Structure-based optimization of a non-β-lactam lead results in inhibitors that do not up-regulate β-lactamase expression in cell culture

Article abstract of DOI:10.1021/ja042984o

Bacterial expression of β-lactamases is the most widespread resistance mechanism to β-lactam antibiotics, such as penicillins and cephalosporins. There is a pressing need for novel, non-β-lactam inhibitors of these enzymes. One previously discovered novel

Full text of DOI:10.1021/ja042984o

Published on Web 03/12/2005
Structure-Based Optimization of a Non-â-lactam Lead Results
in Inhibitors That Do Not Up-Regulate â-Lactamase
Expression in Cell Culture
Donatella Tondi,†,‡ Federica Morandi,†,‡ Richard Bonnet,§ M. Paola Costi,*,‡ and
Brian K. Shoichet*,†
Contribution from the Department of Pharmaceutical Chemistry, UniVersity of Californias
San Francisco, 600 16th Street San Francisco, California 94143-2240, Dipartimento di Scienze
Farmaceutiche, UniVersita` degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100,
Modena, Italy, and SerVice de Bacte´riologie, Faculte´ de Me´decine, UniVersite´ d'AuVergne,
63001 Clermont-Ferrand, France
Received November 20, 2004; E-mail: costimp@unimore.it; shoichet@cgl.ucsf.edu
Abstract: Bacterial expression of â-lactamases is the most widespread resistance mechanism to â-lactam
antibiotics, such as penicillins and cephalosporins. There is a pressing need for novel, non-â-lactam inhibitors
of these enzymes. One previously discovered novel inhibitor of the â-lactamase AmpC, compound 1, has
several favorable properties: it is chemically dissimilar to â-lactams and is a noncovalent, competitive
inhibitor of the enzyme. However, at 26 µM its activity is modest. Using the X-ray structure of the AmpC/1
complex as a template, 14 analogues were designed and synthesized. The most active of these, compound
10, had a Ki of 1 µM, 26-fold better than the lead. To understand the origins of this improved activity, the
structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by
X-ray crystallography to 1.97 and 1.96 Å, respectively. Compound 10 was active in cell culture, reversing
resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In
contrast to â-lactam-based inhibitors clavulanate and cefoxitin, compound 10 did not up-regulate â-lactamase
expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape
from this resistance mechanism derives from its dissimilarity to â-lactam antibiotics.
Introduction
lactam bond in the eponymous â-lactam ring of these drugs,
inactivating them.7,8
Microbial resistance to antibiotics is now a serious threat to
public health.1,2 A pressing problem is resistance to the â-lactam
antibiotics, including the penicillins and cephalosporins, which
are among the most widely used class of antibiotics. Several
mechanisms contribute to this resistance, including mutations
in the target of these drugs, cell-wall biosynthesis transamidases
called penicillin binding proteins, deletion and modification of
the porin channels through which the drugs diffuse, and
expression of pumps that export the drugs out of the bacterial
cells.3-6 The most widespread resistance mechanism remains
the expression of â-lactamase enzymes, which hydrolyze the
To overcome these resistance enzymes, â-lactam molecules
that inhibit (e.g. clavulanic acid) or evade (e.g. aztreonam)
â-lactamases have been introduced. These molecules are
themselves â-lactams and, like the penicillins, most are deriva-
tives of microbial natural products that have been in the
biosphere over evolutionary time. Consequently, resistance to
them has evolved rapidly, often in the â-lactamases themselves.9
Mutant enzymes have arisen that can evade â-lactam-based
â-lactamase inhibitors.10-12 Enzymes that are naturally resistant
to current inhibitors, including class C â-lactamases such as
AmpC, have become prominent in clinical settings.13 Mecha-
† University of California, San Francisco.
‡ Universita` degli Studi di Modena e Reggio Emilia.
(7) Bennett, P. M.; Chopra, I. Molecular Basis of beta-Lactamase Induction
in Bacteria. Antimicrob. Agents Chemother. 1993, 37(2), 153-158.
(8) Essack, S. Y. The Development of beta-Lactam Antibiotics in Response
to the Evolution of beta-Lactamases. Pharm. Res. 2001, 18(10), 1391-
1399.
§ Universite` d'Auvergne.
(1) Rice, L. B.; Bonomo, R. A. beta-Lactamases: Which Ones Are Clinically
Important? Drug Resist. Updates 2000, 3(3), 178-189.
(2) Cosgrove, S.; Carmeli, Y. The Impact of Antimicrobial Resistance on Health
and Economic Outcomes. Clin. Infect. Dis. 2003, 36, 1433-1437.
(3) Chambers, H. F. Penicillin-Binding Protein-Mediated Resistance in Pneu-
mococci and Staphylococci. J. Infect. Dis. 1999, 179(Suppl. 2), 353-359.
(4) Li, X. Z.; Nikaido, H. Efflux-Mediated Drug Resistance in Bacteria. Drugs
2004, 64(2), 159-204.
(9) Medeiros, A. A. Cooperative Evolution of Mechanisms of beta-Lactam
Resistance. Clin. Microbiol. Infect. 2000, 6(Suppl. 3), 27-33.
