Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase

Article abstract of DOI:10.1021/bi100523c

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C₅₅ undecaprenyl pyrophosphate (UPP). It has been demonstr

Full text of DOI:10.1021/bi100523c

5366 Biochemistry 2010, 49, 5366–5376
DOI: 10.1021/bi100523c
Biophysical Investigation of the Mode of Inhibition of Tetramic Acids, the Allosteric
Inhibitors of Undecaprenyl Pyrophosphate Synthase
Lac V. Lee, Brian Granda, Karl Dean, Jianshi Tao, Eugene Liu, Rui Zhang, Stefan Peukert, Sompong Wattanasin,
Xiaoling Xie, Neil S. Ryder, Ruben Tommasi, and Gejing Deng*
Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139
Received April 7, 2010; Revised Manuscript Received May 14, 2010
ABSTRACT: Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight mole-
cules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C55 undecaprenyl
pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of
UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA
binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site
of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates
with UPPS and with the UPPS TA complex was investigated by protein fluorescence spectroscopy. Our results
3
suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free
UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate
binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding
mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS TA complex, but the quaternary complex,
3
UPPS TA FPP IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the
3
3
3
conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading
to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to
quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and
inhibitors produced from a medicinal chemistry program.
Undecaprenyl pyrophosphate synthase (UPPS)1 is an essential
enzyme for bacterial viability. The C55 undecaprenyl pyropho-
sphate (UPP) produced by UPPS reaction is the lipid carrier for
precursors of various cell wall structures, such as peptidoglycan,
teichoic acids, and O-antigens (1). UPPS is highly conserved
among Gram-positive and Gram-negative bacteria. The critical
biological function of UPPS, as well as an active site amenable to
small molecule inhibition as revealed by crystallographic struc-
tures (2-5), makes UPPS an attractive target for the discovery of
novel antibacterial agents.
UPPS catalyzes the condensation of eight molecules of
IPP with FPP (Scheme 1). Chen and co-workers examined the
multiple-step kinetics of the UPPS reaction and defined the
mechanism of the Escherichia coli UPPS reaction pathway using
steady-state and pre-steady-state kinetic approaches (6). E. coli
UPPS binds FPP and then IPP (ordered substrate binding
mechanism). This initial substrate binding triggers eight contin-
uous IPP condensation steps catalyzed by the enzyme leading to
the final product C55-UPP. Different product distributions were
found with various enzyme:substrate and FPP:IPP ratios in the
presence or absence of Triton. Triton is believed to activate UPPS
activity by enhancing the rate of product dissociation and the rate
of a protein conformational change (6).
UPPS inhibitors of the tetramic acid class have been identified
by high-throughput screening (HTS) and a subsequent medicinal
chemistry program (7). The complexity of the UPPS reaction
mechanism makes the characterization of UPPS inhibitors using
traditional enzyme kinetics rather challenging. To overcome this
difficulty, we used biophysical approaches to investigate the mode
of inhibition of this class of inhibitors. In this investigation, we
applied an FPP fluorescent analogue (8) to evaluate the possibility
of TA binding to the FPP binding site. A tetramic acid analogue
containing a photosensitive moiety was used to probe the site of
binding. The inhibition mechanism of tetramic acids was investi-
gated by determining the interaction of a representative tetramic
acid inhibitor with UPPS in the presence and absence of substrate
analogues (S)-farnesyl thiopyrophosphate (FsPP) and (S)-iso-
pentyl thiolopyrophosphate (IsPP) using photo-cross-linking and
protein fluorescence spectroscopy. It was also shown that fluore-
scent probe displacement and competitive photo-cross-linking
using the same photosensitive TA analogue can be used for quick
identification of HTS hits that bind to the FPP or TA site.
*To whom correspondence should be addressed: 500 Technology
Square, Cambridge, MA 02139. Telephone: (617) 871-7889. Fax: (617)
871-5791. E-mail: gejing.deng@novartis.com.
MATERIALS AND METHODS
Reagent Supply. Substrates FPP (Figure 1A) and IPP were
purchased from Sigma. FsPP (Figure 1B) and IsPP were purchased
from Echelon Biosciences Inc. The fluorescent FPP analogue
[Fluoro-probe (Figure 1C)] was chemically synthesized in house
according to the published procedure (8). A photosensitive tetra-
mic acid analogue [Photo-probe (Figure 1D)] was synthesized
1Abbreviations: UPPS, undecaprenyl pyrophosphate synthase; IPP,
isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; UPP, undeca-
prenyl pyrophosphate; TA, tetramic acid; FsPP, (S)-farnesyl thiopyropho-
sphate; IsPP, (S)-isopentyl thiolopyrophosphate; MOI, mode of inhibition;
Tris, tris(hydroxymethyl)aminomethane; PBS, phosphate-buffered saline;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMSO,
dimethyl sulfoxide; HTS, high-throughput screening.
r
pubs.acs.org/Biochemistry
Published on Web 05/17/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 25, 2010 5367
F
IGURE 1: Chemical structures of compounds used in this study.
Scheme 1: Reaction Catalyzed by Undecaprenyl Pyrophosphate Synthase
using the procedure described below. Compound 1 [5-benzyl-4-
hydroxy-5-methyl-2-oxo-2,5-dihydro-1H-pyrrole-3-carboxylic
acid (4-cyclohexylphenyl)amide, a representative tetramic acid
(Figure 1E)] and compound 2 [(4aS,8aS)-4-hydroxy-2-oxo-1,2,
4a,5,6,7,8,8a-octahydro[1,6]naphthyridine-3-carboxylic acid (4-
piperidin-1-ylphenyl)amide, a representative non-tetramic acid
(Figure 1F)] were also chemically synthesized (9, 10). Total E. coli
lipid extract was purchased from Avanti Polar Lipids, Inc. (Ala-
baster, AL). Biomol Green reagent was purchased from Enzo Life
Sciences International, Inc. (formerly Biomol International, Inc.).
