Inhibition of the class C β-lactamase from Acinetobacter spp.: Insights into effective inhibitor design

Article abstract of DOI:10.1021/bi9015988

The need to develop β-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C₃/C₄ carboxylate of β-lactams showed the lowest Ki (11 ± 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla_ADC from 16 to 4 μg/mL and from 8 to 1 μg/mL, respectively. On the other hand, each carbapenem exhibited a K_i of <20 μM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C₆ hydroxyethyl group. To improve our understanding of the interactions between the β-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the β-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADCβ-lactamase. In contrast, theADC-carbapenem models offer a novel role for theR₂ side group and also suggest that elimination of the C₆ hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new β-lactamase inhibitors against these critical drug resistance targets.

Full text of DOI:10.1021/bi9015988

Biochemistry 2010, 49, 329–340 329
DOI: 10.1021/bi9015988
Inhibition of the Class C β-Lactamase from Acinetobacter spp.: Insights into Effective
Inhibitor Design†
Sarah M. Drawz,‡ Maja Babic,@ Chrisrtopher R. Bethel,# Magda Taracila,@ Anne M. Distler, ,O Claudia Ori,r
Emilia Caselli, Fabio Prati,*,r and Robert A. Bonomo*,#,§, ,^
‡Departments of Pathology, §Medicine, Pharmacology, ^Molecular Biology and Microbiology, Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106, @Division of Infectious Disease and HIV Medicine, University Hospitals Case Medical
Center, and #Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, and
rDepartment of Chemistry, University of Modena and Reggio Emilia, 41100 Modena, Italy. OCurrent address: Department of
Chemistry, Cuyahoga Community College, Highland Hills, Ohio 44122.
Received September 11, 2009; Revised Manuscript Received November 19, 2009
ABSTRACT: The need to develop β-lactamase inhibitors against class C cephalosporinases of Gram-negative
pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives,
including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of
Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available
carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a
meta-carboxyphenyl moiety corresponding to the C3/C4 carboxylate of β-lactams showed the lowest Ki (11 (
1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime
minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying blaADC from 16 to 4 μg/
mL and from 8 to 1 μg/mL, respectively. On the other hand, each carbapenem exhibited a Ki of <20 μM, and
timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corre-
sponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C6
hydroxyethyl group. To improve our understanding of the interactions between the β-lactamase and the
inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl
cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this
chiral cephalothin analogue adopts a novel conformation in the β-lactamase active site. Further, the addition
of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the
ADC β-lactamase. In contrast, the ADC-carbapenem models offer a novel role for the R2 side group and also
suggest that elimination of the C6 hydroxyethyl group by retroaldolic reaction leads to a significant
conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and
structures of the boronic acid derivatives and carbapenems provide insights for the development of new
β-lactamase inhibitors against these critical drug resistance targets.
Acinetobacter spp. are Gram-negative pathogens responsible
for an increasing number of serious nosocomial infections,
including hospital-acquired pneumonia, urinary tract infections,
and bacteremia (1-4). This nonfermentive, aerobic pathogen
harbors multiple antibiotic resistance determinants, including
chromosomal AmpC β-lactamase enzymes, OXA carbapene-
mases, metallo-β-lactamases, and multidrug resistance (MDR)1
efflux pumps (5, 6). In addition, changes in outer membrane
proteins decrease the permeability to antimicrobials (7). Besides
intrinsic resistance, Acinetobacter spp. possess the ability to
acquire new resistance determinants through gene mutations,
derepression, and transfer from other organisms. These re-
markable attributes can lead to infections resistant to all avail-
able β-lactam antibiotics (8, 9). Consequently, treatment of
patients with Acinetobacter spp. infections is very challenging,
and therapeutic options are severely limited for MDR
strains (10-12).
One strategy for restoring the efficacy of β-lactam antibiotics is
the development of novel β-lactamase inhibitors. Boronic acid
derivatives are compounds that replace the β-lactam ring with
boronic acid. The boron atom forms a reversible, dative covalent
bond with the active site serine of class A and C β-lactamases,
assuming a geometry that resembles the tetrahedral transition
†This work was supported in part by the Department of Veterans
Affairs Merit Review Program and National Institutes of Health (NIH)
Grant 1R01 A1063517-01. R.A.B. is also supported by the Veterans
Integrated Service Network 10 Geriatric Research, Education, and
Clinical Center. S.M.D. was supported in part by NIH Grant T32
GM07250 and the Case Western Reserve University (CWRU) Medical
Scientist Training Program. M.B. was supported by the Wyeth Fellow-
ship in Antimicrobial Resistance at CWRU. F.P., E.C., and C.O.
gratefully thank Fondazione Cassa di Risparmio di Modena for
financial support.
*To whom correspondence should be addressed. R.A.B.: phone, (216)
791-3800, ext. 4399; fax, (216) 231-3482; e-mail, robert.bonomo@med.
va.gov. F.P.: phone, þ39 059 205 5056; fax, þ39 059 373 543; e-mail,
prati.fabio@unimore.it.
1Abbreviations: ADC, Acinetobacter-derived cephalosporinase; amu,
atomic mass unit; EI MS, electron impact mass spectrometer; ESI-MS,
electrospray ionization mass spectrometry; IPTG, isopropyl β-D-thio-
galactopyranoside; MIC, minimum inhibitory concentration; MDR,
multidrug resistant; NCF, nitrocefin; PBC, periodic boundary condi-
tions; PDB, Protein Data Bank; THF, tetrahydrofuran.
r
2009 American Chemical Society
Published on Web 11/19/2009
pubs.acs.org/Biochemistry

330 Biochemistry, Vol. 49, No. 2, 2010
Drawz et al.
FIGURE 1: Schemes illustrating the interactions of a serine β-lactamase with (A) the cephalosporin ceftazidime, (B) the boronic acid ceftazidime
analogue, compound 2, and (C) the carbapenem imipenem.
state of the β-lactamase hydrolytic reaction (Figure 1) (13, 14). Via
modification of the boronic acid substituents to resemble in
structure, distance, and stereochemical arrangement the R1 side
chains of natural substrates, affinities in the nanomolar range
against class C enzymes of Escherichia coli are achieved (15-17).
A second approach to counteracting β-lactamase-mediated
antibiotic resistance is the design of β-lactams that resist hydro-
lysis. Through the combined efforts of natural product screens
and medicinal chemistry, β-lactamase-stable penem and cephem
derivatives have been modified and synthesized. The most potent
β-lactams are the derivatives of thienamycin (i.e., imipenem,
meropenem, ertapenem, and doripenem). Carbapenems act as
inhibitors of class A, class C, and certain class D β-lactamases
by forming a prolonged acyl-enzyme intermediate with the
β-lactamase that is very slowly hydrolyzed (18-25).