(10) Meroueh, S. O.; Roblin, P.; Golemi, D.; Maveyraud, L.; Vakulenko, S. B.;
Zhang, Y.; Samama, J. P.; Mobashery, S. Molecular Dynamics at the Root
of Expansion of Function in the M69L Inhibitor-Resistant TEM â-Lacta-
mase from Escherichia coli. J. Am. Chem. Soc. 2002, 124, 9422-9430.
(11) Sun, T.; Bethel, C. R.; Bonomo, R. A.; Knox, J. R. Inhibitor-Resistant
Class A â-Lactamases: Consequences of the Ser130-to-Gly Mutation Seen
in Apo and Tazobactam Structures of the SHV-1 Variant. Biochemistry
2004, 43, 14111-14117.
(5) Li, X. Z.; Zhang, L.; Nikaido, H. Efflux Pump-Mediated Intrinsic Drug
Resistance in Mycobacterium smegmatis. Antimicrob. Agents Chemother.
2004, 48(7), 2415-2423.
(6) Thiolas, A.; Bornet, C.; Davin-Regli, A.; Pages, J. M.; Bollet, C. Resistance
to Imipenem, Cefepime, and Cefpirome Associated with Mutation in Omp36
Osmoporin of Enterobacter Aerogenes. Biochem. Biophys. Res. Commun.
2004, 317(3), 851-856.
(12) Bush, K. beta-Lactamases of Increasing Clinical Importance. Curr. Pharm.
Des. 1999, 5(11), 839-845.
9
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J. AM. CHEM. SOC. 2005, 127, 4632-4639
10.1021/ja042984o CCC: $30.25 © 2005 American Chemical Society

Structure-Based Optimization of Non-â-Lactam Lead
A R T I C L E S
in the past,18,19 in a novel series this course is unavailable.
Second, whereas â-lactams are eVolVed for cellular efficacy,
neither the purely synthetic compound 1 nor its analogues need
be active against bacteria. Having discovered a novel lead, the
question became, could we improve its affinity and biological
activity?
Here we describe our preliminary efforts to improve this series
of inhibitors. We continue to use a structure-based approach.
In the structure of the AmpC/1 complex, the inhibitor comple-
ments the core of the active site but leaves a distal region open.
We sought derivatives to take advantage of this region that were
relatively easy to synthesize and would not diminish the
solubility and “leadlike” properties of the inhibitors.20 We
therefore focused on derivates that made new interactions with
polar residues in the distal part of the AmpC site, including
Arg204, or that tested features of the ligands that appeared
important for binding. Fourteen analogues were synthesized,
the best of which bound to AmpC with 26-fold improved affinity
over 1. To understand their improved affinity, X-ray crystal
structures were determined for two of the new analogues in
complex with AmpC. To explore their biological potential,
several of the new inhibitors were tested in bacterial cell culture
against clinical pathogens. We observe intriguing differences
in their biological effects on resistant bacteria compared to
classic, â-lactam-based inhibitors such as clavulanate and
cefoxitin.
Figure 1. Characteristic â-lactam substrate and inhibitor of AmpC, and
the lead compound for the novel inhibitor family discussed here.
nisms that alter the expression levels of the enzymes in the
presence of the drugs or the inhibitors have appeared. There
are strains of pathogenic bacteria that, recognizing the presence
of a â-lactam-based inhibitor, will overexpress the â-lactamase
that these drugs are meant to inhibit.14 There is thus a pressing
need for novel â-lactamase inhibitors, not based on a â-lactam
core structure. Such inhibitors would not be hydrolyzed by
â-lactamases or mutant â-lactamases and would not be recog-
nized by the suite of bacterial resistance mechanisms mobilized
against â-lactams.15
Recently, we reported the structure-based discovery of a
novel, noncovalent inhibitor of the widespread class C â-lac-
tamase AmpC, compound 1 (Figure 1).16 This compound is
dissimilar to penicillins and cephalosporins and binds to the
enzymes noncovalently and reversibly, in contrast to the
â-lactam substrates and inhibitors. Despite these differences,
the X-ray crystal structure of the AmpC/1 complex revealed
that 1 complements the core of the active site, interacting with
key residues involved in â-lactam recognition and hydrolysis
such as Ser64, Lys67, Asn152, and Tyr221. We concluded that
the ligand recognition encoded by the AmpC structure was
plastic enough to accommodate inhibitors genuinely dissimilar
to â-lactams, allowing a new departure in the medicinal
chemistry of their inhibitors.
Results
Synthesis. The synthesis of the methyl ester intermediates
and their corresponding acids was accomplished by reacting the
sulfonyl chloride with the appropriate amine in anhydrous
pyridine (Scheme 1). The only exception was in the synthesis
of compound 4A, where the amine reacted with the monom-
ethylphthalate in the presence of diisopropylamine (DIEA) and
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP) to afford the desired methyl ester (Scheme
2). Each ester was subsequently hydrolyzed by suspending it
in sodium hydroxide and isolating the final acids through
precipitation with dilute HCl.
All intermediate esters (1A-14A) were characterized either
through elementary analysis or monodimensional 1H NMR, or
both, whereas all final carboxylic acids (1-14) were character-
ized through monodimensional 1H NMR and either mass
spectroscopy or elementary analysis, or all three (see Materials
and Methods and Supporting Information, Table S1). The
structures of all compounds were consistent with their analytical
and spectroscopic data.