4-Aminobenzophenone, methyl malonyl chloride, and N-(4-ami-
nophenyl)piperidine were purchased from Aldrich. 4-Cyclohexa-
nylaniline was purchased from Lancaster Synthesis Inc. 2-Amino-
2-methyl-3-phenylpropanoate was purchased from Chem-Impex
International. Solvents and other reagents were purchased from
Sigma-Aldrich or Fisher Scientific Inc. Water used in all experi-
ments was HPLC-grade and was purchased from Sigma.
Expression and Purification of Streptococcus pneumo-
niae UPPS. The construct used for expression of S. pneumoniae
UPPS was created using the pET15b expression vector consisting of
a thrombin-cleavable N-terminal hexahistidine sequence followed
by the S. pneumoniae UPPS sequence. pET15b-SpUPPS was trans-
formed into competent E. coli BL21(DE3) Star host cells (Invitro-
gen), and they were grown at 37 °C on a culture plate containing
imMedia AMP Agar (Invitrogen) until visible colonies were ob-
served. Cells were transferred to a culture medium containing 5%
EZ Mix Terrific Broth (Sigma), 1.5% (w/v) glucose, and 150μg/mL
ampicillin and grown at 37 °C to an OD600 of 2. Then 1 volume of
sterilized glycerol was added to 4 volumes of the E. coli culture,
which resulted in a high-density glycerol stock culture. Cells from
the stock culture were transferred to a starter medium containing
5% EZ Mix Terrific Broth, 1 mM MgSO4, 1.3% (w/v) glucose, and
150 μg/mL ampicillin and grown at 37 °C for 3 h. Following this,
cells were diluted in fresh starter medium and grew at 30 °C over-
night. Cells were harvested by centrifugation, and cell paste was sus-
pended in sterilized Terrific Broth containing 1% glucose and
150 μg/mL ampicillin. Subsequently, the suspended cells was trans-
ferred into an “auto-induction” expression culture medium contain-
ing 1% (w/v) N-Z Amine AS (Sigma-Aldrich), 0.5% (w/v) yeast
extract, 1 mM MgSO4, 25 mM (NH4)2SO4, 50 mM KH2PO4,
50 mM Na2HPO4, 0.5% (v/v) glycerol, 0.05% (w/v) glucose, 0.2%
(w/v) R-lactose, and 150 μg/mL ampicillin and grown to an OD600 of
0.6. Cells were harvested via centrifugation and washed with a buffer
containing 50 mM Tris (pH 7.5) and 100 mM NaCl. Cells were then
resuspended, homogenized, lysed, and centrifuged. The supernatant
was loaded onto a Ni-NTA column and washed with a buffer
containing 50 mM HEPES (pH 7.3), 500 mM NaCl, 10% glycerol,
1 mM TCEP-HCl, EDTA-free protease inhibitor cocktail tablets
(1 tablet/100 mL), and 20 mM imidazole and then washed with the
same buffer containing 50 mM imidazole. His-tagged SpUPPS was
eluted within a linear gradient from 200 to 300 mM imidazole.
Fractions containing S. pneumoniae UPPS were pooled and sub-
jected to centrifugal ultrafiltration against a buffer containing
50 mM HEPES (pH 7.3), 500 mM NaCl, 10% glycerol, 1 mM
TCEP-HCl, and EDTA-free protease inhibitor cocktail tablets
(1 tablet/100 mL). The protein was then loaded onto a Superdex-
200PG HiLoad size-exclusion column (GE Healthcare) and eluted
with a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, and
10% glycerol. The identity of the purified protein was confirmed
by mass spectrometry (measured molecular mass of 30735 Da,
calculated molecular mass of 30737 Da with Met1 cleaved). The
purity of the protein was estimated to be 95% by SDS-PAGE.

5368 Biochemistry, Vol. 49, No. 25, 2010
Lee et al.
Scheme 2: Synthesis of Photo-probe
Synthesis of Photo-probe. Synthesis of Photo-probe was
conducted using a four-step procedure starting from commer-
cially available 4-aminobenzophenone and methyl malonyl
chloride (Scheme 2) as described below.
was poured into 80 mL of an ice/water mixture and was extracted
twice with 100 mL of ethyl acetate each time. The combined
organic phase was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuum. The residue was purified by flash
column chromatography on silica gel with an ethyl acetate/
heptane mixture (20:80 to 80:20) which resulted in 400 mg of
compound 5 (85% yield): 1H NMR (400 MHz, CDCl3) δ 9.73 (s,
1H), 7.74-7.76 (d, 2H), 7.69-7.71 (m, 2H), 7.61-7.64 (d, 2H),
7.49-7.53 (m, 1H), 7.39-7.43 (m, 2H), 7.19-7.23 (m, 2H), 7.12-
7.16 (d, 1H), 7.07-7.12 (m, 2H), 6.56-6.62 (d, 1H), 4.55-4.62 (m,
1H), 4.11-4.19 (q, 2H), 3.21-3.33 (dd, 2H), 2.58-2.66 (m, 2H),
2.15-2.25 (m, 1H), 1.98-2.08 (m, 1H), 1.21-1.26 (t, 3H); calculated
molecular mass of C28H28N2O5, 472 Da; measured MS ESþ m/z
473, MS ES- m/z 471.
