Rational design of a β-lactamase inhibitor achieved via stabilization of the trans-enamine intermediate: 1.28 A crystal structure of wt SHV-1 complex with a penam sulfone

Article abstract of DOI:10.1021/ja063715w

β-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of β-lactamases that are capable of hydrolyzing our most potent β-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the frans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 β-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A β-lactamase inhibitors.

Full text of DOI:10.1021/ja063715w

Published on Web 09/20/2006
Rational Design of a â-Lactamase Inhibitor Achieved via
Stabilization of the trans-Enamine Intermediate: 1.28 Å
Crystal Structure of wt SHV-1 Complex with a Penam Sulfone
Pius S. Padayatti,† Anjaneyulu Sheri,‡ Monica A. Totir,§ Marion S. Helfand,|
Marianne P. Carey,† Vernon E. Anderson,† Paul R. Carey,† Christopher R. Bethel,|
Robert A. Bonomo,|, John D. Buynak,‡ and Focco van den Akker*,†
Contribution from the Departments of Biochemistry, Chemistry, and Pharmacology, Case
Western ReserVe UniVersity, CleVeland, Ohio 44106, Department of Chemistry, Southern
Methodist UniVersity, Dallas, Texas 75275-0314, and Research DiVision, Louis Stokes CleVeland
Veterans Affairs Medical Center, CleVeland, Ohio 44106
Received May 27, 2006; E-mail: focco.vandenakker@case.edu
Abstract: â-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria.
The continuing evolution of â-lactamases that are capable of hydrolyzing our most potent â-lactams presents
a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient
inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural
studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV
variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate.
We report here the 1.28 Å resolution crystal structure of wt SHV-1 in complex with a rationally designed
penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to
tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to
stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1
â-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing
the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based
class A â-lactamase inhibitors.
occurring more frequently than before4 thus stimulating the need
to design novel â-lactamase inhibitors.5-7
Introduction
Resistance to â-lactam antibiotics mediated by bacterial
â-lactamases (EC 3.5.2.6) is a global problem.1,2 â-Lactamases
hydrolyze and inactivate penicillins and cephalosporins, thereby
rendering these â-lactams inactive before they reach their
intended bacterial targets, the penicillin-binding proteins (PBPs).
Hence, â-lactamases ensure bacterial survival against the
therapeutic challenge of â-lactam antibiotics. To overcome this
threat, â-lactam antibiotics are often administered with â-lac-
tamase inhibitors (clavulanate, sulbactam, and tazobactam).3
This successful strategy has prolonged the clinical utility of
penicillin antibiotics, especially against class A â-lactamases.
Unfortunately, not all â-lactamases are susceptible to inhibitors
and, of those that are, resistance against such inhibitors is
The major members of class A â-lactamases are TEM-1,
SHV-1, and CTX-M enzymes. TEM-1 and SHV-1 â-lactamases
hydrolyze the inhibitors through a complex, multistep reaction
pathway. The catalytic mechanism by which â-lactamase
inhibitors react with â-lactamases has been of great interest yet
the identity of the species giving rise to inhibition remains
controversial.8-18 Figure 1a shows a reaction scheme as it has
(4) Therrien, C.; Levesque, R. C. FEMS Microbiol. ReV. 2000, 24, 251-262.
(5) Georgopapadakou, N. H. Expert Opin. InVest. Drugs 2004, 13, 1307-1318.
(6) Buynak, J. D. Curr. Med. Chem. 2004, 11, 1951-1964.
(7) Lee, N.; Yuen, K. Y.; Kumana, C. R. Drugs 2003, 63, 1511-1524.
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(13) Fisher, J.; Charnas, R. L.; Knowles, J. R. Biochemistry 1978, 17, 2180-
2184.
† Department of Biochemistry, Case Western Reserve University.
‡ Department of Chemistry, Southern Methodist University.
§ Department of Chemistry, Case Western Reserve University.
| Research Division, Louis Stokes Cleveland Veterans Affairs Medical
Center.
(14) Fisher, J.; Charnas, R. L.; Bradley, S. M.; Knowles, J. R. Biochemistry
1981, 20, 2726-2731.
(15) Fisher, J.; Belasco, J. G.; Khosla, S.; Knowles, J. R. Biochemistry 1980,
19, 2895-2901.
Department of Pharmacology, Case Western Reserve University.
(1) Helfand, M. S.; Bonomo, R. A. Curr. Drug Targets: Infect. Disord. 2003,
3, 9-23.
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Trans. R. Soc. London, Ser. B 1980, 289, 309-319.
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10.1021/ja063715w CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 13235-13242
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A R T I C L E S
Padayatti et al.