The very novelty of this inhibitor posed several problems,
ones that are perhaps shared by many genuinely new leads. First,
the inhibition constant of compound 1 was, at 26 µM, weak,
and its activity in vivo was poor. Moreover, its novelty
confronted us with an unanticipated design problem. Whereas
one can draw upon 60 years of medicinal chemistry in designing
analogues of â-lactams,17 something we ourselves have done
Design, Enzymology, and Binding Affinity. We wished to
investigate which groups in the lead inhibitor 1 were critical
for binding, and should be maintained, and where we could
improve affinity through chemical elaboration. We first focused
on the 3-(sulfonylamino)-2-carboxylic acid thiophene moiety
of 1 (Figure 1), which in the AmpC complex structure hydrogen
(13) Philippon, A.; Arlet, G.; Jacoby, G. Plasmid-Determined AmpC-Type beta-
Lactamases. Antimicrob. Agents Chemother. 2002, 46(1), 1-11.
(14) Hanson, N. D.; Sanders, C. C. Regulation of Inducible AmpC beta-
Lactamase Expression among Enterobacteriaceae. Curr. Pharm. Des. 1999,
5(11), 881-894.
(15) Powers, R. A.; Blazquez, J.; Weston, G. S.; Morosini, M. I.; Baquero, F.;
Shoichet, B. K. The Complexed Structure and Antimicrobial Activity of a
Non-beta-lactam Inhibitor of AmpC beta-Lactamase. Protein Sci. 1999,
8(11), 2330-2337.
(18) Caselli, E.; Powers, R. A.; Blasczcak, L. C.; Wu, C. Y.; Prati, F.; Shoichet,
B. K. Energetic, Structural, and Antimicrobial Analyses of beta-Lactam
Side Chain Recognition by beta-Lactamases. Chem. Biol. 2001, 8(1), 17-
31.
(19) Trehan, I.; Morandi, F.; Blaszczak, L. C.; Shoichet, B. K. Using Steric
Hindrance To Design New Inhibitors of Class C beta-Lactamases. Chem.
Biol. 2002, 9(9), 971-980.
(20) Oprea, T. I. Current Trends in Lead Discovery: Are We Looking for the
Appropriate Properties? Mol. DiVersity 2002, 5(4), 199-208.
(16) Powers, R. A.; Morandi, F.; Shoichet, B. K. Structure-Based Discovery of
a Novel, Noncovalent Inhibitor of AmpC beta-Lactamase. Structure
(London) 2002, 10(7), 1013-1023.
(17) Konaklieva, M. I. Lactams as Inhibitors of Serine Enzymes. Curr. Med.
Chem.: Anti-Infect. Agents 2002, 1(3), 215-238.
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A R T I C L E S
Tondi et al.
Scheme 1. Synthesis of 3-((4-chloroanilin)sulfonyl)thiophene-2-carboxylic Acid Derivativesa
a Step a: Py dry, 50 °C, N2. Step b: 2 N NaOH, 80 °C, HCl dil.
Scheme 2. Synthesis of N-(4-Chlorophenyl)phthalamic Acid Derivativea
a Step c: DMF, DIEA, PyBOP. 60 °C, N2. Step b: 2 N NaOH, 80 °C, HCl dil.
Table 1. Ki Values of Thiophene-Carboxy Derivatives vs AmpC
Table 2. X-ray Data Collection and Refinement Statistics for
AmpC/10 and AmpC/11
AmpC/10
AmpC/11
cell constants
a (Å)
117.75
118.469
b (Å)
c (Å)
â (deg)
space group
resolution (Å)
unique reflcns
total obs
76.77
97.90
116.63
C2
1.97
55,165
105,106
9.1 (25.8)a
93.7 (94.6)
76.279
97.674
116.37
C2
1.96
53,587
154,202
5.4 (39.3)a
95.4 (90.3)
1.96-20
R
merge (%)
completenessb (%)
resolution range for refinement (Å) 1.97-20
(1.97-2.04)
(1.96-2.03)
I / σI
9.43 (2.14)
11.07 (2.09)
700
no. of protein residues
no. of water molecules
rmsd bond lengths (Å)
rmsd bond angles (deg)
709
516
313
0.0148
1.7413
16.84
21.53
17.71
0.0085
1.472
19.81
22.65
28.57
R
R
cryst (%)
free (%)c
av B factor, protein atoms
(Å2; molecules 1 and 2)
av B factor, inhibitor atoms
(Å2; molecule 1)
39.78
a Highest resolution shell in parentheses. Subsequent values in parentheses
are for that shell. b Fraction of theoretically possible reflections observed.
c Rfree was calculated with 5% of reflections, randomly selected, set aside.
affinity, consistent with the importance of the ion-dipole
interactions made by this carboxylate (e.g., compound 1A, Table
1). Similarly, inverting the order of the sulfonyl-amino group
reduced affinity 10-20-fold (compare compounds 1 and 2 or
11 and 14, Table 1), and replacing the sulfonamide with an
amide reduced affinity 8-fold, consistent with the importance
of the hydrogen bond between this group and Asn152 observed
crystallographically. We also replaced the thiophene of 1 with
a more synthetically tractable benzene. This substitution dimin-
ished activity, but by less than 2-fold (compound 4, Table 1).