Methyl 3-(4-Benzoylphenylamino)-3-oxopropanoate (Com-
pound 3). To a well-stirred solution of 4-aminobenzophenone
(3.95 g, 20 mmol) and triethylamine (5.5 mL, 40 mmol) in 20 mL
of THF and 60 mL of dichloromethane (DCM) at 0 °C was added
methyl malonyl chloride (2.73 g, 20 mmol) in 20 mL of DCM. The
reaction mixture was stirred at 0 °C for 2 h and then stirred at room
temperature for 16 h. The reaction was quenched with 50 mL of an
aqueous solution of NaHCO3. The organic phase was separated,
and the aqueous layer was washed with 50 mL of DCM. The
organic phases were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuum. The residue was
purified by flash column chromatography on silica gel with an ethyl
acetate/heptane mixture (10:90 to 30:70). This procedure produced
3.3 g of compound 3 (56% yield): 1H NMR (400 MHz, DMSO-d6)
δ 11.58 (broad, 1H), 7.76-7.81 (m, 4H), 7.71-7.74 (m, 2H),
7.61-7.63 (m, 1H), 7.51-7.55 (m, 2H), 3.76 (s, 3H), 3.21 (bs, 2H);
calculated molecular mass of C17H15NO4, 297 Da; measured MS
ESþ m/z 298 and MS ES- m/z 296.
N-(4-Benzoylphenyl)-4-hydroxy-2-oxo-5-phenethyl-
2,5-dihydro-1H-pyrrole-3-carboxamide (Photo-probe). To
a solution of compound 5 (400 mg, 0.85 mmol) in 10 mL of ethanol
was added 21% NaOEt in ethanol (830 mg, 2.54 mmol). The
mixture was stirred at room temperature for 24 h. Ethanol was
removed by vacuum; 10 mL of an ice/water mixture was added,
and the residue was acidified to pH <5 with 1.0 N aqueous HCl. A
solid was formed, filtered, and dried in vacuum. The procedure
1
3-(4-Benzoylphenylamino)-3-oxopropanoic Acid (Com-
pound 4). To a solution of methyl 3-(4-benzoylphenylamino)-3-
oxopropanoate (3.2 g, 10.8 mmol) in 100 mL of methanol was
added 1.0 N aqueous NaOH (32.4 mL, 32.4 mmol). The resulting
mixture was stirred at room temperature for 16 h. Organic solvent
was removed under reduced pressure, and the residue was cooled
with an ice bath. A 1.0 N HCl solution was added to adjust the pH
to <5. The resulting solid was filtered and dried. The procedure
produced 2.6 g of compound 4 (85% yield): 1H NMR (400 MHz,
DMSO-d6) δ 11.58 (broad, 1H), 7.74 (s, 4H), 7.71 (m, 2H), 7.66
(m, 1H), 7.55 (m, 2H), 3.21 (s, 2H); calculated molecular mass of
C16H13NO4, 283 Da; measured MS ESþ m/z 284, MS ES- m/z 282.
Ethyl 2-[3-(4-Benzoylphenylamino)-3-oxopropanamido]-
4-phenylbutanoate (Compound 5). To a stirred mixture that
contained 3-(4-benzoylphenylamino)-3-oxopropanoic acid (283 mg,
1.0 mmol), ethyl-2-homophenylalanine (243 mg, 1.0 mmol),
diisopropylethylamine (DIPEA, 0.53 mL, 3.0 mmol), and di-
methylformamide (DMF, 8 mL) was added 1-ethyl-3-(3-dimethy-
laminopropyl)carbodiimide (EDCI, 350 mg, 1.1 mmol). The
mixture was stirred at room temperature for 16 h. The mixture
produced 340 mg of a white solid (94% yield): H NMR (400
MHz, DMSO-d6) δ 11.24 (broad, 1H), 7.70-7.75 (m, 5H), 7.67-
7.69 (m, 2H), 7.62-7.66 (m, 1H), 7.53-7.57 (m, 3H), 7.27-7.30
(m, 2H), 7.21-7.23 (m, 2H), 7.15-7.20 (m, 1H), 3.60-3.67
(s, 1H), 2.62-2.68 (m, 2H), 1.96-2.02 (m, 1H), 1.63-1.69
(m, 1H); calculated molecular mass of C26H22N2O4, 426 Da;
measured MS ESþ m/z 427, MS ES- m/z 425.
Biomol Green Assay. UPPS activity was analyzed by a
Biomol Green assay after the pyrophosphate generated by the
UPPS reaction had been converted to inorganic phosphate by
pyrophosphatase. The purified S. pneumoniae UPPS used in this
assay was mixed with liposome made from total E. coli lipid
extract in a 1:2 ratio (w/v). The enzyme reaction was conducted in
100 mM Tris-HCl (pH 7.3), 50 mM KCl, 1 mM MgCl2, 0.01%
Triton X-100, 20 μg/mL BSA, 3 μM FPP, 16 μM IPP, and
2 units/mL E. coli pyrophosphatase. The inorganic phosphate
was then quantified with Biomol Green reagent according to the
vendor’s instructions.
Photo-Cross-Linking. Photo-cross-linking was performed
using an RPR-100 Rayonet Photochemical Chamber Reactor

Article
Biochemistry, Vol. 49, No. 25, 2010 5369
(Southern New England Ultra Violet Co.). UPPS (30 μM) was
preincubated with 30 μM Photo-probe for 15 min at room tem-
perature followed by UV exposure with the 350 nm lamps for
10-15 min. Dose-dependent competitive photo-cross-linking ex-
periments were conducted using the same procedure except that
unlabeled compound (0-200 μM) was included in the preincu-
bation mixture. For studies involving prebound substrate, the
substrate analogue was preincubated with UPPS for 10 min prior
to addition of Photo-probe. Samples were prepared in 1ꢀ PBS (pH
7.4). The total volume of the sample mixture was 20 μL. To prevent
self-cross-linking of UPPS induced by short UV light, the sample
mixture of UPPS and Photo-probe were incubated in a borosilicate
mass spectrometry (MS) sample tube. The MS sample tube was
next lowered into a 15 mm ꢀ 160 mm borosilicate test tube and
then placed in the carousel of the Rayonet Reactor for photo-
cross-linking.