The Acinetobacter-derived cephalosporinases (ADCs) are class
C β-lactamases found in Acinetobacter baumannii and Acineto-
bacter genomospecies 3 and are responsible for resistance to
penicillins, cephalosporins, and β-lactam-β-lactamase inhibitor
combinations (26). These AmpC β-lactamases demonstrate a
remarkable kcat for first-generation cephalosporins and relatively
low affinity for the commercially available β-lactamase inhibi-
tors (26). Therefore, ADC enzymes can serve as important targets
for the design of new mechanism-based inactivators. To date, the
search for effective inhibitors for the ADC β-lactamase remains
challenging. To this end, we synthesized and tested a panel
of boronic acid derivatives with specific side chains to serve as
chemical probes. We also designed a novel boronate that contained
the R1 side chain of cefoperazone. Concurrently, we explored the
role of the R2 side chain of four different carbapenems in the
inhibition of ADC. Taken together, our results indicate that the
interactions between the ADC β-lactamase and inhibitors’ scaf-
folds and side chains yield important insights into the properties of
class C enzyme active sites.
of cephalothin and inhibitors studied are shown in Figure 2. The
boronic acid ceftazidime analogue and cephalothin analogues
were synthesized as previously described (15, 17). The chiral
cephalothin analogues 4 and 5 were obtained in the enantiomeri-
cally pure form. Imipenem and ertapenem were obtained from
Merck & Co. Inc. (Whitehouse Station, NJ). Meropenem was
purchased from AstraZeneca Pharmaceuticals (Wilmington,
DE) and doripenem from Ortho-McNeil Pharmaceutical Inc.
(Raritan, NJ).
Synthesis of the Cefoperazone Analogue. Cefoperazone
analogue 1 was synthesized according to the general protocol for
the other boronic acid derivatives by acylation of pinacol bis-
(trimethylsilyl)aminomethaneboronate with the commercially
available cefoperazone acid, promoted by isobutyl chlorofor-
mate (17). Triethylamine (231 μL, 1.66 mmol) and isobutyl
chloroformate (216 μL, 1.66 mmol) were added to a solution
of (2R)-2-[(4-ethyl-2,3-dioxopiperazinyl)carbonylamino]-2-(4-
hydroxyphenyl)acetic acid (530 mg, 1.66 mmol) in anhydrous
tetrahydrofuran (THF, 60 mL) at 0 ꢀC and allowed to react
under an argon atmosphere for 1 h. A solution of bis-
(trimethylsilyl)aminomethaneboronate (500 mg, 1.66 mmol) in
anhydrous THF (5 mL), previously treated for 30 min with
anhydrous methanol (1.74 mmol), was added at the same
temperature. After 20 min, the cooling bath was removed and
the mixture was allowed to react overnight at room temperature.
Thereafter, the reaction mixture was diluted with diethyl ether
(60 mL), and the precipitate (triethylammonium chloride) was
removed by filtration. The solvent was distilled under reduced
pressure, and the solid residue crystallized from ethyl acetate,
affording the title compound as a whitish solid (54% yield): [R]D
-59.0 (c 0.8, CH3OH). The pinacol ester spontaneously hydro-
lyses in the phosphate buffer, generating the corresponding
cefoperazone boronic acid.
1H and 13C NMR spectra of compound 1 were recorded on a
Bruker DPX-200 spectrometer. The chemical shifts (δ) are
reported in parts per million downfield from the internal
standard, tetramethylsilane (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet). Mass fragmentations were determined on
MATERIALS AND METHODS
Antibiotics and Inhibitors. Ceftazidime and cefotaxime were
purchased from Sigma (St. Louis, MO). The chemical structures

Article
Biochemistry, Vol. 49, No. 2, 2010 331
FIGURE 2: Chemical structures of (A) commercially available inhibitors and cephalosporin substrate cephalothin, (B) boronic acid derivatives,
and (C) carbapenems used in this study. The cephalothin structure is labeled withthe accepted ring numbering system. The C6 hydroxyethyl group
of imipenem, which may be eliminated after formation of the acyl-enzyme intermediate, is circled in dashed lines.
a Finnigan MAT SSQ A electron impact mass spectrometer (EI
MS, 70 eV): 1H NMR (200 MHz, DMSO-d6) δ 1.07 (3H, t, J =
7.2 Hz, CH2CH3), 1.15 (12H, s, CH3 pic), 2.44 (2H, d, J = 3.7
Hz, NCH2B), 3.38 (2H, q, J = 7.2 Hz, CH2CH3), 3.48-3.62 (2H,
m, CH2 pip), 3.82-3.96 (2H, m, CH2 pip), 5.32 (1H, d, J = 7.3
Hz, CHPh), 6.71 (2H, d, J = 8.5 Hz, meta), 7.19 (2H, d, J = 8.5
Hz, ortho), 8.41 (1H, t, J = 3.7 Hz, NH), 9.14 (1H, s, OH), 9.64
(1H, d, J = 7.3 Hz, NH); 13C NMR (50 MHz, DMSO-d6) δ 12.3,
25.1, 25.2, 40.8, 42.1, 43.3, 56.5, 83.3, 115.5, 128.7, 129.2, 152.3,
155.9, 157.5, 159.8, 170.6; EI MS m/z (%) 474 (Mþ, 0.2), 458
(0.4), 400 (2), 374 (2), 332 (14), 317 (10), 275 (16), 274 (96), 273
(36), 216 (19), 173 (9), 148 (18), 142 (58), 121 (35), 120 (46), 99
(100), 83 (15).
Genetic Constructs and Host Strains. For large-scale
protein expression and β-lactamase characterization, the blaADC
gene (specifically, blaADC-7) was cloned into the pET24a(þ)
vector (kanamycin resistant, Novagen, Madison, WI) following
a previously published method (26). After sequencing verifica-
tion, the correct construct was maintained in E. coli DH10B cells
and transformed into E. coli BL21(DE3) cells for protein
expression. For MIC determinations, blaADC was directionally
cloned into the pBC SK(þ) phagemid vector (chloramphenicol
resistant, Stratagene, La Jolla, CA) as previously described (26).
Briefly, the pET24a(þ) blaADC construct was digested with XbaI
and BamHI in Multi-Core buffer (Promega, Madison, WI),
preserving the 50 upstream flanking region from the pET24a(þ)
vector in front of the insert when ligated into pBC SK(þ).