In an attempt to increase the affinity of compound 1, we
considered derivates that improved either the apolar or polar
complementarity in the distal chloro-phenyl ring of the
inhibitor. This functionality is located in a more open region of
the active site, flanked by the conserved Tyr221 on the “floor”
of the active site, bulk solvent at the “top” of the inhibitor, and
polar and apolar residues such as Arg204, Gly320, Thr319, and
Val211 to its side. Introducing bulk or hydrophobicity, such as
a Estimated error margins on the Ki values are (50%. b No detectable
inhibition at 100 µM concentration of compound.
bonds with active site residues. For instance, the carboxylate
of 1 binds in the “oxyanion” hole of AmpC, hydrogen bonding
with main chain amide nitrogens, Ser64, and a conserved water
molecule (Wat401), while the sulfonamide hydrogen bonds with
the conserved Asn152 of AmpC. Derivates that replaced the
thiophene carboxylate with a methyl ester substantially reduced
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Structure-Based Optimization of Non-â-Lactam Lead
A R T I C L E S
Figure 2. X-ray crystal structure of the AmpC/10 complex. (A) Stereoview of 2Fo - Fc electron density maps contoured at 1σ of the refined model. Carbon
atoms are gray for the protein and orange for the ligand; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, yellow. (B) AmpC/10 complex in the active
site. Dashed blue lines represent hydrogen bonds; red spheres represent water molecules. Interaction distances are listed in Table S2, Supporting Information.
Figures 2 and 3 were generated with Pymol (http://pymol.sourceforge.net/).
in 5, 6, and 13, affected affinity only modestly. More polar
derivates, such as carboxylate and hydroxyl groups, improved
affinity more substantially, up to 26-fold in compounds 7-10
(Table 1).
with the conserved Wat401. There are two enzyme monomers
in the asymmetric unit in the AmpC structures, and in one of
them, monomer 2, a fifth hydrogen bonds is formed to Wat481.
As with the lead compound 1, the thiophene ring is within van
der Waals distance of residues Leu119 and Leu293, which form
a hydrophobic patch on AmpC. A sulfonamide oxygen interacts
with the Oγ of Ser64 and the Nú of Lys67; the other hydrogen
bonds with the Nδ2 of Asn152. As in the AmpC/1 complex,
the nitrogen atom of the sulfonamide interacts with the main-
chain oxygen of Ala318.
X-ray Crystallographic Structure Determination. To in-
vestigate the structural bases for the higher affinities found for
the new series, we determined the crystal structure of AmpC in
complex with compound 10 and with compound 11 at 1.97 and
1.96 Å resolution, respectively (Table 2). Proline and glycine
residues excluded, 92.2% of the amino acids were in the most
favored regions of the Ramachandran plot and 7.8% were in
the additionally allowed regions for the crystal structure of
AmpC/10. For AmpC/11, 92.5% of the nonproline and nong-
lycine amino acids were in the most favored regions of the
Ramachandran plot and 7.5% were in the additionally allowed
regions. The positions of both inhibitors in the active sites of
each structure were unambiguously identified in the initial Fo
- Fc difference map contoured at 3σ. In the AmpC/10 complex,
the inhibitor was observed and modeled in both monomers in
the asymmetric unit, whereas in the AmpC/11 complex, the
inhibitor was only observed and modeled in one of the two
monomers.
Overall, the complexes with 10 and 11 resemble that of the
AmpC/1 complex, with a few interesting differences. Key
hydrogen bond interactions in the conserved parts of all three
structures, the thiophene-carboxylate and the sulfonamide
functionalities, are maintained (Tables S2 and S3, Supporting
Information; Figures 2 and 3). The thiophene carboxylate in
both 10 and 11 hydrogen bonds with both the Oγ and the main-
chain NH of the hydrolytic Ser64, with a third hydrogen bond
formed with the main-chain nitrogen of Ala318 and a fourth
Differences emerge in the 4′-carboxylate-2′-hydroxy benzene
ring, which is the point of substitution for compound 10. The
ring itself appears to form edge-to-face quadrupole interactions
with Tyr221 via the introduced 2′-hydroxyl of 10; the distance
between this hydroxyl and Cδ1 of Tyr221 is 3.43 Å. This
interaction is also present in the structure of the lead 1, but in
this case it appears to be a ring-ring or quadrupolar interaction,
whereas with 10 the intercession of the hydroxyl makes it a
dipole-quadrupolar interaction. The presence of the 2′-hydroxyl
also displaces 10 slightly “up” in the site, moving the center of
the ring away from Tyr221 and toward polar residues such
Arg204 and Thr319, by about 0.2 Å in monomer 2. In both
crystallographic monomers 1 and 2 of AmpC the 4′-carboxylate
points toward the solvent accessible entry of the binding site.
In monomer 1, this carboxylate hydrogen bonds with an ordered
water molecule, Wat521 (distance 2.9 Å), which in turn
hydrogen bonds with the Nꢀ of Arg204 (distance, 2.8 Å). In
monomer 2, Arg204 adopts a different conformation and these
interactions are not observed. Instead, a new interaction is
observed between Gln120 and the sulfonamide of 10, with the
Gln120 in this monomer pointing toward the inhibitors rather
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A R T I C L E S
Tondi et al.