F
ing. Excitationat 336 nm and emission at 460 nm. The concentrations
of Fluoro-probe and UPPS were fixed at 2 and 3 μM, respectively.
IGURE 2: Competition of Fluoro-probe with FsPP for UPPS bind-
The true dissociation constant (KD) of the UPPS inhibitor
complex was calculated using eq 3 [Cheng-Prusoff equation (12)].
Mass Spectrometry. Intact protein mass spectrometry was
conducted using an Agilent 1100 HPLC system interfaced with a
Waters ZQ4000 single-quadrupole mass spectrometer with an
electrospray ionization source operated in positive ion mode.
Mass spectra were analyzed using MassLynx (Waters Inc.).
Peptide mapping was conducted using a capillary HPLC system
(Agilent 1200) coupled to a QTrap4000 mass spectrometer (Applied
Biosystems) operated in data-dependent mode. Protein was sub-
jected to tryptic digestion after the protein had been denatured,
reduced, and alkylated and extra alkylation reagent had been
removed by dialysis. A Thermo LTQ ion trap mass spectrometer
(Thermo Electron Corp.) was used to conduct MS3 experiments.
Determination of the Equilibrium Binding Constant (KD)
of the Fluorescent Probe. The KD of the fluorescent FPP
analogue (named Fluoro-probe thereafter) for binding to S.
pneumoniae UPPS was determined by titration of the UPPS
enzyme (final concentrations of 0-15 μM) into a fixed amount of
Fluoro-probe (final concentration of 2 μM) in a buffer containing
0.01% Triton X-100, 100 mM Tris-HCl (pH 7.5), 50 mM KCl,
and 1 mM MgCl2. The fluorescence intensity was measured with
excitation at 336 nm and emission at 460 nm using a BMG
PHERAstar Microplate Reader (BMG LABTECH GmbH).
Data were fit using GraFit 5 to eq 1 (Morrison equation) (11)
to produce the KD value for Fluoro-probe binding.
3
KDapp
KD
¼
ð3Þ
½probeꢁ
1 þ
KDprobe
where KpDrobe and [probe] are the dissociation constant and con-
centration of Fluoro-probe, respectively.
The fluorescent probe displacement assay can also be con-
ducted via titration of UPPS into a mixture of a fixed concentra-
tion of Fluoro-probe and testing compound. In the presence of a
competitive binder, the apparent dissociation constant of the
Fluoro-probe increases. The apparent dissociation constant of
the UPPS probe complex can be calculated using eq 1.
3
Fluorescence Binding Experiments. The protein fluores-
cence was determined using an Infinite M1000 microplate reader
(Tecan Group Ltd.) or an RSM1000 spectrofluorometer (Olis,
Inc.). Emission spectra were scanned in the range of 300-500 nm
with an excitation wavelength of 285 nm. For studies involving
prebound inhibitor, inhibitor was preincubated with UPPS for 15
min prior to addition of FsPP, IsPP, or both. All samples were
prepared in buffer containing 100 mM HEPES (pH 7.3), 0.5 mM
MgCl2, 50 mM KCl, and 1.25% DMSO.
ꢂꢀ
ꢁ
ꢃ
ð1Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
F ¼ RI þ ðβ - RÞ E þ I þ KDprobe
-
ðEþIþKDprobeÞ - 4EI =2
2
RESULTS
Fluorescent Probe Displacement. Chen and co-workers
designed a fluorescent FPP analogue to study the interaction of
the ligand with E. coli UPPS (8). We utilized this tool compound,
i.e., Fluoro-probe, to study possible TA binding at the FPP binding
site. This probe, when excited at its maximum absorbance wave-
length (336 nm), emitted light at 460 nm linearly with the increase
in probe concentration. Fluorescence self-quenching was not
observed up to 10 μM probe (data not shown). Binding of the
fluorescent probe to UPPS resulted in a decreased fluorescence
intensity. This measurable change in intensity is the basis for the
probe displacement assay (8). First, the dissociation constant of
Fluoro-probe for binding to S. pneumoniae UPPS was determined
via titration of UPPS into a fixed concentration of Fluoro-probe.
Data were fit to eq 1 (ligand depletion considered) which resulted
in a dissociation constant (KD) of 0.25 μM.
where F is the measured fluorescence intensity, I is the concen-
tration of Fluoro-probe, E is the concentration of UPPS, and R
and β are scaling factors for low and high fluorescence intensities,
respectively.
Fluorescent Probe Displacement. The fluorescent probe
displacement assay was conducted at a fixed concentration of UPPS
(3 μM) and fluorescent probe (2 μM), and varying inhibitor concen-
trations in a buffer containing 100 mM Tris-HCl (pH 7.5), 50 mM
KCl, 1 mM MgCl2, and 0.01% Triton X-100. Probe fluorescence
increases with increasing concentrations of a competitive binder due
to the displacement of Fluoro-probe from UPPS. Data were fit to eq
2 to yield an apparent KD (KaDpp) value for inhibitor binding.
ꢂꢀ
ꢁ
ꢃ
ð2Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
F ¼ RE þ ðβ - RÞ E þ I þ KDapp
-
ðEþIþKDappÞ - 4EI =2
The apparent dissociation constant of Fluoro-probe is ex-
pected to increase in the presence of increasing concentrations
of competing inhibitors. To experimentally demonstrate this, we
measured the fluorescence intensity of the probe at fixed con-
centrations of the probe and UPPS with varied concentrations
2
where I is the concentration of unlabeled inhibitor. Please note
that in eq 2, the first term is RE instead of RI, as in eq 1.