Antimicrobial Susceptibility (MICs). E. coli DH10B cells
expressing the blaADC gene were phenotypically characterized by
lysogeny broth agar dilution MICs. The MICs for various
antibiotics were determined using a Steers Replicator that
delivered 10 μL of a diluted overnight culture containing 104
colony forming units. The cephalothin analogues 3 and 5 were
tested at a constant concentration of 4 μg/mL in combination
with either ceftazidime or cefotaxime.
β-Lactamase Purification. The ADC β-lactamase was pre-
pared from E. coli BL21(DE3) cells after induction with iso-
propyl β-D-thiogalactopyranoside (IPTG). Cultures (500 mL)
were induced at an optical density at 600 nm of 0.5-0.8 (final
IPTG concentration of 0.2 mM) at 37 ꢀC for 4 h in lysogeny
broth. These cells were pelleted and resuspended in 50 mM Tris
(pH 7.4) and β-lactamase liberated with lysozyme and EDTA per
established methods (28). Accordingly, the ADC protein was
purified by preparative isoelectric focusing and fast protein
liquid chromatography with a Sephadex Hi Load 26/60 column
(Pharmacia, Uppsala, Sweden) (29). The enzyme was quantified,
purity assessed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and size verified by mass spectrometry (26).
Kinetics. Steady state kinetics were performed on an
Agilent (Palo Alto, CA) 8453 diode array spectrophotometer.
Each continuous assay was performed in 10 mM phosphate-
buffered saline (pH 7.4) at room temperature. Ki values were
calculated by measurement of the initial velocity in the
presence of a constant concentration of enzyme (3 nM), and
increasing concentrations of the inhibitors (ranging from
50 nM to 500 μM) competed against the indicator substrate
nitrocefin (NCF) (BD Biosciences, San Jose, CA) (Δε482
=
17400 M-1 cm-1). The Ki values were corrected to account for
the affinity of NCF for ADC using the following equa-
tion (30):
KiðcorrectedÞ ¼ KiðobservedÞ=ð1 þ ½Sꢀ=KmNCF
Þ
ð1Þ
Due to time-dependent inhibition of chiral boronic acid
derivatives, compounds 4 and 5 were preincubated with enzyme

332 Biochemistry, Vol. 49, No. 2, 2010
Drawz et al.
for 5 min in phosphate-buffered saline before the reaction was
initiated with the addition of substrate, as described pre-
viously (15, 27, 31-33). In earlier experiments, preincubation
of the achiral compound 3 with enzyme did not affect the Ki
determination (data not shown).
Table 1: Ki Values of Inhibitors in Direct Competition Assays with ADC
inhibitor
Ki (μM)
commercially available class A inhibitors
clavulanate
4275 ( 253a
109 ( 3a
Electrospray Ionization Mass Spectrometry (ESI-MS).
Mass spectrometry was performed to determine products of
inactivation. We incubated 14 μM ADC for 15 min with and
without compounds 2 and 5 and each carbapenem at an
inhibitor:enzyme ratio of 20:1. Each reaction was terminated
by the addition of 0.1% trifluoroacetic acid and each mixture
immediately desalted and concentrated using a C18 ZipTip
(Millipore, Bedford, MA) according to the manufacturer’s pro-
tocol. Samples were then placed on ice and analyzed within 1 h.
Spectra of the intact ADC-inhibitor proteins were generated
on a Q-STAR XL Quadrupole-Time-of-Flight mass spectro-
meter (Applied Biosystems, Framingham, MA) equipped with a
nanospray source. Experiments were performed by diluting the
protein sample with a 50% acetonitrile/0.1% trifluoroacetic acid
mixture to a concentration of 10 μM. This protein solution was
then infused at a rate of 0.5 μL/min, and the data were collected
for 2 min. Spectra were deconvoluted using Analyst (Applied
Biosystems). All measurements have an error of (3 atomic mass
units (amu).
sulbactam
tazobactam
91 ( 21a
boronic acid derivatives
compound 1
0.60 ( 0.06
0.31 ( 0.03
compound 2
compound 3
0.78 ( 0.02
compound 4
0.036 ( 0.008
0.011 ( 0.001
compound 5
carbapenems
imipenem
1.3 ( 0.1
5.8 ( 0.2
12.2 ( 0.4
19 ( 2
ertapenem
doripenem
meropenem
aKi values obtained previously by the same methodology (26).
E. coli AmpC (PDB entry 2BLS). The program uses a method
based on CLUSTAL W which aligns multiple sequences using
a progressive pairwise alignment algorithm (38). A multiple-
sequence alignment is generated, and secondary structure
matches graded as identical, strong, weak, or nonmatching are
based on the calculated alignment score.
The minimized and equilibrated ADC model was used for
constructing the acylation complexes of the ADC β-lactamase
and the chiral cephalothin analogue 5, imipenem, and merope-
nem ligands. The ligand structures were built using Discovery
Studio Fragment Builder tools. The CHARMm force field was
applied; the molecule was solvated with PBC and minimized
using a Standard Dynamics Cascade protocol (one minimization
using the steepest descent algorithm, followed by the adopted
basis Newton-Raphson algorithm and three subsequent
dynamics stages at NVT and 300 K).
The minimized ligands were docked in the active site of the
enzyme using LibDock (39). The generated conformations
(30-40) were manually analyzed and the most favorable ones
chosen. The complex between the ligand and the enzyme was
created, solvated, and energy-minimized. The acyl-enzyme
complex was created by making a bond with Ser64, and the
assembly was further minimized using the conjugate gradient
algorithm with PBC to 0.001 minimum derivative. To reach the
minimum equilibrium, the complexes were equilibrated using
molecular dynamics simulations.
Molecular Representations. The ADC model was gene-
rated by the SWISS-MODEL automated protein structure
homology modeling server (available at http://swissmodel.
expasy.org) using the deposited GenBank ADC-7 protein se-
quence (AY648950) and the Enterobacter aerogenes CMY-10
β-lactamase as a template (Protein Data Bank entry 1ZKJ)
(34, 35). We optimized the generated model by energy minimiza-
tion using Discovery Studio version 2.1 (Accelrys, San Diego,
CA). The minimization was performed in several steps, using a
steepest descendent and conjugate gradient algorithm to reach
the minimum convergence (0.02 kcal mol-1 A-1). The protein
˚
was immersed in a water box, 7 A from any face of the box, and
the solvation model used was with periodic boundary conditions
(PBC). The force field parameters of CHARMm were used for
minimization, and the particle mesh Ewald method was used to
treat long-range electrostatics. The bonds that involved hydrogen
atoms were constrained with the SHAKE algorithm. Following
equilibration, two separate 2 fs molecular dynamics simulations
(heating-cooling and production) at a constant pressure and
temperature (300 K) were conducted for the ADC model. The
trajectories were analyzed, and the minimum energy conforma-
tion was chosen.