Figure 1. (a) Proposed reaction scheme for inhibitors with class A â-lactamases. The transient inactivation of SHV-1 involves the trans-enamine intermediate
which energetically is more favorable than the cis-enamine intermediate. To escape the transient inhibition, the reaction needs to proceed back to the imine
intermediate before it can continue and result in either the reactivation of the active SHV-1 enzyme or irreversible inactivation. The kreact measured corresponds
to (kinact + kdeacyl)(kcfi)(ktfc)/[kcftkifc + (kcft + kcfi)(kinact + kdeacyl)]. (b) Schematic diagram of SA2-13 and tazobactam.
been proposed for tazobactam:19,20 Following the noncovalent
association step, S70 is acylated opening the â-lactam ring of
the inhibitor. This is followed by cleavage of the C5-S bond
opening the thiazolidine ring with concomitant formation of a
C5dN imine. This imine intermediate partitions between three
pathways: tautomerization to form both cis- and trans-enamines;
covalent modification resulting in irreversibly inactivated
enzyme; hydrolysis which liberates active enzyme. If the rate
constant for conversion to the trans-enamine competes with the
other two processes and if the rate constant for the conversion
of the trans-enamine to imine is slow in comparison to the assay
time, it can be detected as “reactivation” of the “dead” enzyme.
An approach to improving the efficiency of â-lactamase
inhibitors is to slow one or more of the rate constants by
stabilizing a particular intermediate. Inhibitor resistant â-lac-
tamase variants have been found to have decreased affinity for
the inhibitors, increased turnover numbers and decreased
complexity of inhibitory intermediates25 and decreased trans-
enamine formation.26 One approach to overcoming this problem
is to decrease the rate constant for the deacylation step of
â-lactamase inhibitors. For example, it was recently reported
that a designed substrate penicillanic acid analogue, with a 6R-
hydroxylmethyl group in place of the “usual” 6â-acylamino
substituent, showed decreased deacylation.27 This modification
was designed to displace a nucleophilic water (activated by the
general base E166) needed for deacylation. Our groups recently
interfered with the deacylation step of the reaction by mutating
E166 to an alanine such that the nucleophilic deacylation water,
normally positioned by this amino acid, is displaced. Through
the use of Raman crystallography the resulting inhibition
reaction28 was tracked and showed the importance of the trans-
enamine intermediate for sulbactam, clavulanic acid, and
tazobactam, the latter being the most conformationally or-
dered.29,30
kinact.
21-24 Additionally these resistant enzymes display decreased
(25) Pagan-Rodriguez, D.; Zhou, X.; Simmons, R.; Bethel, C. R.; Hujer, A.
M.; Helfand, M. S.; Jin, Z.; Guo, B.; Anderson, V. E.; Ng, L. M.; Bonomo,
R. A. J. Biol. Chem. 2004, 279, 19494-19501.
(26) Sulton, D.; Pagan-Roderiguez, D.; Zhou, X.; Liu, Y.; Hujer, A. M.; Bethel,
C. R.; Helfand, M. S.; Thomson, J. M.; Anderson, V. E.; Buynak, J. D.;
Ng, L. M.; Bonomo, R. A. J. Biol. Chem. 2005, 280, 35528-35536.
(27) Maveyraud, L.; Massova, I.; Birck, I.; Miyashita, K.; Samama, J. P.;
Mobashery, S. J. Am. Chem. Soc. 1996, 118, 7435-7440.
(28) Helfand, M. S.; Totir, M. A.; Carey, M. P.; Hujer, A. M.; Bonomo, R. A.;
Carey, P. R. Biochemistry 2003, 42, 13386-13392.
(29) Padayatti, P. S.; Helfand, M. S.; Totir, M. A.; Carey, M. P.; Carey, P. R.;
Bonomo, R. A.; van den, A. F. J. Biol. Chem. 2005, 280, 34900-34907.
(30) Padayatti, P. S.; Helfand, M. S.; Totir, M. A.; Carey, M. P.; Hujer, A. M.;
Carey, P. R.; Bonomo, R. A.; van den, A. F. Biochemistry 2004, 43, 843-
848.
(19) Bush, K.; Macalintal, C.; Rasmussen, B. A.; Lee, V. J.; Yang, Y.
Antimicrob. Agents Chemother. 1993, 37, 851-858.
(20) Yang, Y.; Janota, K.; Tabei, K.; Huang, N.; Siegel, M. M.; Lin, Y. I.;
Rasmussen, B. A.; Shlaes, D. M. J. Biol. Chem. 2000, 275, 26674-26682.
(21) Chaibi, E. B.; Peduzzi, J.; Farzaneh, S.; Barthelemy, M.; Sirot, D.; Labia,
R. Biochim. Biophys. Acta 1998, 1382, 38-46.
(22) Lin, S.; Thomas, M.; Shlaes, D. M.; Rudin, S. D.; Knox, J. R.; Anderson,
V.; Bonomo, R. A. Biochem. J. 1998, 333 (Pt 2), 395-400.
(23) Delaire, M.; Labia, R.; Samama, J. P.; Masson, J. M. J. Biol. Chem. 1992,
267, 20600-20606.
(24) Meroueh, S. O.; Roblin, P.; Golemi, D.; Maveyraud, L.; Vakulenko, S. B.;
Zhang, Y.; Samama, J. P.; Mobashery, S. J. Am. Chem. Soc. 2002, 124,
9422-9430.