5370 Biochemistry, Vol. 49, No. 25, 2010
Lee et al.
F
IGURE 3: Identification of UPPS inhibitors that bind to the FPP binding site using Fluoro-probe. Excitation at 336 nm and emission at 460 nm.
Each titration was performed via titration of UPPS into a fixed concentration of Fluoro-probe (2 μM) and compound (concentration indicated in
the figure): (A) FPP and (B) compound 1.
F
IGURE 4: Identification of the peptide to which Photo-probe is covalently attached by mass spectrometry. (A) Mass spectra of intact UPPS before
(a) and after (b) UV exposure (350 nm). The small peaks shown at 30919 Da in inset a and 31349 Da in inset b represent the small population of corres-
ponding UPPS which contained a post-translational modification at its N-terminal His tag. The extracted ion chromatogram (XIC) of the modified
peptide is shown in inset c. (B) Amino acid sequence of N-terminally His-tagged S. pneumoniae UPPS. The probe-modified peptide is underlined.
Scheme 3: Photo-Cross-Linking of a Compound That Contains a Benzophenone Moiety (circled) to a Protein (thick gray bar)
of FsPP. An increase in FsPP concentration resulted in the
enhancement of the fluorescence of Fluoro-probe because
of its displacement from UPPS by FsPP (Figure 2). Fitting the
data to eq 2 resulted in an apparent KD of 3.7 μM for FsPP
binding. The true KD value of FsPP is 0.41 μM as calculated
using eq 3.

Article
Biochemistry, Vol. 49, No. 25, 2010 5371
F
IGURE 5: Identification of the site of Photo-probe attachment by tandem mass spectrometry. (A) MS2 and MS3 spectra. The Photo-probe-
modified peptide (peptide þ Photo-probe = 1148 Da þ 426 Da = 1574 Da) was subjected to a low-energy fragmentation (MS2). This
fragmentation caused a bondcleavage within the Photo-probe structure, leaving part of the Photo-probe (179 Da) still attached to the peptide (red
circle). The doublycharged ion of thisfragment([M þ 2H]2þ m/z 664.5) was subjected to a further fragmentation (MS3). The resulting y and b ions
(labeled in blue and red, respectively) revealed the peptide sequence as well as the site of Photo-probe attachment (red letter in the sequence). (B)
X-ray structure of S. pneumoniae UPPS in complex with FPP (5) shown in cartoon representation. Protein is shown in ribbon and the substrate in
sticks; part of helix R2 (AGMEALQTV) is colored green, of which the side chain of Met49 is shown.
Whereas the displacement of the probe can be measured at
fixed concentrations of the probe and UPPS with varying com-
peting compound concentrations, the reverse experiment could
be conducted when the probe and competing compound con-
centrations were fixed and the UPPS concentration was varied.
The advantage of the reverse titration is that any possible
probe quenching by a competing compound can be factored in,
because the interference is constant during UPPS titration. As an
example, the competition between FPP and Fluoro-probe for
UPPS is shown in Figure 3A. The apparent dissociation constant
of Fluoro-probe changed from 0.26 μM at 0 μM FPP to 0.84 μM
at 4 μM FPP and to 1.64 μM at 10 μM FPP.
the fluorescence signal produced by 2 μM Fluoro-probe was
quenched by 0.01% Triton and the signal completely quenched
by 1% Triton. To balance between maintaining enzyme activity
and obtaining robust fluorescence signals, we used 0.01% Triton
X-100 for the fluorescent probe displacement assay. At this
concentration, UPPS maintained ∼70% activity (14).
The possibility of tetramic acid inhibitors binding to the FPP
binding site was examined via titration of UPPS into a fixed
concentration of Fluoro-probe and TA compound. The data
showed that tetramic acids did not compete with Fluoro-probe,
indicating that TA class inhibitors are not competitive inhibitors
with respect to FPP. The result for a representative TA inhibitor,
compound 1 (IC50 = 0.45 μM as determined by the Biomol
Green assay), is shown in Figure 3B. Within experimental error,
the dissociation constant of Fluoro-probe did not change with an
increased concentration of compound 1.
Previous studies showed that Triton, a nonionic detergent, was
essential for the in vitro enzymatic activity of UPPS, probably
by mimicking the lipidic environment in which this enzyme
may operate in vivo (6, 13). Our study showed that ∼25% of

5372 Biochemistry, Vol. 49, No. 25, 2010
Lee et al.
Identification of the TA Binding Site Using Photo-Cross-
Linking and Mass Spectrometry. Benzophenone-based
photolabeling has been extensively applied, especially for the
determination of receptor-ligand binding sites (15). Upon light
irradiation, a target specific compound containing a benzophe-
none photoreactive group can covalently attach to the protein to
which it binds (Scheme 3).
We discovered that tetramic acids containing 1-phenylpiper-
idine or biphenyl groups showed enzyme inhibitory activity
comparable to that of compound 1. This led us to believe that
a benzophenone substitution would create a photoreactive probe
that binds and inhibits UPPS. A Photo-probe compound
(Figure 1D) was designed and synthesized. It inhibited UPPS
with an IC50 of 0.63 μM (as determined by the Biomol Green
assay). Intact protein mass spectrometry showed that this probe
compound (molecular mass of 426 Da) covalently modified
UPPS with 1:1 molar ratio after the UPPS and Photo-probe
mixture was exposed to long UV light in the 350 nm region (inset
b in Figure 4A). As indicated in Materials and Methods, shielding
by multiple layers of borosilicate glass was determined to be
critical for the photo-cross-linking experiment as bleeding over of
short UV light from the 350 nm lamps promoted self-cross-
linking of two interacting UPPS monomers [UPPS forms a
functional dimer in solution (13)]. Without borosilicate shield-
ing, the covalent dimer was detected by SDS-PAGE after UV
exposure (data not shown). This covalent dimer made mass
spectrum deconvolution problematic.