To verify the quality of the ADC β-lactamase model, we used
the Protein Structure and Model Assessment Tools available at
http://swissmodel.expasy.org (see Figures 1-3 of the Supporting
Information). The atomic empirical mean force potential
(ANOLEA) evaluation of the model’s packing quality showed
that 98% of the amino acids were in the favorable energy
environment (36). We validated the stereochemical quality of
the ADC model using Procheck which compares the geometry of
protein residues with the stereochemical parameters of well-
refined, high-resolution structures (37). Additionally, 97.5% of
the non-proline, non-glycine residues in the ADC model were in
the most favorable region of a Ramachandran plot.
RESULTS
Kinetics. Table 1 summarizes our kinetic analysis of the
inhibition of ADC β-lactamase. To establish a comparison, we
list the previously reported Ki values of the commercially avail-
able β-lactamase inhibitors against ADC; these Ki values are not
in the range that would translate into effective inhibition in MIC
testing (26).
In contrast, we found that the boronic acid derivatives
containing the R1 side chain of cephalosporins bind the class C
ADC with Ki values in the nanomolar range. The cefoperazone
analogue and the ceftazidime analogue, compounds 1 and 2,
respectively, exhibit Ki values of e1 μM. Compound 5 with the
cephalothin R1 side chain and the meta-carboxyphenyl ring,
which has a carboxylate that is designed to mimic the geometry
and distances of the conserved C4 carboxylate of cephalosporin
The Align Multiple Sequences function of Discovery Studio
version 2.1 allowed us to compare the generated ADC protein
structure with that of the deposited crystal structure coordinates
for the Enterobacter cloacae P99 enzyme (PDB entry 1XX2) and

Article
Biochemistry, Vol. 49, No. 2, 2010 333
F
IGURE 3: Overlay of the molecular coordinates for the E. coli AmpC covalently bound to the cephalothin substrate (colored by atom, PDB entry
1KVM) and boronic acid chiral cephalothin analogue, compound 5 (colored green, PDB entry 1MXO). The position of cephalothin’s
dihydrothiazine ring and C4 carboxylate is shown relative to the meta-carboxyphenyl group of compound 5, which is designed to mimic in
stereochemistry and geometry the conserved β-lactam carboxylate.
β-lactams (Figure 3), had the lowest Ki for ADC (11 ( 1 nM). We
Table 2: ESI-MS Analysis (amu) of ADC Alone and Incubated with
Inhibitorsa
interpret the 70-fold difference in Ki values between compounds 3
and 5 to mean that the meta-carboxyphenyl moiety contributes
significantly to the binding of this inhibitor with the ADC
β-lactamase. However, compound 4, which lacks only the meta-
carboxylate as compared to 5, also had a low Ki of 36 ( 8 nM.
Because inhibition reactions with the boronic acid derivatives
are reversible, we quantified the binding energy contribution of
these substituents by using Ki as an equilibrium constant in the
Gibbs free energy equation (15):
predicted
molecular weight species observed Δdifference from
of β-lactamase
or inhibitor
in deconvoluted
spectra
the β-lactamase
molecular weight
ADC alone
40631
40638
7
ADC with inhibitor
compound 2
compound 5
imipenem
330
319
299
40637
40638
40936
40893
41112
41069
41058
41015
41021
40979
1
0
298
255
474
431
420
377
383
341
ΔΔG ¼ -RT ln½ðKi5Þ=ðKi3Þꢀ
ð2aÞ
ertapenem
doripenem
meropenem
476
420
383
Compared to the achiral cephalothin analogue 3, we deter-
mined that the meta-carboxyphenyl group on the chiral cepha-
lothin analogue 5 contributes 2.5 kcal/mol in binding energy to
ADC. The presence of the meta-carboxylate provides 0.7 kcal/
mol of the 2.5 kcal/mol provided by this substituent:
aAll measurements have an error of (3 amu.
ΔΔG ¼ -RT ln½ðKi5Þ=ðKi4Þꢀ
ð2bÞ
ESI-MS and the Nature of Inactivation Products. We
performed timed ESI-MS with ADC, compounds 2 and 5, and
the carbapenems to detect covalent intermediates in the inactiva-
tion pathway. As shown in Table 2 and Figure 4, analysis of the
ADC-2 and ADC-5 reactions using ESI-MS shows that the
β-lactamase is unmodified. This result is expected as boronates
undergo reversible inhibition. In contrast, when ADC was
reacted with the carbapenems, the predominant mass adduct
formed corresponded to the sum of the molecular weights of the
enzyme and the inhibitor, suggesting the formation of a non-
fragmented covalent acyl-enzyme product. This result is con-
sistent with MS data of the carbapenems forming acyl-enzyme
intermediates with the class A SHV-1 β-lactamase (21). In
addition, the ESI-MS analysis of each ADC-carbapenem spec-
trum included a small adduct which was the mass equivalent of
the carbapenem and β-lactamase minus 43 ( 3 Da, an observa-
tion made previously (40). We advance that there is a retroaldol
elimination of the ligand’s C6 hydroxyethyl substituent (see
Figure 5 and discussion below).
On the basis of these calculations, we maintain that the
presence of the phenyl group, which approximates the cephalos-
porin’s dihydrothiazine ring, is largely responsible for the low Ki
values of compounds 4 and 5.
Carbapenems are highly effective β-lactam antibiotics in the
treatment of Gram-negative bacteria. Furthermore, carbape-
nems form prolonged acyl-enzyme intermediates with class
C β-lactamases which can effectively inhibit the enzyme. We
chose the four commercially available carbapenems to explore
the determinants that contribute to the inactivation of ADC
β-lactamase. Carbapenems, as ADC AmpC inhibitors, demon-
strated low Ki values (ranging from 1.3 ( 0.1 to 19 ( 2 μM).
Comparing the carbapenems with different R2 side chains,
we see that the least substituted, imipenem, has the lowest Ki.
The penem scaffold on which these β-lactams are constructed is
similar; thus, we assign the differences in Ki values among these
carbapenems to the interactions of the β-lactamase with the R2
side chain.

334 Biochemistry, Vol. 49, No. 2, 2010
Drawz et al.
Molecular Representations. To understand the interactions
between the carbapenems and high-affinity cephalothin analogue
5 in the absence of a crystal structure, we constructed a molecular
model of ADC β-lactamase from a homology modeling server.
Accurate high-resolution protein models can be generated from
templates with >50% sequence similarity; our model shared
66% sequence similarity with the template (41).