Figure 3. X-ray crystal structure of the AmpC/11 complex. (A) Stereoview of 2Fo - Fc electron density maps (blue, contoured at 1σ) of the refined model.
Carbon atoms are colored gray for the protein and pink for the ligand; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, yellow. (B) AmpC/11 complex
in the active site. Dashed blue lines represent key hydrogen bonds. Red spheres represent water molecules. Interaction distances are listed in Table S3,
Supporting Information.
Table 3. Synergy of Compounds 10, 11, and 1 with Ceftazidime
against Clinical Strains Producing AmpC-type â-lactamase
than out of the site, as it does in monomer 1. Also in monomer
2, difference electron density suggests that a second conforma-
MICa
(
µg/mL)
tion of the inhibitor may exist, with the distal phenyl ring rotated
by 180°, allowing the 2′-hydroxyl group to hydrogen bond with
the side chain of Asn343 through an ordered water molecule.
To investigate how important these polar and quadrupolar
interactions might be, we also determined the structure of the
AmpC/11 complex by X-ray crystallography. Compound 11
replaces the 4′-carboxylate of 10 with a 3′-nitro and correspond-
ingly loses an order of magnitude in affinity (Table 1). In the
structure of the AmpC/11 complex, the inhibitor is moved
slightly down in the site, keeping the quadrupole edge-to-face
stacking with Tyr221, but this time without the intercession of
the 2′-hydroxyl, which is no longer present. The 3′-nitro is
relatively distant from Arg204, and we observe no interaction
with an ordered water; the loss of these polar interactions may
be consistent with the diminished affinity for this inhibitor.
Microbiology. To investigate the potential of these com-
pounds to reverse antibiotic resistance, we undertook antimi-
crobial activity studies in bacterial cell culture. Compounds 10
and 11 were tested for synergy with the third generation
cephalosporin ceftazidime (CAZ) against pathogenic bacteria
from clinical isolates from the Clermont-Ferrand University
Hospital (France), and the results were compared with those of
the lead compound 1. Strains tested were E. aerogenes 298, E.
cloacae P99, C. freundii 63, K. pneumoniae Kus, K. pneumoniae
704, and P aeruginosa CF34. All were resistant to CAZ because
of the expression of class C â-lactamases. The minimum
inhibitory concentration (MIC) of CAZ against these pathogens
ranged from 256 to 64 µg/mL (Table 3; Figure 4).
strains
CAZb
CAZ/10c
CAZ/11c
CAZ/1c
E. aerogenes 298
E. cloacae P99
C. freundii 63
K. pneumoniae Kus
K. pneumoniae 704
P. aeruginosa CF34
128
128
128
256
64
16
16
16
32
16
64
64
128
64
256
64
32
64
64
256
64
64
64
64
a Minimum inhibitory concentration. b CAZ ) ceftazidime. c The in-
hibitor:CAZ ratio was 1:1, and the concentration of DMSO was always
less than 1%.
Compound 10 increased bacterial susceptibility to CAZ,
improving its MIC values by 4- to 8-fold. This was sufficient
to partially restore clinically relevant susceptibility for E.
Aerogenes 298, E. cloacae P99, C. freundii 63, and K.
pneumoniae 704. Conversely, the lead compound 1 and
compound 11, both of which have lower affinities than does
10, had relatively little effect on the potency of CAZ. Consistent
with this result, 10 synergized the activity of CAZ and the
penicillin piperacillin (PIP) in disk diffusion assays, as can be
seen by the characteristic tear-drop shapes of the zones of
bacterial growth inhibition (Figure 5). This contrasts dramati-
cally with the same experiment performed with the â-lactam-
based inhibitors clavulanate and cefoxitin (IC50 of 340 nM vs
AmpC; not shown), both of which induce AmpC expres-
sion.15,21,22 Although clavulanate and cefoxitin inhibit the AmpC-
(21) Kadima, T. A.; Weiner, J. H. Mechanism of Suppression of Piperacillin
Resistance in Enterobacteria by Tazobactam. Antimicrob. Agents Chemoth-
er. 1997, 41(10), 2177-2183.
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Structure-Based Optimization of Non-â-Lactam Lead
A R T I C L E S
Figure 4. Activity in cell culture of the lead compound 1 and compound
10 vs bacteria from clinical isolates that express AmpC â-lactamase.
type â-lactamase expressed by these cells, they nevertheless
antagonize the activity of CAZ and PIP in the disk diffusion
studies. This results in flattened zones of bacterial grown
inhibition, reflecting the up-regulation of â-lactamase production
in the E. cloacae bacteria.