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13236 J. AM. CHEM. SOC. VOL. 128, NO. 40, 2006

A Penam Sulfone Bound to wt SHV-1
â
-Lactamase
A R T I C L E S
Table 1. Kinetic Measurements for SA2-13 and Tazobactam
% resid v
30 min
1
1
1
compd
KD
(
µM)
k
inact (s-
)
k
inact/KD
k
react(5) (s-
)
k
react(30) (s-
)
5 min
24 h
tn
tazobactam
SA2-13
0.1 ( 0.025
1.7 ( 0.1
0.10 ( 0.006
0.05 ( 0.001
1.0 ( 0.3
0.030 ( 0.002
0.034 ( 0.001
0.004 ( 0.001
0.040 ( 0.016
0.004 ( 0.001
4
4
4
4
0
47
5
120
increasing concentrations of inactivators. A fixed concentration of
enzyme, nitrocefin, and increasing nM concentrations of inactivator
(tazobactam or SA2-13) were used in each assay. The kobs for
inactivation was determined graphically as the reciprocal of the ordinate
of the intersection of the straight lines obtained from the initial, V0,
and final, Vf, steady-state velocities. Each kobs was plotted versus
In an effort to slow the deacylation step by improving the
stability of the trans-enamine intermediate in wt SHV-1, the
tazobactam bound SHV-1 E166A structure30 was used as the
starting point for rational inhibitor design. Tazobactam makes
numerous interactions within the active site. However, the
triazolyl moiety of tazobactam is an obvious candidate for
alteration since it does not make a direct hydrogen bond, only
a water-mediated hydrogen bond with S130.30 On the basis of
a previous crystal structure, it is hypothesized that this triazolyl
moiety might be beneficially replaced by a negatively charged
carboxylate attached through an appropriately sized linker. This
penam sulfone compound (SA2-13, Figure 1b) was synthesized
and found to be a good inhibitor of SHV-1 â-lactamase. We
report here the 1.28 Å resolution crystal structure of a designed
tazobactam analogue SA2-13 bound to wt SHV-1. The wt
SHV-1 structure contains the inhibitor in a trans-enamine
conformation stabilized via the carboxylate group positioned
in close proximity to K234, S130, and the catalytic S70. This
structure-based penam sulfone inhibitor captured in the active
site offers new opportunities to exploit the trans-enamine
intermediate for successful class A â-lactamase inhibition.
inhibitor concentration, [I], and fit to eq 2 to determine kinact and kinact
/
KI (the second-order rate constant for reaction of free enzyme with
free inhibitor to give inactive enzyme):
kobs ) kinact[I]/(KI + [I])
(2)
The partitioning of the initial enzyme inhibitor complex between
hydrolysis and enzyme inactivation, i.e., the turnover number (tn )
kcat/kinact), was obtained in the following manner. First, increasing
amounts of inhibitor (SA2-13 or tazobactam) were incubated with a
fixed concentration of SHV-1 â-lactamase in a total volume of 600
µL of 20 mM phosphate-buffered saline, pH 7.4, at RT. After 24 h, an
aliquot (40 µL) was removed from the mixture and the steady-state
velocity was measured and compared with a control sample with no
inactivator added. The proportion of SA2-13 or tazobactam relative to
SHV-1 that resulted in 90% inactivation after 24 h was designated the
turnover number, tn (as previously defined19).
These values served as a guide to determine the ratio of inhibitor
(I) to enzyme (E) in an experiment to determine kreact, the apparent
first-order rate constant for reactivation of the â-lactamase. To determine
Materials and Methods
Design and Synthesis of SA2-13. The starting point for the SA2-
13 design are the coordinates of the trans-enamine intermediate of
tazobactam trapped in the deacylation deficient E166A mutant of SHV-
1.30 The position and interactions of the triazolyl moiety of tazobactam
within the active site seemed suboptimal since no direct hydrogen bonds
were made. Tazobactam’s triazolyl moiety assumes a similar position
to that of an ordered HEPES molecule observed in previous SHV-1
structures.29,31 The ability of SHV-1 to bind a negatively charged group
within this active site region, such as the sulfonate group of HEPES,
generated the basis for the modification of tazobactam. The triazolyl
group was therefore replaced by a carboxylate moiety attached via a
short linker (Figure 1). Such a linked-fragment approach32 would allow
the negatively charged group to interact with K234, S130, and T235
in a fashion similar to that for the sulfonate moiety of HEPES.
Experimental details of the synthesis and spectral data of SA2-13 are
in the Supporting Information. Tazobactam was a kind gift of Wyeth
Pharmaceuticals.
Kinetics of Inactivation. Kinetic constants of the inactivation of
the SHV-1 â-lactamases were determined at 25 °C (room temperature,
RT), using an Agilent 8453 diode array spectrophotometer. Each
experiment was performed in a 1 mL final volume using 20 mM
phosphate-buffered saline at pH 7.4. Measurements were obtained using
nitrocefin (BD Biosciences) (∆ꢀ 482 ) 17 400 M-1 cm-1). A direct
competition assay was performed to determine the dissociation constant
for the preacylation complex, KI or KD, of the inhibitors (SA2-13 and
tazobactam). A final concentration of 100 µM nitrocefin was used as
the indicator substrate along with 7 nM SHV-1 â-lactamase in these
determinations. The data were analyzed to account for the affinity of
nitrocefin for the SHV-1 â-lactamase:
kreact, 1.39 µM SHV-1 was mixed with 0.125 mM SA2-13 or 0.125
mM tazobactam in a total volume of 80 µL for 30 min at RT (I/E ratio
of SA2-13/SHV-1 ) 90/1 and I/E ratio of tazobactam/SHV-1 ) 90/1)
forming a combination of inactive enzyme along with enzyme bound
trans-enamine. This mixture was placed on a Macro Spin Column G10
(The Nest Group, Southborough, MA) and spun in an Eppendorf 5415D
table-top centrifuge at 2000 rpm at RT for 30 s to remove unbound
inactivator. Reactivation of the enzyme, corresponding to conversion
of the enamine to free enzyme, was detected by incubating the 80 µL
flow thru volume for 5, 30, and 1440 min at RT. At these designated
time points, a 5 µL aliquot was removed and the transient recovery of
activity monitored in the presence of 100 µM nitrocefin as an indicator
substrate (final concentration in a 1 mL cuvette is 7 nM SHV-1, 630
nM SA2-13, and 630 nM tazobactam). To serve as a control, 13.9 µM
of SHV-1 without inhibitor was treated in an identical manner and
measured at each time point. Enzfitter and Microsoft Excel were used
as software programs for analysis, and data are shown in Table 1.