Mass spectrometric analysis of UPPS alone indicated that the
protein’s N-terminal Met1 was processed (calculated change in
mass of -131 Da), and a small population of the protein
contained a post-translational modification [the small peak with
178 Da of extra mass relative to UPPS (inset a in Figure 4A)].
This post-translational modification is likely due to a sponta-
neous R-N-6 gluconoylation often seen in His-tagged pro-
teins (16). To identify the modification site of Photo-probe, the
sample, after UV exposure, was subjected to trypsin digestion
followed by peptide mapping using LC-MS. The analysis
identified a peptide with a mass consistent with addition
of Photo-probe [m/z 1574.8 (inset c in Figure 4A)]. The location
of the peptide is underlined in the amino acid sequence of
S. pneumoniae UPPS (Figure 4B).
The site of covalent attachment of Photo-probe was identified
by tandem mass spectrometry. At a low collision energy, the
amide bond present in the Photo-probe structure was cleaved
while other amide bonds present in the peptide backbone re-
mained intact. This resulted in a doubly charged fragment ion at
m/z 664.5 (red circle in Figure 5A). This ion was subjected to
further fragmentation (MS3). This experiment not only confirmed
the sequence of the probe-modified peptide but also revealed the
site of probe attachment [Met49 (Figure 5A)]. According to the
crystal structure of S. pneumoniae UPPS (5), Met49 is located in
helix R2 close to the FPP binding site (Figure 5B). A systematic
study of the photo-cross-linking of various amino acids with
benzophenone showed that methionine is particularly favored in
terms of reactivity and product stability (17). On the basis of this
and the fluorescent probe displacement results, the binding site of
TA appeared to be allosteric but adjacent to the FPP binding site.
F
IGURE 6: Fluorescence emission spectra of S. pneumoniae UPPS:
(A) substrate binding to UPPS and (B) substrate binding to UPPS
which was prebound with compound 1. Final concentrations were as
follows: 1 μM UPPS, 15 μM FsPP, 40 μM IsPP, and 5 μM compound
1. Spectra are averages of two scans. Signals contributed by buffer
blank and unbound compound 1 (4.1 μM, estimated on the basis of
the IC50 value) have been subtracted.
IPP with the UPPS FsPP complex was observed. The addition of
3
IPP alone to UPPS failed to produce fluorescence change unless
FPP (or FsPP) was preincubated with the enzyme (18). These
data suggested that E. coli UPPS follows an ordered substrate
binding mechanism. We conducted a similar substrate binding
study with S. pneumoniae UPPS and found that in the presence of
Mg2þ both FsPP (or FPP) and IsPP (or IPP) could bind and
enhance the fluorescence of the enzyme in the absence of the
other substrate (Figure 6A). This suggests that, unlike E. coli
UPPS, S. pneumoniae UPPS likely follows a random-sequential
substrate binding mechanism. Under the condition used, addi-
tion of both substrate analogues caused a reduction in fluores-
cence intensity relative to the fluorescence produced by the
addition of one substrate (FPP or IPP) to UPPS (Figure 6A).
Inhibition Mechanism of Tetramic Acids. To study the
inhibition mechanism of TA, the interactions of TA with various
enzyme forms, including apo and substrate-bound S. pneumoniae
UPPS, were investigated using the Photo-probe. Results showed
that the probe molecule bound to and modified UPPS in the
absence of substrate (Figure 7B) but did not do so when S.
pneumoniae UPPS was prebound with either substrate analogue
(Figure 7C,D). To investigate whether the formation of the
Substrate Binding Order of S. pneumoniae UPPS.
A
UPPS TA complex can prevent the substrate from binding, the
3
fluorescence binding study conducted by Chen and colleagues
showed that addition of FPP (or FsPP) to E. coli UPPS caused a
reduction in the protein intrinsic fluorescence. In the presence
of Mg2þ, the enhancement of fluorescence upon the binding of
interaction between substrate analogues and the UPPS TA
3
complex was examined using protein fluorescence spectroscopy.
With excitation at 285 nm, the intrinsic fluorescence of S.
pneumoniae UPPS increased upon addition of compound 1,

Article
Biochemistry, Vol. 49, No. 25, 2010 5373
F
IGURE 7: Interaction of tetramic acid with free or substrate-bound UPPS. Samples were analyzed by mass spectrometry after UV exposure (350 nm)
at room temperature for 10 min: (A) 30 μMUPPS, (B) 30μMUPPSand30μM Photo-probe, (C) 30 μM Photo-probe added to a preincubated mixture
containing 30 μM UPPS and 100 μM FsPP, and (D) 30 μM Photo-probe added to a preincubated mixture containing 30 μM UPPS, 100 μMFsPP, and
100 μM IsPP. The small peak shown at ∼30919 Da represents UPPS with a post-translational modification (PTM) at its N-terminal His tag.
indicating an interaction of tetramic acid with UPPS (Figure 6B).
using the Photo-probe. At a fixed concentration of Fluoro-probe,
inhibitors that bind to the FPP binding site should compete with
Fluoro-probe for binding to UPPS and show a competition profile
similar to that illustrated in Figure 3A. Inhibitors that bind to the
TA binding site should compete with Photo-probe for binding to
UPPS, resulting in a reduced level of photo-cross-linking by the
Photo-probe. As expected, compound 1 competed with Photo-
probe for binding to UPPS in a dose response manner (Figure 8).
To date, we have not identified a competitive inhibitor with respect
to FPP. However, a bicyclic heterocycle compound (compound 2)
identified from a HTS follow-up effort was found to bind at the TA
binding site (Figure S2 of the Supporting Information).