We first compared our ADC model to the defined crystal
structures of En. cloacae P99 and E. coli AmpC. An alignment
based on the predicted, and known, secondary structures of
ADC, P99, and E. coli AmpC shows that ADC shares 63%
sequence similarity with both the P99 and E. coli AmpC
β-lactamases (37 and 40% amino acid identity, respectively)
(Figure 4 of the Supporting Information).
Using the representation of the ADC-5 acyl-enzyme, we
gained insight into how the Acinetobacter cephalosporinase
interacts with compound 5. As the crystal structure of the E. coli
AmpC in complex with the same boronic acid derivative has
been determined (Ki = 1 nM; PDB entry 1MXO), we overlaid
this structure on our generated model (Figure 6) (15). The overall
tertiary structures of ADC and the E. coli AmpC are similar with
conservation of the R-helix and β-sheet domains. The loops and
turns between these secondary structures follow slightly different
paths, but we note that these deviations may be part of the model
construction and are allowable (e.g., the permission of increased
flexibility for these strand regions).
In the active site, the backbone amides of Ser64 and Ser318,
which form the β-lactamase oxyanion hole or electrophilic center,
˚
are ∼1 A farther apart in the ADC model than in the E. coli
AmpC structure (42, 43). The position of the backbones and side
chains of the catalytically important Tyr150 and Lys67 also
˚
varies by approximately 2 A between the enzymes (44, 45). Thus,
our model suggests that ADC may harbor a unique binding
region as compared to the E. coli AmpC. These tertiary features
are reflected in the disposition of compound 5 in the ADC
acyl-enzyme model; in Figure 7, we show that the inhibitor may
adopt different conformations in these class C β-lactamases.
Comparing equivalent atoms of the boronic acid derivatives (e.g.,
the meta-carboxylate carbons or thiophene sulfur atoms) reveals
F
IGURE 4: Deconvoluted mass spectra of (A) ADC β-lactamase
alone, (B) ADC after incubation with compounds 2 and 5 for
15 min, and (C) ADC β-lactamase after incubation with imipenem,
ertapenem, doripenem, and meropenem for 15 min. The peak in each
ADC-boronate spectrum corresponds to the unmodified ADC
enzyme. The major peak in each of the ADC-carbapenem spectra
indicates covalent attachment of the β-lactam with a minor addi-
tional peak corresponding to the acyl-enzyme intermediate without
the carbapenem’s C6 hydroxyethyl substituent. All measurements
have an error of (3 atomic mass units (amu).
˚
a >5 A deviation in the configuration of the compounds in the
overlaid structures. Crystal structures of boronic acid deriva-
tives with AmpC enzymes typically show that one boronic acid
oxygen atom is placed in the oxyanion hole formed by residues
64 and 318, and the other oxygen atom forms a hydrogen bond
with Tyr150 (15, 17, 44, 46). In the ADC-5 model, one of the
boronic acid hydroxyl groups interacts with the Ser64 back-
bone carbonyl oxygen, but as the boronic acid and chiral
substituents on the inhibitor are rotated approximately 120ꢀ
compared to those of the E. coli β-lactamase structure, both
Susceptibility Testing. Nanomolar affinity inhibitors are
clinically useful only if they can penetrate the outer cell wall of
Gram-negative organisms and restore susceptibility to partner
β-lactams. To this end, we performed MIC testing using com-
pounds 3 and 5. Our results show that when ADC β-lactamase is
expressed in the uniform E. coli DH10B background, the
cephalothin analogues 3 and 5 lower MICs to ceftazidime and
cefotaxime (from 16 to 8 and 4 μg/mL and from 8 to 2 and 1 μg/
mL, respectively) (Table 3).
Our previous data showed that E. coli DH10B harboring
blaADC have MICs of 0.06 μg/mL with respect to meropenem,
ertapenem, and imipenem-cilistatin (26). Thus, we did not per-
form MIC testing with the carbapenems in combination with the
cephalothin analogues as a reduction in susceptibility would be
difficult to detect.
˚
oxygens are approximately 5 A from Ser318 or Tyr150.
˚
Instead, Tyr150 is within ∼3 A of both the carbonyl oxygen
and thiophene ring sulfur atom from the cephalothin R1 group
of the boronic acid derivative. Our model also shows a
hydrogen bond between this R1 carbonyl oxygen and Lys67.
Thus, the residues contributing to the high affinity of this
inhibitor for these two β-lactamases may play different roles in
each AmpC.
In our kinetic studies, we noted 15-fold differences between the
Ki values of the highest-affinity carbapenem (imipenem) and the
lowest-affinity carbapenem (meropenem). As these compounds
differ primarily in their R2 side chains, we created models of
the ADC-imipenem and ADC-meropenem acyl-enzyme

Article
Biochemistry, Vol. 49, No. 2, 2010 335
F
IGURE 5: Proposed mechanism of the retroaldolic reaction leading to elimination of the C6 hydroxethyl substituent from the β-lactama-
se-carbapenem acyl-enzyme intermediate. Glu272, supported by Lys315, may serve as the base to deprotonate the alcoholic functionβ-hydroxy
carbonyl moiety of the C6 substituent. Alternatively, Glu272 may abstract a proton from Lys315 which subsequently deprotonates the C6 group
(mechanism colored red). The incipient negative charge on the β-lactam carbonyl could be supported by Tyr150 and Lys67.
DISCUSSION
Table 3: MIC Values (micrograms per milliliter) of Ceftazidime and
Cefotaxime in Combination with 4 μg/mL Cephalothin Analogues
Our analysis shows that high-affinity inhibition of the ADC
β-lactamase, a class C cephalosporinase of increasing medical
importance, is a realistic goal. We assayed two types of inhibitors
against ADC: (i) compounds that resemble the natural substrate
for the Acinetobacter cephalosporinase and (ii) the currently
available carbapenems. This approach teaches us important lessons
about the inhibition of this clinically challenging β-lactamase and
elucidates contributions of R1 and R2 side chains. We begin with an
examination of the data revealing the low micromolar Ki values of
the ADC β-lactamase by the carbapenems and then discuss how
the boronic acid derivatives, as chemical probes, yield important
insights into the nature of class C enzyme active sites.
After incubation of ADC and each carbapenem, our ESI-MS
data reveal the formation of two molecular species. The mass of
the predominant species corresponds to the intact carbapenem
acylating ADC; the mass of the minor species corresponds to
that of the acyl-enzyme intermediate minus 43 Da (Table 2).
