shown in the predicted model of HTS04781 with
H. pylori UPPS, the sulfonamide group forms H-bonds with Gly16 and Arg26 and the N atom in the tetracyclic ring is hydrogen bound to the main chain
of Met12. Extensive hydrophobic interactions were found between the tetracyclic ring with the surrounding residues including Met12, His30, Gly33 and Val34.Computer virtual screening of the H. pylori UPPS inhibitors. Two compounds, BTB06061 shown in (a) and HTS04781 in (b), were identified from the computer fitting of the Maybridge compounds into the active site of E. coli and H. pylori UPPS. The data of enzyme activities in the presence of different concentrations of the inhibitors were used to determine the
IC50 values of the inhibitors. The compounds displayed IC50 of 350 and 363 μM, respectively, in inhibiting
H. pylori UPPS activity. However, the IC50 of BTB06061 became 71 μM in inhibiting E. coli UPPS and HTS04781 was
almost inactive against the enzyme. The modeled structures of the inhibitor bound in
the active site of H. pylori UPPS are shown at the bottom.
(a)(b)Surprisingly, BTB06061 showed 5-fold smaller IC50 (71 μM)
against E. coli UPPS and HTS04781 almost did not inhibit
E. coli UPPS, although two compounds inhibited
H. pylori equally. From the modeling (not
shown), the smaller entrance in E. coli UPPS compared to
H. pylori UPPS at the top of the tunnel due to the partial blockage by the amino acids such as Trp75 from the flexible loop might restrict, or at least partially limit the access of bulky compound HTS04781 that contains four rigid aromatic rings to the active site, thereby leading to the loss of inhibitory activity when
competing with the substrate for binding.


## 4. Discussion

In this paper, we describe the crystal structures of UPPS fromH. pyroli, a wide-spreading and life-threatening pathogen, and the first structure-derived inhibitors from computer virtual screening.
Although a high-throughput
screening has been performed for UPPS by a pharmaceutical company
[24], none of the inhibitors have been
reported. So far, a series of IPP analogues with a dicarboxylate moiety in
place of the diphosphate were synthesized and the E-pentenylbutanedioic acid
showed inhibition of UPPS with an IC50 of 135 μM [25]. Based on the known structure of UPPS (9),
two carboxylate groups may
coordinate with the catalytic Mg2+ ion which was bound with the
pyrophosphate group of the substrates. Recently, we reported some bisphosphonates, which inhibited trans-type FPPs, which could also inhibit cis-type UPPS with sub-μM IC50
when containing suitable hydrophobic side-chains [26]. The crystal structures show that four molecules of inhibitors are bound in the active site and one of them occupies the FPP site with a phosphoate group chelating with the Mg2+. Here, we report the first two novel inhibitors identified from a randomized compound library through virtual screening. These two inhibitors likely occupy the FPP site of H. pylori UPPS based on computer modeling. Two inhibitors displayed similar inhibition against H. pylori UPPS,
but very different inhibition on E. coli UPPS. The one with bulky skeleton did not inhibit E. coli UPPS, likely owing to the partially blocked opening at the top of tunnel by the flexible loop in the E. coli UPPS active site. Our results shed light on the possibility of developing antibiotics specially targeting pathogenic H. pylori
without killing the intestinal E. coli.



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*Source: 841312-2008-02-25.xml*