Discussion
There is a pressing need for novel inhibitors to combat
antibiotic resistance, but the discovery of such confronts one
with new problems. In the case of lead compound 1, a non-â-
lactam inhibitor of â-lactamases, the challenges were to improve
the binding affinity in a series where one could not simply apply
60 years worth of â-lactam structure-activity relationships and
to improve the biological efficacy in a scaffold that, unlike
â-lactams, had not been evolved for in vivo activity. Three
important results emerge from our preliminary efforts to improve
this series of compounds. First, a cycle of structure-based design
and testing led to small, soluble inhibitors whose improved
affinities could be explained on the basis of the X-ray structures
subsequently determined for their complexes. Second, the
improved affinity is mirrored by the improved activity in cell
culture. Third, new biology follows from the new chemical
structures in this series, which, not being â-lactams, simply
inhibit â-lactamase and do not up-regulate its expression in cell
culture, as do classic â-lactam-based inhibitors such as clavu-
lanate (Figure 5).7
It is encouraging that modest changes in this series can
improve inhibition significantly (compare the lead 1 with 10,
Table 1). Also, the resulting analogues remain relatively soluble
and small, leaving further room for chemical elaboration. Indeed,
an encouraging aspect of this series is that increased affinity
comes from polar groups that act to maintain the overall
solubility of the compounds, most particularly the addition of
a carboxylate on the distal phenyl ring. Exactly what interactions
are responsible for the improved affinity is less certain. The
Figure 5. Effects on up-regulation of a class C â-lactamase in E. cloacae
by â-lactamase inhibitors, each with piperacillin (PIP) and ceftazidime
(CAZ): (A) â-lactam-based inhibitor clavulanate (center); (B) â-lactam-
based inhibitor cefoxitin (FOX); (C) non-â-lactam inhibitor 10.
crystal structures of the 1.0 µM inhibitor 10 and the 14 µM
inhibitor 11 in complex with AmpC suggest that water-mediated
interactions with Arg204 are responsible for the improved
affinity of 10. This conclusion must be seen as quite tentative
at this point, since this interaction is only seen in one of the
(22) Sanders, C. C.; Bradford, P. A.; Sanders, W. E. J. Penicillin-Binding Proteins
and Induction of AmpC â-Lactamase. Antimicrob. Agents Chemother. 1997,
41, 2013-2015.
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A R T I C L E S
Tondi et al.
mmol) was dissolved in 10 mL of anhydrous pyridine, and the
3-(chlorosulfonyl)thiophene-2-carboxylic acid methyl ester (100 mg,
0.42 mmol) was added dropwise to the solution under stirring. The
reaction was stirred for 24 h, at 50 °C, under nitrogen. The resulting
solution was treated with water and ice; the crude product, correspond-
ing to the 3-((4-chlorophenyl)sulfamoyl)thiophene-2-carboxylic acid
methyl ester (1A) was formed and isolated through filtration. Yield:
73 mg, 80%; mp 87-90 °C. Anal. Calcd for C12H10ClNO4S2: C, 43.44;
H, 3.04; N, 4.22. Found: C, 43.51; H, 3.10; N, 4.28.
3-((4-Chlorophenyl)sulfamoyl)thiophene-2-carboxylic Acid (1).
Compound 1A (60 mg, 0.18 mmol) was suspended in 10 mL of 2 N
NaOH, and the reaction was stirred for 1 h at 80 °C, until the starting
material was completely dissolved. Thereafter, the solution was cooled,
and dilute HCl was added until the solution reached pH 2. A precipitate,
corresponding to 3-((4-chlorophenyl)sulfamoyl)thiophene-2-carboxylic
acid (1) was formed and isolated through filtration. Yield: 42 mg, 73%;
mp 195-198 °C. 1H NMR: 7.97 (H1, d, J 5.4 Hz); 7.49 (H2, d, J 5.4
Hz); 7.21 (H3, d/o, J 8.8 Hz); 7.40 (H4, d/o, J 9 Hz); 7.40 (H5, d/o, J
9 Hz); 7.21 (H6, d/o, J 8.8 Hz); 10.20 (H7, s). Anal. Calcd for C11H8-
ClNO4S2: C, 41.58; H, 2.54; N, 4.41. Found: C, 41.45; H, 2.48; N,
4.51.
two monomers of the AmpC/10 complex. From a molecular
recognition standpoint, it is surprising that a water-mediated
hydrogen bond between the carboxylate and Arg204, in a
relatively solvent-exposed region, can significantly improve
affinity, though this sort of effect is not unprecedented in
AmpC.23 Other explanations are possible; for instance the
improved affinity may derive from an overall improved comple-
mentarity to the highly electropositive active site of AmpC.
Detailed conclusions as to the importance of these interactions
must remain tentative pending more rigorous analyses, such as
double-perturbation thermodynamic cycles;23,24 what we can say
is that the X-ray structures of these complexes provide templates
for designing and understanding such experiments.
What might justify this effort is that the improved enzyme
affinity of inhibitor 10 results in 4- to 8-fold improvement in
antibiotic efficacy. In bacterial cell culture, a 1:1 ration of 10
to CAZ synergizes the activity of the latter against widespread
hospital pathogens such as E. cloacae and C. freundii (Table 3
and Figure 4) and is significantly more potent than the lead
compound 1 or compound 11. This antibiotic effect suggests
that this series of inhibitors, whose Ki values remain relatively
modest, may be useful leads for further optimization. The X-ray
crystal structures of compound 10 in complex with AmpC
provide atomic resolution templates for future design efforts.
Perhaps the most striking result to emerge from this study,
however, comes from comparing the effects of the classic
â-lactam-based inhibitors clavulanate and cefoxitin with those
of compound 10 on an inducible strain of E. cloacae. These
bacteria up-regulate the expression of class C â-lactamase in
the presence of â-lactams such as clavulanate, thereby over-
whelming these â-lactams with the very enzyme they are meant
to inhibit (Figure 5A,B). Conversely, compound 10, which is
not a â-lactam, simply inhibits the â-lactamase expressed by
these bacteria, it does not lead to its up-regulation. Thus, the
novel chemical structures represented by this series of inhibitors
leads to novel, therapeutically relevant biological effects.