On the basis of previous MS studies in our and other labora-
tories, we assign the major peak to the carbapenem acyl-
enzyme species (21, 40). Formation of a stable acyl-enzyme
intermediate is supported by previously defined crystal structures
of carbapenems and class A and C β-lactamases (18, 20, 21). Our
ADC-imipenem and ADC-meropenem acyl-enzyme models
show conformations in which the β-lactam carbonyl oxygen is not
found in the oxyanion hole formed by the backbone nitrogens of
residues Ser64 and Ser318. This observation is consistent with the
X-ray crystal structure of the E. coli AmpC β-lactamase with
imipenem where the carbonyl was positioned approximately 180ꢀ
outside of the oxyanion hole (18). Additional crystal structures of
class A β-lactamases in complex with carbapenems have also
shown this repositioning of the β-lactam carbonyl (20, 21). This
displacement is likely precipitated by steric interactions induced
by the carbapenems’ C6 hydroxyethyl groups, producing a
conformational change that forces the carbonyl away from
the oxyanion hole and into a position unfavorable for hydro-
lysis (20, 42, 43). This reasoning offers an explanation for the
inhibition of the Acinetobacter cephalosporinase by the car-
bapenems.
E. coli DH10B
E. coli DH10B blaADC
ceftazidime
1
16
8
ceftazidime-compound 3
ceftazidime-compound 5
cefotaxime
1
1
4
0.06
0.06
0.06
8
cefotaxime-compound 3
cefotaxime-compound 5
2
1
intermediates to explore the Ki contributions of these substituents
(Figure 8A,B). On the basis of the MS results indicating the
presence of species corresponding to the acyl-enzyme inter-
mediate both with and without the C6 hydroxyethyl group of the
carbapenems, we constructed representations of intact imipenem
and meropenem as well as these compounds without their C6
hydroxyethyl group. Our models predict that when the hydro-
xyethyl group is present, the carbonyl oxygen from the β-lactam
ring of both imipenem and meropenem is located outside of the
enzyme’s electrophilic, or oxyanion, hole created by the back-
bone nitrogen atoms of Ser64 and Ser318 (47, 48). Rather, the
imipenem β-lactam carbonyl is hydrogen bonded to Lys67, and
˚
the meropenem β-lactam carbonyl is only 1.5 A from Tyr150.
The R2 side chain for both intact carbapenems is oriented out of
the active site in the acyl-enzyme intermediate.
In contrast, when the C6 hydroxyethyl group is removed, the
conformation of the carbapenem is significantly changed. Most
notably, the β-lactam carbonyl rotates toward the oxyanion hole,
approximately 90ꢀ in imipenem, and entirely into the electrophilic
pocket for meropenem. Further, the R2 side chain of imipenem
flips back toward the active site so that the terminal amide group
˚
is ∼11 A from its position in the complex with the C6 group. The
imipenem R2 group now interacts with Tyr150, Asn152, Lys67, and
Gln120. Modeling without the hydroxyethyl group for meropenem
changes the conformation of the R2 group, but it remains oriented
outside of the binding site. For both imipenem and meropenem, the
model without the C6 hydroxyethyl group is more energetically
favorable, as calculated by the final potential energy of the
complexes (Δ - 520 and Δ - 80 kcal/mol, respectively).

336 Biochemistry, Vol. 49, No. 2, 2010
Drawz et al.
F
IGURE 6: Overlay of molecular coordinates for the E. coli AmpC-5 complex colored yellow (PDB entry 1MXO) and generated ADC-5 model
colored by secondary structure. The position ofR-helices and β-sheets is generally preserved between the two proteins, but deviations are observed
in the strand turns between these secondary structures. Active site differences are illustrated by the altered conformation of 5 (colored green) in
ADC as compared to 5 (colored yellow) bound to the E. coli AmpC.
F
IGURE 7: Comparison of the binding site interactions between the E. coli AmpC-5 (left) and ADC-5 (right) models. Figures have a perspective
view to show positions of the residues in relation to the inhibitor. The boronic acid derivative is bound to Ser64 in both structures, but the relative
rotation of the inhibitor in ADC changes the relationships with other active site residues. Specifically, the boronic acid oxygens (circled in dashed
blue lines) interact with Ala318 and Tyr150 in E. coli AmpC, but the hydrogen bond with Ser318 is lost in ADC. Also in ADC, the meta-
carboxylate of the dihydrothaizine ring analogue has no clear interaction with previously identified carboxylate binding residues (e.g., Asn346,
Arg349, or Asn289 inE. coli AmpC). Instead, the group may form a long hydrogen bond with Asn287. The carbonyl oxygen of the cephalothin R1
sidechain interacts withAsn152inE. coliAmpC but isreoriented towardLys67 in ADC(seedashedred lines). Lastly, the R1 thiophene ringsulfur
in the ADC-5 model is moved toward Tyr150 as compared to the E. coli AmpC-5 structure. Overall, these significant active site differences
suggest that while ADC may possess novel architecture, the ability to recognize inhibitors and substrates is preserved because of the versatile
functions of the binding site residues.
Second, we observed a minor peak in each ADC-carbapenem
spectrum that reflects the elimination of the carbapenem C6
hydroxyethyl group. This observation was reported previously in
the class A Mycobacterium tuberculosis blaC (40). Our molecular
representations of imipenem and meropenem in complex with
ADC give us insights into how the carbapenems are behaving in

Article
Biochemistry, Vol. 49, No. 2, 2010 337
speculate that removal of the C6 group is an alternative mechan-
ism of alleviating the steric clashes induced by this substi-
tuent, allowing repositioning of the acyl-enzyme intermediate.
When the β-lactam carbonyl is aligned in the oxyanion hole, the
conformation is more compatible with hydrolysis, and an
increased level of turnover is likely, consistent with the relatively
small amount of this species evident on ESI-MS. Furthermore,
we observe this process after a 15 min incubation, which is within
the bacterial generation time (i.e., 20 min) and suggests that this
elimination may be occurring in cells.
The mechanism of elimination of the hydroxyethyl group is
likely to be a retroaldolic-type reaction of the β-hydroxyethyl
moiety of the β-lactamase acyl-enyzme intermediate (Figure 5).
We propose that Glu272, supported by Lys315, may serve as the
base to deprotonate directly the alcoholic function of the C6
substituent. Alternatively, Glu272 may abstract a proton from
the amine side chain of Lys315, and then Lys315 would then
deprotonate the carbapenem C6 alcohol group. In either case, the
negative charge on the β-lactam carbonyl could be stabilized by
Tyr150 and Lys67. Interestingly, this proposes another role for
Tyr150, which is already implicated in both acylation and
“substrate-activated catalysis” in AmpC enzymes (44, 45, 50, 51).
This mechanism is consistent with our molecular representations
which show Glu272 within hydrogen bonding distance of the C6
hydroxyethyl group for both imipenem and meropenem; the
Lys315, Tyr150, and Lys67 residues are also positioned to
support this reaction (see Figure 8A,B). Further investigation
of these residues and their potential roles in the retroaldolic-type
reaction will help elucidate the C6 elimination mechanism.