Crystallization and Soaking. SHV-1 â-lactamase was expressed
and purified according to previously described protocols.28 Crystals of
SHV-1 were grown using the sitting drop method as described
previously.33 The final 10 µL drop was prepared using a 4 µL protein
solution, 1 µL of 5.6 mM Cymal-6 (Hampton Research), and 5 µL of
30% w/v PEG 6000, in 100 mM HEPES pH 7.0. The reservoir solution
contained 30% w/v PEG-6000 in 100 mM HEPES pH 7.0 and crystals
grew in 2-3 days at RT. To determine the best soaking time to trap
the intermediate, Raman crystallography was used to identify and track
the intermediate (which led us to increase the soaking concentration to
50 mM to obtain maximal occupancy). SHV-1 crystals were transferred
(31) Nukaga, M.; Mayama, K.; Hujer, A. M.; Bonomo, R. A.; Knox, J. R. J.
Mol. Biol. 2003, 328, 289-301.
KD(corrected) ) KD(observed)/(1 + [S]/Km)
(1)
(32) Verlinde, C. L.; Rudenko, G.; Hol, W. G. J. Comput.-Aided Mol. Des.
1992, 6, 131-147.
The first-order rate constant for enzyme inactivation, kinact, was
determined by monitoring the reaction time courses in the presence of
(33) Kuzin, A. P.; Nukaga, M.; Nukaga, Y.; Hujer, A.; Bonomo, R. A.; Knox,
J. R. Biochemistry 2001, 40, 1861-1866.
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A R T I C L E S
Padayatti et al.
Table 2. Data Collection and Refinement Statistics for wt SHV-1
Complexed to SA2-13
enzyme and inhibitor complex inactivation (kinact) are 2-fold
different. The slower kinact for SA2-13 compared to tazobactam
is also apparent when performing Raman measurements on wt
SHV-1 crystals (data not shown). This results in a 33-fold
difference in the second-order rate constant of inactivation. In
contrast, the kreact following 5 and 30 min incubations are
markedly different. SA2-13 dissociates from the enzyme:
inhibitor complex 8.5-fold slower at 5 min and 10-fold slower
at 30 min. The consequence of this behavior is an equal residual
velocity determination at 5 and 30 min (4%). When measure-
ment was extended out to 1440 min, there was 47% recovery
of activity in the SA2-13:SHV-1 inactivation mixture, while
the tazobactam:SHV-1 mixture resulted in complete inactivation.
Structure Refinement. The refined model of the wt SHV-1
â-lactamase soaked with SA2-13 bound to tazobactam contains
residues 26-292, a covalently bound trans-enamine conforma-
tion of SA2-13 molecule, one partial and one entire Cymal-6
molecule, and 344 water molecules. Nine of the protein side
chains had alternate conformations, and one entire residue,
N170, had an alternate conformation. The final model yielded
an R-factor of 15.4% and R-free of 16.7% for data between 50
and 1.28 Å (Table 2). The stereochemistry of the refined model
was excellent with 93% of the residues falling within the most
favored core of the Ramachandran plot and none within the
disallowed region.
Like other inhibitor-SHV-1 complexes, the protein structure
is very similar to that of previous SHV-1 structures. The root-
mean-square difference for superposition of the CR atoms of
residues 26-292 is 0.33 Å between the uncomplexed wt SHV-1
â-lactamase and this SA2-13 complexed structure. The co-
valently bound SA2-13 has induced virtually no changes in the
active site with the exception of residue N170 which has 2
alternate conformations: the “inward” wt conformation pointing
into the active site as observed in the uncomplexed SHV-1
structure where it binds the nucleophilic deacylation water;40
an “outward” conformation where it interacts with the carboxy-
late moiety attached to C3 of SA2-13 similarly to how
tazobactam interacts in the E166A SHV-1 structure (Figures 2
and 3).30 The alternate N170 conformations cause the nucleo-
philic deacylation water, which is normally held by E166 and
N170, to have partial occupancy (waters w1 and w2 in Figure
3).