Addition of FsPP or IsPP to the preformed UPPS TA complex
3
caused a large increase of fluorescence relative to that of the
UPPS TA complex, suggesting the interaction of FsPP or IsPP
3
with the UPPS TA complex. Addition of IsPP to the preformed
3
UPPS TA FsPP complex or addition of FsPP to the preformed
3
3
3
3
UPPS TA IsPP complex did not produce a fluorescence change
relative to the spectrum of the ternary complex, suggesting that
the UPPS TA FsPP IsPP quaternary complex was not formed
3
3
3
(Figure 6B).
With excitation at 285 nm, compound 1 alone exhibited a low
level of fluorescence at maximum emission ∼40 nm above the
maximum emission of UPPS (Figure 6B). To account for any
fluorescence signal contributed by the unbound compound, the
emission spectrum of compound 1 was subtracted from the emi-
ssion spectra of UPPS samples containing compound 1. The
amount of unbound compound 1 in UPPS samples was estimated
using the Morrison equation, assuming KD = IC50. The 20-22 nm
blue shift observed in emission spectra obtained using an Infinite
M1000 microplate reader (Tecan) compared to the emission
spectra from the RSM1000 spectrofluorometer (Olis) is due to a
light source intensity calibration engineered into the Infinite
M1000 microplate reader. When uncalibrated data were retrieved
by application of an instrument specific calibration file provided
by Tecan, an emission spectrum similar to that obtained using
RSM1000 was obtained (Figure S1 of the Supporting Infor-
mation). With the microplate reader, we obtained very repro-
ducible data. No photobleaching effect was observed.
DISCUSSION
UPPS is an essential enzyme for bacterial viability and is highly
conserved among Gram-positive and Gram-negative bacteria.
The critical biological function of UPPS as well as an active site
amenable to small molecule inhibition makes UPPS an attractive
target for the discovery of novel antibacterial agents.
It has been demonstrated by HTS and a subsequent medicinal
chemistry program that tetramic acids are potent and selective
UPPS inhibitors (7). UPPS catalyzes the condensation of eight
molecules of IPP with FPP. Depending on reaction conditions
and the ratio of enzyme to substrate or ratio of FPP to IPP,
reaction products of varying lengths can be generated (6). The
dynamics of interactions of these products with UPPS has not
been fully understood. The complexity of the reactions impedes
the characterization of UPPS inhibitors by traditional enzyme
kinetics. In this study, we used biophysical approaches to
investigate the inhibition mechanism of TA class inhibitors.
Our data showed that TA did not compete with Fluoro-probe
for binding to UPPS (Figure 3B), indicating that TA is not a com-
petitive inhibitor with respect to FPP. The interactions between
Studying the Mode of Binding of New Compounds Using
Fluoro-probe and Photo-probe. A number of HTS hits as well
as compounds generated from a medicinal chemistry program
were evaluated by performing fluorescent probe displacement
using the Fluoro-probe and competitive photo-cross-linking

5374 Biochemistry, Vol. 49, No. 25, 2010
Lee et al.
F
IGURE 8: Compound 1 competes with Photo-probe for binding to UPPS in a dose response fashion. Samples were analyzed by mass
spectrometry after UV exposure (350 nm) at room temperature for 15 min: (A) 30 μM UPPS and 30 μM Photo-probe, (B) 30 μM UPPS,
30 μM Photo-probe, and 100 μM compound 1, and (C) 30 μM UPPS, 30 μM Photo-probe, and 200 μM compound 1.
FIGURE 9: Proposed substrate binding mechanism of S. pneumoniae UPPS (dotted rectangle) and inhibition mechanism of tetramic acid. Dotted
arrows represent the subsequent events after substrate binding needed for the first substrate turnover.
TA and various enzyme forms were investigated by photo-cross-
linking and intact protein mass spectrometry. Our results indi-
cated that the photosensitive TA analogue bound to and modified
UPPS only when the enzyme was not prebound with substrates
(Figure 7). Since the photo-cross-linking method monitors covalent
modification of UPPS by Photo-probe, it cannot be used to study
located near the substrate binding site and may be used to probe the
interaction of the substrate with UPPS and with the preformed
UPPS TA complex. Unlike E. coli UPPS which follows an ordered
3
substrate binding mechanism with FPP binding prior to IPP bind-
ing, our data for S. pneumoniae UPPS are consistent with a
random-sequential substrate binding mechanism, with each sub-
strate binding in a manner independent of the other (described
by the dotted rectangle in Figure 9). TA enhanced the fluorescence
of apo-UPPS, confirming the interaction between TA and
the interaction between the substrate and the preformed UPPS TA
3
complex. According to the crystal structure of S. pneumoniae
UPPS (5), three tryptophan residues (W33, W77, and W227) are

Article
Biochemistry, Vol. 49, No. 25, 2010 5375
F
conformational change (red dotted line) upon binding of tetramic acid prevents IPP from binding to UPPS.
IGURE 10: Proposed mode of binding of tetramic acid inhibitors: (A) substrate-bound UPPS and (B) the UPPS TA FPP ternary complex. The
3
3
apo-UPPS. Addition of FsPP or IsPP to the preformed UPPS
TA complex caused a large enhancement in fluorescence relative
TA did not show binding to any preformed enzyme-substrate
complex, it does not qualify to be, by definition, an uncompetitive
or noncompetitive inhibitor. On the basis of the location of the
Photo-probe attachment in the S. pneumoniae UPPS structure, TA
likely binds to a deeper position in the hydrophobic tunnel relative
to IPP. One substrate (FPP or IPP) can still bind to UPPS when it
is bound with TA, but the second substrate cannot, suggesting that
binding of TA to UPPS causes a conformational change that alters
the substrate binding site, therefore preventing substrate turnover,
leading to the inhibition of UPPS activity. On the basis of TA’s
binding site being distinct from the substrate binding site and its
ability to alter enzyme activity, we classify TA into the allosteric
inhibitor category. However, we do not know if UPPS activity is
regulated by any allosteric mechanism related to the site where TA
binds. On the basis of all the data presented above, we propose that
TA inhibits UPPS by a mechanism as described in Figure 9.