Structural and kinetic studies provide evidence that, following
acylation of carbapenems by β-lactamases, the acyl-enzyme
intermediate formed can tautomerize between a Δ2- and
Δ1-pyrroline species that have differing rates of deacylation
(19, 52-55). We modeled both the tautomers in our molecular
representations with ADC, but the interactions between the
enzyme and the carbapenems were not significantly different
for either the Δ1 or Δ2 species. We anticipate that the Δ2-Δ1
tautomerization exists as both a separate and integrated pathway
to C6 hydroxyethyl group elimination and plan further examina-
tion of the reaction and its implications for inhibition.
We next turn our attention to the contribution of the R2 side
chain to the differing Ki values of the carbapenems for the ADC
β-lactamase. The four carbapenems tested share a common
β-lactam ring scaffold and vary by their R2 substituents, yet we
observed up to 15-fold differences in Ki values. Our models offer
insights into how these side groups interact with the ADC enzyme
and suggest that each carbapenem may behave uniquely. In both
the imipenem and meropenem models including the C6 hydro-
xyethyl group, the R2 side chain is oriented out of the active
site and does not engage in significant interactions with the
enzyme. This outward conformation is also seen in the E. coli
AmpC-imipenem, TEM-imipenem, and SHV-meropenem
crystal structures (PDB entries 1LL5, 1BT5, and 2ZD8, res-
pectively) (18, 20, 21). However, upon removal of the C6
hydroxyethyl group, the R2 side chain of imipenem rotates
toward the binding pocket and interacts with several active site
residues, including Tyr150, Asn152, Lys67, and Gln120. In
contrast, the R2 group of meropenem modeled without the C6
hydroxyethyl group is still positioned away from active site
residues. In light of the Ki measurements for these two carbape-
nems, the molecular representations predict that the interactions
between the R2 group of imipenem and ADC may stabilize this
F
IGURE 8: Molecular representation of (A) the ADC-imipenem
acyl-enzyme model and (B) the ADC-meropenem acyl-enzyme
model. The intact carbapenem is colored green and the carbapenem
without the C6 hydroxyethylgrouporange. Hydrogens are not shown
except on the carbapenem C6 hydroxyethyl which is likely deproto-
nated byGlu272, leading to elimination ofthe group. Removalof this
C6 substituent may lead to reorientation of the compound in the
active site. Specifically, the β-lactam carbonyl moves back toward the
oxyanion hole formed by the backbone nitrogens of Ser64 and
Ser318, an approximately 90ꢀ rotation for imipenem and entirely
into the hole for meropenem. Also after C6 elimination, the R2 group
of imipenem is repositioned from outside of the binding pocket into a
network of interactions with Tyr150, Asn152, Lys67, and Gln120.
Active site interactions are not observed for the R2 group of
meropenem, which may have implications for the differing Ki values
of these carbapenems.
the active site of ADC following this elimination. When the C6
hydroxyethyl group is removed from the carbapenem, both
compounds adopt new positions in which the β-lactam carbonyl
moves to be either entirely in the oxyanion hole (meropenem) or
rotated back toward the hole approximately 90ꢀ (imipenem).
This prediction is similar to the conclusion drawn from the X-ray
crystallographic evidence of the class A Asn132Ala TEM enzyme
variant which demonstrated that the substitution allowed the
β-lactam carbonyl to rotate back into the oxyanion hole (49). We

338 Biochemistry, Vol. 49, No. 2, 2010
Drawz et al.
form of the compound in the active site, while for meropenem,
the interactions with the enzyme are less favorable (e.g., electro-
statically or sterically). Notably, the R2 group of imipenem is the
least substituted of those of the carbapenems, and the additional
atoms and ring structures of meropenem, doripenem, and
ertapenem may affect the Ki values by various mechanisms,
e.g., limiting conformational flexibility necessary for rotating
back toward the active site to make favorable interactions. We
note that the R2 side chain of imipenem in the crystal structure
with TEM Asn132Ala remains oriented out of the active site,
despite the alleviation of the steric strain caused by the C6
substituent (PDB entry 1JVJ) (49). That our model of imipenem
without the C6 hydroxyethyl group leads to significant R2
conformational change may reflect inherent differences between
the inhibition of class A and C β-lactamases by carbapenems,
perhaps partly due to the increased size of the active site in class C
enzymes (56).
the ADC-5 complex to search for other residues that could be
hydrogen bonding with the meta-carboxylate. Previous structural
and functional studies of AmpC enzymes suggest that the more
conserved sites Xaa343, Asn346, Arg349, and Thr316 interact
with the C3/C4 carboxylate of the substrate, although none of
these residues were involved with the meta-carboxylate in the
E. coli-5 crystal complex (15, 18, 46-48, 59-61). Similarly, each
˚
of these residues is at least 5 A from the meta-carboxylate in our
ADC-5 representation, an unlikely distance for a high-energy
hydrogen bond with the group. Asn287 is an “ancillary” ADC
residue which may be capable of engaging the meta-carboxylate
in a hydrogen bond with ion-dipole character but is positioned
˚
approximately 8 A away in our model (Figure 7). The molecular
explanations of the ADC inhibition by the cephalothin analogues
4 and 5 remain to be validated by further study with boronic acid
derivatives as molecular probes, site-directed mutagenesis of
ADC, and/or crystallography.
We now highlight the versatile β-lactamase inhibitory activity
of the rationally designed boronic acid derivatives (13, 14). All the
cephalosporin analogues had Ki values of e1 μM, and these
values likely reflect the naturally high affinity of AmpC enzymes
for cephalosporin substrates (24). Interestingly, compounds 1-3
which contain only the cephalosporin R1 side chains have very
similar Ki values, suggesting limited differences in the affinity
gains of these R1 structures. The kinetic substrate profile of ADC,
and AmpCs in general, includes increasing affinities for the larger
side chains of “third-generation” cephalosporins, which would
include ceftazidime and cefoperazone (24, 26, 57). “First-genera-
tion” cephalosporins, such as cephalothin, typically have lower
affinities but higher hydrolytic rates. Thus, despite these differ-
ences in affinity and hydrolytic rates, the first- and third-genera-
tion cephalosporin analogues have comparable Ki values as
inhibitors.
The introduction of chirality and the substituent resembling
the dihydrothiazine ring of the cephalosporin nucleus leads to a
22-fold increase in affinity (compound 3 vs 4). The further
addition of the C4 carboxylate leads to a 70-fold increase over
that of the achiral counterpart (compound 3 vs 5). Compound 5,
which incorporates these multiple structural features of the
cephalosporins, displays the lowest Ki for the ADC enzyme.