Data Collection
space group
P212121
unit cell dimensions (Å)
wavelength (Å)
resolution (Å)
redundancy
49.8, 55.2, 84.4
1.0332
50-1.28 (1.33-1.28)
5.9
unique reflections
I / σ(I)
59, 323
41.5/10.4
3.6 (16.6)
97.7 (96.5)
R
merge (%)
completeness (%)
Refinement
resolution range (Å)
R-factor (%)
50-1.28
15.4
R-free (%)
16.7
rmsd deviation from ideality
bond lengths (Å)
angles (deg)
0.0076
1.50
average B-factor (Å2)
protein
8.3
inhibitor
water molecules
16.6
20.4
to 10 µL drops containing 30% PEG 6000 in 0.1 M HEPES pH 7.0
with 0.56 mM Cymal-6 and 50 mM SA2-13 compound and allowed
to soak for 10 min. This was immediately followed by cryoprotection
of the crystals via transfer to mother liquor containing 25% 2-methyl-
2,4-pentanediol (MPD) for 1-2 min including the 50 mM SA2-13 prior
to cryoquenching by immersing the crystal in liquid nitrogen using a
cryoloop (Hampton Research, Inc.). Data were collected at the
Advanced Photon Source APS (SBC beamline 19-ID) at 1.0332 Å
wavelength to 1.28 Å resolution and were processed using SCALEPACK
and DENZO34 (Table 2).
Crystallographic Refinement. Structure determination of the SA2-
13-soaked SHV-1 crystal was carried out using the isomorphous crystal
structure of wt SHV-1 â-lactamase (PDB 1G56).33 The program CNS35
was used for crystallographic refinement. The starting model, consisting
of only the protein coordinates, was subjected to initial rigid body
refinement, followed by model minimization, temperature factor
refinement, and simulated annealing. After several rounds of refinement,
the Cymal-6 and water molecules were placed into difference density
and included in additional rounds of refinement. Finally, the compound
SA2-13 in the trans-enamine form was readily built into clear electron
density (Figure 2) and added to the model for refinement. Throughout
the refinement, an overall anisotropic B-factor was applied. Refinement
topology and parameter files for SA2-13 and Cymal-6 were generated
using the PRODRG2 webserver,36 and the program O was used for
interactive graphical model rebuilding.37 Crystallographic refinement
was monitored using the programs DDQ38 and PROCHECK,39 and the
final refinement statistics are shown in Table 2.
SA2-13 Conformation. The omit electron density is of
excellent quality showing clear density for all SA2-13 moieties
such as the sulfone, both carboxyl groups, and the carbonyl
oxygen in the linker (Figure 2). This is remarkable since repeated
attempts to soak in tazobactam, or other inhibitors, using
identical soaking protocols have generated only empty active
sites in wt SHV-1 (unpublished results). The linearized SA2-
13 structure is observed in the trans-enamine conformation in
an almost ringlike structure. The SA2-13 molecule is at one
end covalently attached to S70, and at its other end, is the
carboxylate linker, anchored via a salt bridge and hydrogen
bonds, bringing this end in close proximity to its covalent
attachment point (Figures 2-4). The middle of the SA2-13 is
observed to “loop” out of the active site. The torsion angle of
the enamine bond defined by atoms C7-C6dC5-N4 refined
to a value of 166° which is similar to that observed for
Results
Kinetic Analysis. The kinetics of inactivation of SA2-13 and
tazobactam are summarized in Table 1. The KD for SA2-13
compared to tazobactam is 17-fold greater (0.100 ( 0.025 vs
1.70 ( 0.1 µM, respectively). The first-order rate constants for
(34) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326.
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Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu,
N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta
Crystallogr., Sect. D 1998, 54, 905-921.
(36) van Aalten, D. M.; Bywater, R.; Findlay, J. B.; Hendlich, M.; Hooft, R.
W.; Vriend, G. J. Comput.-Aided Mol. Des. 1996, 10, 255-262.
(37) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard. Acta Crystallogr.,
Sect. A 1991, 47 (Pt 2), 110-119.
(38) van den Akker, F.; Hol, W. G. Acta Crystallogr., Sect. D 1999, 55 (Pt 1),
206-218.
(39) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J.
Appl. Crystallogr. 2001, 26, 283-291.
(40) Kuzin, A. P.; Nukaga, M.; Nukaga, Y.; Hujer, A. M.; Bonomo, R. A.;
Knox, J. R. Biochemistry 1999, 38, 5720-5727.
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A Penam Sulfone Bound to wt SHV-1
â
-Lactamase
A R T I C L E S
Figure 2. Electron density of the SA2-13 compound bound in the active site of wt SHV-1 â-lactamase. A side-by-side stereoview of 1.28 Å resolution |Fo|
- |Fc| omit electron density contoured at 2.5 σ is depicted in green. Atom types are color coded accordingly: oxygen (red); nitrogen (blue); sulfur (yellow);
carbon (light and dark gray for the protein and SA2-13, respectively). Waters labeled W1-4 are waters numbered 339A, 339B, 154, and 203, respectively,
in the deposited coordinate set.