Further investigations are needed to confirm this mechanism.
To expand the use of these biophysical methods, we used
Fluoro-probe and Photo-probe to determine the mode of inhibi-
tion of other interesting UPPS inhibitors identified from HTS as
well as from a medicinal chemistry program. Inhibitors with the
same binding mode as FPP and TA can be quickly identified by
performing fluorescent probe displacement and competitive
photo-cross-linking, respectively. As an example, compound 2
(a non-TA compound) was shown to bind at the TA binding site
(Figure S2 of the Supporting Information).
3
to that of the UPPS TA complex, suggesting that FPP or IPP can
3
interact with the UPPS TA complex and form the UPPS TA
3
3
3
FsPP or UPPS TA IsPP complex. However, addition of the
second substrate to the preformed UPPS TA FsPP or UPPS
TA IsPP complex did not cause a fluorescence change relative to
the spectra of the complexes (Figure 6B). This suggests that only
3
3
3
3
3
3
one substrate can bind to the UPPS TA complex but nonpro-
3
ductively. The contribution of each of the tryptophan residues in
UPPS to the observed interactions remains unknown and needs
to be further investigated.
The crystal structures of S. pneumoniae UPPS and E. coli UPPS
are significantly structurally similar (2, 5). Substrates FPP and IPP
bind in an elongated hydrophobic tunnel surrounded by four β-
strands (βA-βB-βD-βC) and two helices (R2 and R3). The
lower part of the tunnel hosts the elongated reaction product as a
result of consecutive condensation reactions. Our results showed
that the Photo-probe bound and modified Met49 in helix R2,
suggesting that TA binds to an allosteric site adjacent to the FPP
binding site in the elongated hydrophobic tunnel (Figures 5B and
10B). On the basis of the crystal structure of E. coli UPPS, the loop
containing amino acids 72-82 (corresponding to amino acids
74-84 in S. pneumoniae UPPS), as illustrated by the red dots in
Figure 10, was highly flexible and its electron density could not be
seen in an apo-UPPS structure (19, 20) but could be seen when
Triton or FPP was bound (3, 4). This loop is proposed to be asso-
ciated with IPP binding and is responsible for bridging the interac-
tion of IPP with FPP needed to initiate the condensation reaction
and serving as a hinge to control the interchange between the
closed (substrate-bound) protein conformation and the open (apo-
enzyme and product-bound) protein conformation (18, 19). A
large protein conformational change upon substrate binding was
revealed using fluorescence quenching and stopped-flow meth-
ods (18). TA bound to apo-UPPS but not to the substrate-bound
S. pneumoniae UPPS; we propose that this class of inhibitors binds
to only the open conformation of S. pneumoniae UPPS. When
UPPS is bound with substrates, the UPPS is locked to a closed
conformation, preventing TA from entering the hydrophobic
tunnel. TA did not compete with Fluoro-probe for binding and
therefore is not a competitive inhibitor with respect to FPP. Since
Taken together, these biophysical approaches, including photo-
cross-linking, fluorescent probe displacement, and protein fluore-
scence spectroscopy, have been successfully applied in studying
the mode of binding and the inhibition mechanism of UPPS
inhibitors identified from HTS. These tools are especially useful in
cases where the cocrystal structures of UPPS and the inhibitor are
not available.
ACKNOWLEDGMENT
We thank a few Novartis colleagues for their contributions to
this work: Dr. James Koehn’s lab for producing S. pneumoniae
UPPS, Dr. Jovita Marcinkeviciene for critical review and helpful
discussion of the manuscript, and Dr. Patricia Bradford for
critical reading and editing of the manuscript.

5376 Biochemistry, Vol. 49, No. 25, 2010
Lee et al.
SUPPORTING INFORMATION AVAILABLE
9. Hurley, T. B., Lee, K., Peukert, S., and Wattanasin, S. (2008)
Preparation of bicyclic heteroaryl amides as inhibitors of undecapre-
nyl pyrophosphate synthase. WO 2008014307.
Comparison of emission spectra of S. pneumoniae UPPS
scanned using a Tecan Infinite M1000 microplate reader and
an Olis RSM1000 spectrophotometer and mass spectra showing
competitive photo-cross-linking of Photo-probe with compound
2. This material is available free of charge via the Internet at
http://pubs.acs.org.
10. Hurley, T. B., Peukert, S., and Wattanasin, S. (2008) Preparation of
pyrrolecarboxamides and pyridinecarboxamides as inhibitors of un-
decaprenyl pyrophosphate synthase (UPPS). WO 2008014311.
11. Copeland, R. A. (2000) Protein-Ligand Binding Equilibria. In En-
zymes. A Practical Introduction to Structure, Mechanism, and Data
Analysis, 2nd ed., pp 76-108, Wiley-VCH, Inc., New York.
12. Cheng, Y., and Prusoff, W. H. (1973) Relationship between the
inhibition constant (K1) and the concentration of inhibitor which
causes 50% inhibition (I50) of an enzymatic reaction. Biochem.
Pharmacol. 22, 3099–3108.
13. Allen, C. M. (1985) Purification and characterization of undecapre-
nylpyrophosphate synthetase. Methods Enzymol. 110, 281–299.
14. Li, H., Huang, J., Jiang, X., Seefeld, M., McQueney, M., and
Macarron, R. (2003) The effect of triton concentration on the activity
of undecaprenyl pyrophosphate synthase inhibitors. J. Biomol.
Screening 8, 712–715.
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