Our results indicating the contribution of the meta-carboxyphe-
nyl ring on the Ki of compound 5 are consistent with previous
data demonstrating the importance of this functional group for
molecular recognition in class C AmpC β-lactamases (15, 31).
The crystal structure of compound 5 in complex with the E. coli
AmpC β-lactamase shows a hydrogen bond between the carbox-
ylate of the inhibitor and the amide of Asn289 (15). This
interaction is well-studied, and thermodynamic cycle experiments
revealed that the hydrogen bond contributes 1.7 kcal/mol to the
overall binding affinity, a value within the range for ion-
dipole interaction (58). However, Asn289 is not a well-conserved
residue among class C β-lactamases. The Ki of this cephalothin
analogue increases from 1 nM with E. coli AmpC to 29 nM with
En. cloacae P99 AmpC which has Ser289, suggesting that the
amino acid at position 289 plays a role in the affinity of
compound 5 (47, 58).
AmpC enzyme binding site “hot spots” were previously
identified by a comparison of crystal structures in complex with
both boronic acid inhibitors and β-lactam substrates (46). Our
ADC acyl-enzyme representation reveals that the recognition
elements may differ for the ADC β-lactamase. For example, the
E. coli AmpC hydroxyl binding site was defined by Tyr150 and its
hydrogen bond with one of the boronic acid hydroxyls, displa-
cing the deacylation water (15, 44). Our ADC-5 model shows
significant repositioning of the boronic acid group, making this
interaction with Tyr150 unlikely. Rather, the ADC Tyr150 is
˚
within ∼3 A of the thiophene sulfur and carbonyl oxygen found
in the R1 side chain of compound 5. Further, the R1 amide
recognition site formed by the interaction of the R1 carbonyl and
Asn152 in the E. coli AmpC differs from our ADC-5 represen-
tation, as this same side chain carbonyl forms a hydrogen bond
with Lys67 (46). These consensus binding sites were compiled
exclusively from crystal structures of E. coli AmpC, and our
kinetic data and modeling analyses indicate the ADC β-lacta-
mase may interact differently with boronic acid inhibitors versus
other class C enzymes (15, 46, 58). Our molecular representations
of ADC in complex with the inhibitors were useful for developing
hypotheses; however, we remain cognizant of modeling limita-
tions, such as the lack of active site flexibility and the removal of
water molecules during the ligand docking protocol.
That the chiral cephalothin analogue 5 can maintain a low
nanomolar Ki for several phylogenetically divergent AmpC
β-lactamases reflects not only the potency of this inhibitor but
also what may be an important plasticity of AmpCs (26, 46, 58).
The significant repositioning of the boronic acid derivative
revealed in our ADC-5 model may be an indication of this
enzyme’s versatility, causing the β-lactamase-ligand interactions
to have different molecular correlates. We posit that compounds
4 and 5 benefit from the presence of an additional side chain
which more closely resembles the dihydrothiazine ring of the
natural substrate, cephalosporins. The stereochemistry and con-
formation of the chiral inhibitors may create a better “fit” for this
enzyme. In part, this improved fit may be due to approximation
of the deacylation transition state of the cephalosporinase, a
theory which has been previously offered to explain the high
affinity of chiral boronic acid derivatives (33, 44).
On the basis of amino acid sequence, the Acinetobacter ADC
cephalosporinase is not closely related to other AmpCs and has
a Glu residue at position 289 (Figure 4 of the Supporting
Information). Glu is not a hydrogen bond donor, and our model
shows the Glu289 side chain is well outside hydrogen bond
Hence, the notion of dedicated AmpC enzyme R1 and R2
binding sites may be especially fluid and adaptable in ADC,
permitting the β-lactamase to change recognition elements
depending on the ligand (46, 56). These novel interactions may
reflect the fact that differences in primary sequence can be
˚
distance (∼7 A). Thus, we used our molecular representation of

Article
Biochemistry, Vol. 49, No. 2, 2010 339
compensated by common secondary and tertiary structures,
allowing the enzyme to use multiple ancillary residues to make
contact with substrates and inhibitors. Alternatively, ADC may
have subtle differences in its deacylation mechanism which is
suggested by the unanticipated position of the boronic acid
oxygen atoms in our model. This AmpC structure-function
redundancy merits further study, as it could both lie at the core
of why these β-lactamases have evolved as versatile “traps” of
cephalosporin substrates but also aid the careful design of broad-
spectrum inhibitors (62, 63).
Despite the description of boronates as β-lactamase inhibitors
since the 1970s, boronic acid derivatives have not yet been
developed for clinical use in combination with a β-lactam (64).
Concerns about the safety and efficacy of boron-containing
therapeutics are currently being addressed by clinical studies
sponsored by the pharmaceutical industry (65). The data pre-
sented in this paper encourage the in vivo study of boronates as
β-lactamase inhibitors.
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CONCLUSION
In summary, we provide important insights into the interaction
of two types of inhibitors with the Acinetobacter and other
clinically relevant cephalosporinases. First, we present kinetic
data and molecular representations that explain why carbape-
nems are effective inhibitors of class C enzymes, including
formation of a stable acyl-enzyme intermediate and a role for
the compounds’ R2 side groups. Our results add to a growing
body of evidence supporting the activity of carbapenems as
broad-spectrum β-lactam antibiotics, “slow substrates”, and
inactivators of class A and C β-lactamases (18-21, 23-25).
Second, our ADC model suggests that inhibitors designed to
mimic the structure of natural substrates (i.e., boronic acid
derivatives) may adopt unique conformations in different class
C active sites. Despite significant sequence and structure dissimi-
larity between ADC and the E. coli AmpC, the chiral cephalothin
analogues attain similar Ki values for both enzymes. This
versatility may reflect an important plasticity of this cephalos-
porinase β-lactamase. Our data offer promise for the develop-
ment of compounds that have an extended inhibition profile
across, and within, β-lactamase classes, and specifically against
this challenging Acinetobacter spp. target.
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ACKNOWLEDGMENT
We thank Dr. Andrea Endimiani and Ms. Andrea Hujer for
careful review of the manuscript.
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SUPPORTING INFORMATION AVAILABLE
ADC β-lactamase model validation reports and multiple-
sequence protein alignment of crystal structure coordinates for
En. cloacae P99 (PDB entry 1XX2), E. coli AmpC (PDB entry
2BLS), and the ADC model. This material is available free of
charge via the Internet at http://pubs.acs.org.
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between active-site-serine β-lactamases and mechanism-based inacti-
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