Figure 3. Stereo diagram depicting the interactions of SA2-13 within the active site of SHV-1 â-lactamase. Hydrogen bonds are depicted as black dashed
lines. Water molecule are highlighted as red spheres. Residue N170 has two alternative conformations (shown as nos. 1 and 2). This causes the catalytic
deacylation water, in close proximity to N170 and E166, to also have two alternate positions that pair with each of the N170 conformations (indicated as
W1 and W2, respectively).
tazobactam (168°), which confirms its presence as a trans-
enamine bond. The occupancy of the SA2-13 compound in the
active site after the 10 min soak was g90%, as estimated using
occupancy/B-factor refinements carried out similarly to our
previous tazobactam bound structure.30 The occupancy was
therefore fixed at 1.0.
To explain the difference in kinetic behavior between SA2-
13 and tazobactam, the following model illustrated in Figure
1a is proposed. Our data show that forming the Michaelis
complex (E:I, Figure 1a) is favored for tazobactam over SA2-
13 since tazobactam’s KD is 17-fold lower. The first-order rate
constant for acylation of S70 is also twice as fast with
tazobactam vs SA2-13. Nevertheless, SA2-13 and tazobactam
SHV-1 complexes have the same residual activity (4%) at 5
and 30 min time points once the Michaelis complex is
established. This is likely a result of SA2-13 being able to form
more favorable trans-enamine interactions in the active site of
SHV-1 than tazobactam. This is manifested by the longer time
required to regenerate the free enzyme (kreact 8-10-fold longer).
In a biologically relevant time scale (up to 30 min, the half-life
of bacterial cell division), these results are significant. But
whether these inhibitory properties are yet of clinical utility,
since antibiotic:inhibitor combinations are dosed every 6-8 h,
remains to be established since a larger proportion of free
enzyme is generated after 24 h with SA2-13 compared to
tazobactam (see Table 1). This indicates that the partitioning
ratio for imine intermediate to form enamine or inactivate, the
Discussion
The trans-enamine intermediate of â-lactamase inhibitors,
termed the “waiting-room” intermediate, has previously been
observed in class A â-lactamases using spectroscopic measure-
ments,8,19,26 Inhibitor resistant mutations can negatively affect
this intermediate26 making the stabilization of this intermediate
a potential therapeutic strategy for inhibitors that are less
receptive to â-lactamases with an inhibitor resistant phenotype.
The structure-based linked-fragment design of the SA2-13
â-lactamase inhibitor has resulted in stabilization of the trans-
enamine intermediate of the transient inhibited state such that
the enzyme’s recovery (kreact) via deacylation is decreased by
about 10-fold within the first 30 min. This decreased deacylation
rate constant for SA2-13 allows in vitro activity similar to that
of tazobactam for time points up to 30 min despite a 17-fold
drop in affinity.
k
ift/kinact, is greater for SA2-13. The turnover number is also
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A R T I C L E S
Padayatti et al.
thought to be involved in the deacylation step of the reaction41
since it is in close proximity to the S70-acyl bond. One of the
N170 conformations found in this SA2-13 complex (conf no. 1
in Figure 3) is in the “standard” inward conformation priming
the nucleophilic water which is also present, albeit at partial
50% occupancy water (w1 in Figure 3). The second conforma-
tion of N170 (no. 2 in Figure 3) is facing outward and interacts
with the carboxylate moiety attached to C3 of SA2-13. This
second conformation allows room for w1 to move 1.34 Å to
the w2 position which is also partially occupied. Both the w2
position and the outward conformation of N170#2 have been
previously observed in the E166A SHV-1 tazobactam bound
structure.30 Since the N170 and E166 residues (indirectly)
interact with each other to prime the nucleophilic water, the
ability of N170 to adopt a different conformation in the E166A
structure was not too surprising since E166 had been mutated.
However, the ability of the novel SA2-13 compound to partially
change the conformation of N170 in wt SHV-1 is unexpected
and might have implications for inhibitor design as will be
discussed later. N170 mutations have a drastic effect on activity
and deacylation in class A â-lactamases42 suggesting that, by
extrapolation, inhibitor induced conformational changes in N170
could similarly have an effect on activity and deacylation.
Figure 4. Schematic diagram depicting the interactions of SA2-13 when
bound to wt SHV-1 â-lactamase. Hydrogen bonds are depicted as dashed
lines (distances are shown in Å). van der Waals interactions are shown as
a curved line with perpendicular stripes. The double bond between atoms
C5 and C6 is in the trans configuration which, together with the
intramolecular hydrogen bond involving the N4 atom, identify the trans-
enamine intermediate for the covalently bound SA2-13. Interactions and
active site changes that might contribute to enhanced stabilization of the
trans-enamine intermediate of SA2-13 are indicated by i-iv and are
discussed in the text.
higher for SA2-13 indicating that the kdeacyl/kinact is greater as
well for SA2-13. This suggests that the pathway of irreversible
inhibition is less “traveled” by SA2-13 compared to tazobactam.
A possible explanation is that the irreversible pathway might
need the acylated inhibitor to be more dynamic in the active
site to either fragment or react with nearby side chains such as
S130. SA2-13 is more ordered in the trans-enamine intermediate
state favoring the transient inhibitory species, thereby possibly
limiting its irreversible inhibition pathway. Alternatively, the
triazolyl moiety of tazobactam might make the inhibitor more
prone to fragmentation and irreversible inhibition compared to
the carboxyl linker of SA2-13. The addition of the carboxyl
linker in SA2-13 has thus two effects, lengthening the trans-
enamine state and decreasing the irreversible inhibition state,
but these effects seem to cancel out at time points of 30 min
and less.