Intermediate ester derivatives (2A-14A) and final carboxylic acids
(2-14) were synthesized according to the method followed for 1A and
1. Details are reported in the Supporting Information.
Enzymology. Inhibitors were dissolved in DMSO at a concentration
of 50 mM; more dilute stocks were subsequently prepared as necessary.
Kinetic measurements were performed using nitrocefin as substrate in
50 mM Tris buffer, pH 7.0, and monitored in an HP8453 UV-vis
spectrophotometer. The Km of nitrocefin for AmpC in this buffer was
127 µM. The concentration of AmpC was determined spectrophoto-
metrically in concentrated stock solutions made from lyophilized
powder and subsequently diluted; this enzyme had been previously
expressed and purified, as described.15 The concentration of enzyme
in all reactions was 1.75 nM. Inhibition Ki values were obtained from
IC50 plots assuming competitive inhibition, an assumption consistent
with both previous inhibition patterns in this series16 and with
experiments investigating the effect of increasing substrate concentra-
tions (not shown). For compound 10, the Ki value was also obtained
by comparison of progress curves in the presence and in the absence
of inhibitor;18,25,26 the result was consistent with the value determined
from the IC50 plots.
Materials and Methods
Crystal Growth and Structure Determination. Cocrystals of
AmpC in complex with compounds 10 and 11 were grown by vapor
diffusion in hanging drops equilibrated over 1.8 M potassium phosphate
buffer (pH 8.7) using microseeding techniques. The initial concentration
of the protein in the drop was 3.8 mg/mL, and the concentrations of
compounds 10 and 11 were 0.8 mM. The compounds were added to
the crystallization drops in a 1.2% DMSO, 1 M potassium phosphate
buffer (pH 8.7) solution. Crystals appeared a few weeks after equilibra-
tion at 21 °C. Before data collection, crystals were immersed in a
cryoprotectant solution of 20% sucrose, 1.8 M potassium phosphate,
pH 8.7, for about 30 s, and were flash-cooled in liquid nitrogen.
Diffraction data were collected on frozen crystals at the Advance
Light Source (ALS, Lawrence Berkeley Laboratory, CA). Both data
sets were measured from single crystals. Reflections were indexed,
integrated, and scaled using the HKL software package.27 For both
structures, the space group was C2, with two molecules in the
asymmetric unit. For AmpC/10, molecule 1 of the asymmetric unit
was modeled with 352 residues, including the inhibitor (7 residues,
Chemistry. All reagents were purchased from Sigma-Aldrich or
Fluka and were reagent grade. Progress of the reaction was monitored
by thin-layer chromatography (TLC) on silica gel plates (Riedel-de-
Haen, Art. 37341). Merck silica gel (Kieselgel 60) was used for flash
chromatography (230-400 mesh) when required. Extracts were dried
over MgSO4, and solvents were removed under reduced pressure.
Melting points were determined on a Bu¨chi 510 capillary melting
point apparatus and are uncorrected. Elemental analyses were performed
1
on a Perkin-Elmer Elemental Analyzer 240C. H NMR spectra were
recorded on a Bruker DPX 200 MHz spectrometer (Centro Interdipar-
timentale Grandi Strumenti Universita` di Modena) with trimethylsilane
(TMS) as internal standard; the values of the chemical shifts (δ) are
given in parts per million and coupling constants (J) in hertz. Dimethyl
sulfoxide-d6 (DMSO-d6) was used as the solvent. Mass spectra were
determined on a Finnigan MAT SSQ 710 A mass spectrometer (EI, 70
eV).
3-((4-Chlorophenyl)sulfamoyl)thiophene-2-carboxylic Acid Meth-
yl Ester (1A). The starting amine, 4-chlorophenylamine (35 mg, 0.28
(25) Waley, G. S. A Quick Method for the Determination of Inhibition Constants.
Biochem. J. 1982, 205, 631-633.
(23) Roth, T.; Minasov, G.; Shoichet, B. Thermodynamic Cycle Analysis and
Inhibitor Design against â-Lactamase. Biochemistry 2003, 42, 14483-
14491.
(24) Fersht, A. R.; Shi, J. P.; Knill-Jones, J.; Lowe, D. M.; Wilkinson, A. J.;
Blow, D. M.; Brick, P.; Carter, P.; Waye, M. M. Y.; Winter, G. Hydrogen
Bonding and Biological Specificity Analysed by Protein Engineering.
Nature 1985, 314, 235.
(26) Weston, G. S.; Blazquez, J.; Baquero, F.; Shoichet, B. K. Structure-Based
Enhancement of Boronic Acid Inhibitors of AmpC â-Lactamase. J. Med.
Chem. 1998, 41, 4577-4586.
(27) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data Collected
in Oscillation Mode. Methods Enzymol., Macromol. Crystallogr., Part A
1997, 276, 307-326.