SA2-13 Active Site Interactions. The SA2-13 forms a
linearized acyl-enzyme trans-enamine intermediate in which
both the â-lactam ring and thiazolidine ring are opened (shown
schematically in Figures 1 and 4). The compound makes a
number of stabilizing interactions (Figures 3 and 4): its O8
atom, positioned in the oxyanion hole, forms hydrogen bonds
with the backbone nitrogens of S70 and A237, the standard
carboxylate group attached to the C3 atom makes a hydrogen
bond with N132 as well as with N170 in one of the conforma-
tions observed (conf no. 2 in Figure 3), and the sulfone moiety
makes an intramolecular hydrogen bond with N4 of SA2-13.
The designed addition of SA2-13, the carboxyl-linker moiety,
makes a number of intended interactions: a salt-bridge with
K234, hydrogen bonds with S130 and T235, and a water-
mediated interaction (Wat#3) with R244 (Figure 3 and 4). As
intended, these latter interactions are similar to interactions that
the sulfonate of HEPES makes in some of the SHV crystal
structures (Figure 5). Although not predicted, one of the oxygens
in the linker makes a water-mediated interaction with S130 (via
Wat#4, Figure 4).
The atomic structure of the SHV-1 â-lactamase complexed
with SA2-13 is surprisingly similar to those of the E166A:
tazobactam structure and the wt SHV-1 structure except for a
critical change involving residue N170 and a water molecule.
The remarkable ease of obtaining a well-defined SA2-13
compound in the wt SHV-1 active site is in agreement with the
decreased kreact since such relatively short soaks did not yield
occupied active sites for tazobactam, or other inhibitors, unless
much higher soaking concentrations in combination with a much
longer time point was used33 or a deacylation deficient E166A
variant was used.29,30 The SA2-13 inhibitor structure bound to
wt SHV-1 has possible implications for enzyme function and
inhibitory mechanisms that will be discussed next.
Comparison of the trans-Enamine SA2-13 and Tazobac-
tam Structures. The interactions of SA2-13 in wt SHV-1 are,
for the atoms in common, quite similar to those observed for
tazobactam in E166A (Figure 6). These shared interactions
include the intramolecular hydrogen bond between the sulfone
and N4 atom, the oxyanion hole interactions, and the carboxyl-
C3 moiety:N170 hydrogen bond. In addition, the newly added
carboxyl moiety of SA2-13 generates a salt-bridge with K234
and hydrogen bonds with S130 and T235 as was designed. A
minor change involves the angle of the compound coming off
S70 which is slightly different for SA2-13 compared to
tazobactam leading to a slight shift of the sulfone/carboxyl part
of about 1.0 Å (Figure 6). This angle has also been shown to
Active Site Conformational Changes. The only significant
difference in the active site is that N170 adopts two alternate
conformations upon SA2-13 binding (Figure 3). This N170
residue in wt SHV-1 normally adopts a single conformation
involved in interacting with a nucleophilic water molecule held
in place by both N170 and E166,33,40 which is also observed in
numerous other class A â-lactamase structures. This water is
(41) Hermann, J. C.; Ridder, L.; Holtje, H. D.; Mulholland, A. J. Org. Biomol.
Chem. 2006, 4, 206-210.
(42) Zawadzke, L. E.; Chen, C. C.; Banerjee, S.; Li, Z.; Wasch, S.; Kapadia,
G.; Moult, J.; Herzberg, O. Biochemistry 1996, 35, 16475-16482.
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13240 J. AM. CHEM. SOC. VOL. 128, NO. 40, 2006

A Penam Sulfone Bound to wt SHV-1
â
-Lactamase
A R T I C L E S
Figure 5. Overlay of SA2-13 and HEPES when bound to SHV-1. The active sites of the HEPES bound SHV-2 structure and that of our SA2-13 wt SHV-1
structure were superimposed. The figure shows the active site of wt SHV-1 depicting both SA2-13 (blue stick) and HEPES (yellow stick) illustrating the
similar position of the sulfone + linker moiety of HEPES and that of the carboxyl + linker moiety of SA2-13.
Figure 6. Superposition of the SA2-13 and tazobactam bound structures. Shown are SA2-13 (blue) when bound to wt SHV-1 and tazobactam (magenta)
when bound to the deacylation deficient E166A variant of SHV-1. Only the protein atoms of the wt SHV-1:SA2-13 complex are shown. The water molecule
involved in forming a hydrogen bond (yellow dashed line) with the triazolyl moiety of tazobactam when complexed to the E166A SHV-1 variant is depicted
as a red sphere.
vary more prominently in the sulbactam and clavulanic acid
bound structures where there are fewer inhibitor:protein interac-
tions.29
S130 whose side chains might play a role in catalysis25,43,44 and
whose properties might be altered by interacting with the
charged carboxyl group. (iii) The newly added carboxyl moiety
is positioned such that it is within ∼3.5 Å of the acyl-enzyme
bond and might therefore electrostatically alter this bond’s
susceptibility to the water’s nucleophilic attack needed for
deacylation. (iv) The additional stabilizing interactions of the
carboxylate force the intermediate in the trans-enamine con-
formation such that it has less opportunity to tautomerize back
to the imine intermediate (Figure 1). The latter intermediate is
Implications for Class A â-Lactamase Inhibitor Design.
The unprecedented ability of SA2-13 to form a stable, long-
lived trans-enamine intermediate in wt SHV-1 is a novel and
promising direction for future â-lactamase inhibitor design.
There are 4 possible reasons for the lower deacylation efficiency
of SA2-13 (shown in Figure 4 labeled i-iv): (i) The newly
added carboxyl group provides a stabilizing force that keeps
the original C3 attached carboxyl moiety in place such that N170
can swing out to interact so that the nucleophilic water is no
longer catalytically primed 100% of the time by N170. (ii) The
newly added carboxyl moiety interacts with residues K234 and
(43) Ellerby, L. M.; Escobar, W. A.; Fink, A. L.; Mitchinson, C.; Wells, J. A.
Biochemistry 1990, 29, 5797-5806.
(44) Thomas, V. L.; Golemi-Kotra, D.; Kim, C.; Vakulenko, S. B.; Mobashery,
S.; Shoichet, B. K. Biochemistry 2005, 44, 9330-9338.
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thought to be the form that undergoes deacylation since the
carbonyl atom is no longer (stabilized) conjugated with the
enamine double bond since this double bond has moved in this
imine intermediate (Figure 1a). All four possible reasons could
significantly contribute to the decreased kreact and trans-enamine
stabilization of SA2-13 although the first reason could maxi-
mally only contribute roughly a factor 2 since the N170 side
chain is displaced only part of the time since it is in alternate
conformations that are roughly equally occupied.
In summary, using the linked fragment design approach, we
have stabilized the trans-enamine intermediate in wt SHV-1
â-lactamase by replacing the triazolyl moiety of tazobactam with
a carboxyl-linker moiety. Although one aspect of tazobactam
was improved, enhancing its trans-enamine intermediate lon-
gevity, SA2-13 has lost some of tazobactam’s efficiency
regarding KD and inhibition efficiency at the 24 h time point.
Both these latter inhibitor characteristics are important since
tazobactam/piperacillin is dosed every 6-8 h. SA2-13’s phar-
macodynamic and pharmacokinetic properties are unknown and
beyond the scope of this manuscript yet our mechanistic studies
demonstrate a proof of concept of stabilizing this intermediate
to obtain a good in vitro inhibitor. Whether this approach will
lead to a potent in vivo inhibitor is unknown, and it is likely
that SA2-13 would need to be further improved. Future
experiments are planned to test the inhibitory efficiency of SA2-
13, and derivatives thereof, against other â-lactamases including
inhibitor resistant and extended spectrum variants of class A
â-lactamases.
Our linked fragment approach in stabilizing the trans-enamine
conformation has some intended features that suggest that this
could be further exploited for general use in optimizing class
A â-lactamase inhibitors. First, the SA2-13 compound makes
few, if any, interactions with residues that are not critical for
catalysis since that might potentially limit its utility due to
increased likelihood of resistance development. The residues
that interact with SA2-13 are conserved (except for S130 which
is a glycine in several inhibitor resistant SHV variants). Second,
a bound negatively charged group in the vicinity of K234 and
S130 is quite often observed in class A â-lactamase crystal
structures, not only in SHV-1 (via a HEPES molecule, Figure
5) but also in PER-1 (via a bound sulfate ion)45 and TEM-1
(via a sulfate ion)46 â-lactamase crystal structures. These
structures suggests that this anion binding site is conserved
among class A â-lactamases and could potentially be further
exploited via rational inhibitor design. Furthermore, â-lactamase
inhibitor design studies focusing on stabilizing the transition
state, via boronic acid analogues, have placed a carboxylate
moiety in this region to improve binding.47 These studies suggest
that this design feature of SA2-13 could be of general interest
to other class A â-lactamases since it involves a very conserved
active site region that can attract negatively charged groups.
Acknowledgment. M.S.H. is supported by a Department of
Veterans Affairs Advanced Career Development Award. R.A.B.
is supported by the Merit Review Program and NIH Grant
R01AI063517-01. J.D.B. acknowledges the support of the
Robert A. Welch Foundation (Grant N-0871) and the Centers
for Disease Control (Grant H75/CCH623342). P.R.C. is sup-
ported by NIH Grant GM54072. F.v.d.A. is supported by NIH
Grant AI062968. We thank Vivien Yee for critical reading of
the manuscript and the personnel at the SBC beamline 19 at
Advanced Photon Source for help with data collection.
Supporting Information Available: Synthetic procedures and
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org. The coordinates and structure factor files
are deposited with the Protein Data Bank (PDB 2H5S).
(45) Tranier, S.; Bouthors, A. T.; Maveyraud, L.; Guillet, V.; Sougakoff, W.;
Samama, J. P. J. Biol. Chem. 2000, 275, 28075-28082.
(46) Jelsch, C.; Lenfant, F.; Masson, J. M.; Samama, J. P. FEBS Lett. 1992,
299, 135-142.
(47) Ness, S.; Martin, R.; Kindler, A. M.; Paetzel, M.; Gold, M.; Jensen, S. E.;
Jones, J. B.; Strynadka, N. C. Biochemistry 2000, 39, 5312-5321.
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