9
4638 J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005

Structure-Based Optimization of Non-â-Lactam Lead
A R T I C L E S
284-290, were left out due to poor density in this region, which is
often disordered in AmpC structures), and molecule 2 was modeled
with 359 residues, including the inhibitor. For AmpC/ 11, molecule 1
was modeled with 342 residues (16 residues were left out), and molecule
2 of the asymmetric unit was modeled 359 residues, including the
inhibitor. The initial phasing models were an apo AmpC structure (PDB
entry 1KE4) for AmpC/10 and an AmpC/boronic acid complex (PDB
entry 1MY8) for AmpC/11, with inhibitor, water molecules, and ions
removed. The models were positioned using rigid body refinement and
refined using the maximum likelihood target in CNS including:
simulated annealing, positional minimization, and individual B-factor
refinement, with a bulk solvent correction.28 σ A-weighted electron
density maps were calculated using CNS and used in further steps of
manual model rebuilding and placement of water molecules with the
programs XtalView and O.29,30 The inhibitors were built into the 2Fo
- Fc and Fo - Fc electron density maps in each active site of the
asymmetric unit. Subsequent refinement cycles consisted of positional
minimization and B-factor refinement in CNS.
with Mueller-Hinton broth using the microdilution method according
to NCCLS guidelines.34 To test the inhibitory activity, compounds were
dissolved in DMSO and dilutions were performed using growth
medium. An adequate final concentration in which to determine the
MIC was obtained where the concentration of DMSO was <1%. The
ratio Inhib/CAZ was 1. A control done with DMSO showed that DMSO
did not have an effect in the bacteria growth.
For the â-lactamase induction experiments, plates of Mueller-Hinton
agar were inoculated with a clinical strain of E. cloacae in which
production of AmpC is inducible by â-lactams (Clermont-Ferrand
hospital, unpublished strain). Inhibitors were added to blank disks, and
the final content of inhibitor per disk was 128 µg for compound 10
and 8 µg for clavulanic acid. Disks of ceftazidime, cefoxitin, and
piperacillin contained 30, 30, and 75 µg, respectively.35
Data Deposition. The coordinates and structure factors have been
deposited in the Protein Data Bank under accession codes 1XGJ for
AmpC in complex with compound 10 and under accession code 1XGI
for AmpC in complex with 11.
Microbiology. Compounds 10 and 11 were tested for synergy with
the â-lactam ceftazidime against pathogenic bacteria from clinical
isolates at the Clermont-Ferrant Hospital (France); these bacteria were
resistant to â-lactams because of the expression of class C â-lactamases.
Strains of bacteria tested were E. aerogenes 298, E. cloacae P99, C.
freundii 63, K. pneumoniae Kus, K. pneumoniae 704, and P. aeruginosa
CF34.31-33 Minimum inhibitor concentration values were determined
Acknowledgment. This work was supported by Grant
GM63815 (to B.K.S.). We thank Piergiorgio Pecorari for his
valuable suggestions and Alberto Venturelli for conversations,
Yu Chen and Veena Thomas for reading this manuscript, and
Jesus Blazquez for introducing some of us to synergy and
antagonism tests for â-lactamase inhibitors.
Supporting Information Available: Detailed information
related to the synthesis and chemical characterization of
compounds 2-14 and a table listing all compounds character-
ized through 1H NMR and their numbering (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
(28) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.;
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. Crystallography
and NMR System: A New Software Suite for Macromolecular Structure
Determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54(Pt.
5), 905-921.
(29) McRee, D. E. XtalView/XfitsA Versatile Program for Manipulating Atomic
Coordinates and Electron Density. J. Struct. Biol. 1999, 125(2-3), 156-
165.
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(30) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Improved Methods
for Building Protein Models in Electron Density Maps and the Location
of Errors in These Models. Acta Crystallogr., Sect. A: Found. Crystallogr.
1991, 47, 110-119.
(33) Fleming, P. C.; Goldner, M.; Glass, D. G. Observations on the Nature,
Distribution, and Significance of Cephalosporinase. Lancet 1963, 29(1),
1399-1401.
(31) Bauernfeind, A.; Schneider, I.; Jungwirth, R.; Sahly, H.; Ullmann, U. A
Novel Type of AmpC beta-Lactamase, ACC-1, Produced by a Klebsiella
Pneumoniae Strain Causing Nosocomial Pneumonia. Antimicrob. Agents
Chemother. 1999, 43(8), 1924-1931.
(34) NCCLS, National Committee for Clinical Laboratory Standards. Methods
for Dilution Antimicrobial Tests for Bacteria That Grow Aerobically
ApproVed Standard, 4th ed.; NCCLS document M7-A5; NCCLS: Wayne,
PA, 2000.
(32) De Champs, C.; Poirel, L.; Bonnet, R.; Sirot, D.; Chanal, C.; Sirot, J.;
Nordmann, P. Prospective Survey of beta-Lactamases Produced by Ceftazi-
dime-Resistant Pseudomonas aeruginosa Isolated in a French Hospital in
2000. Antimicrob. Agents Chemother. 2002, 46(9), 3031-3034.
(35) Jarlier, V.; Nicolas, M.; Fournier, G.; Philippon, A. Extended Broad-
Spectrum beta-Lactamases Conferring Transferable Resistance to Newer
beta-Lactam Agents in Enterobacteriaceae: Hospital Prevalence and
Susceptibility Patterns. ReV. Infect. Dis. 1988, 10(4), 867-878.
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