Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases

Article abstract of DOI:10.1021/bi500970f

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of

Full text of DOI:10.1021/bi500970f

Article
pubs.acs.org/biochemistry
Interactions of “Bora-Penicilloates” with Serine β‑Lactamases and
DD-Peptidases
Liudmila Dzhekieva, S. A. Adediran, and R. F. Pratt*
Department of Chemistry, Wesleyan University, Lawn Avenue, Middletown, Connecticut 06459, United States
S
* Supporting Information
ABSTRACT: Specific boronic acids are generally powerful
tetrahedral intermediate/transition state analogue inhibitors of
serine amidohydrolases. This group of enzymes includes bacterial
β-lactamases and DD-peptidases where there has been considerable
development of boronic acid inhibitors. This paper describes the
synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are
closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously
described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is
a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of
class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of
a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the
instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases
(penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of
β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.
nzyme inhibitors remain important as drug leads.1 Boronic
Scheme 2
E
acids, 1, have for quite some time now been designed and
used as sources of active site-specific, anionic, tetrahedral
transition state analogue complexes, 2, of serine amidohy-
drolases (Scheme 1). They are thus very effective inhibitors of
Scheme 1
tetrahedral intermediate/transition state 5 would be 6, arising
from reaction between the enzyme and boronic acid 7. A
number of approximations to the structure 7 have been
described, for example, initially, amidoalkyl boronic acids such
as 8.13,14 Subsequently, closer analogues, such as 9 and 10, were
found to be very powerful β-lactamase inhibitors.15,16 Crystal
structures showed them to form the anticipated tetrahedral
adducts 2 at the β-lactamase active site. To complement these
developments, we describe here the syntheses of the boronic
acids 11 and 12. We follow this with a description and analysis
of their inhibitory activity against representative serine
β-lactamases and DD-peptidases.
these enzymes and potential drug candidates.2−4 Among the
enzymes that are inhibited by these compounds are the
β-lactam-recognizing enzymes, the serine β-lactamases and DD-
peptidases. Boronic acid inhibition of serine β-lactamases has
been recognized for many years,5,6 but only more recently have
such inhibitors of DD-peptidases been identified.7,8 The time
gap between these developments may reflect the increasing
awareness of the evolutionary relationship between DD-
peptidases and β-lactamases and thus their close structural
and functional similarity.9−11
β-Lactamases catalyze the hydrolysis of β-lactam antibiotics
and are thus an important source of bacterial resistance to these
molecules.12 The reaction (Scheme 2; shown with a penicillin)
proceeds by way of a covalent acyl enzyme intermediate 4 and,
therefore, through tetrahedral intermediates 3 and 5. Acyl-
enzymes, analogous to 4, are formed on reaction of DD-
peptidases with β-lactams but in this case hydrolyze very slowly
leading to effective inhibition of these enzymes and thus
interruption of bacterial cell wall synthesis. One would expect
that the closest boronate analogue to a β-lactamase deacylation
MATERIALS AND METHODS
The boronic acids 11 and 12 were synthesized as described in
detail in Supporting Information. The Actinomadura R39 and
■
Received: August 5, 2014
Revised: September 24, 2014
Published: October 10, 2014
© 2014 American Chemical Society
6530
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
Scheme 3
previously described.21,22 The crystal structure of the ampC
β-lactamase, inhibited by 15 [PDB entry 1MXO23], including
the crystallographic water molecules, was modified to construct
the adduct of 12 with the active site Ser 64. The pH was set to
7.0, and the total charge on the complex was zero. At this pH,
the side chain of Tyr 150 was neutral, and those of Lys 67 and
315 were cationic. The partial charges of the enzyme were
assigned by Insight II. Partial charges (MNDO) of the inhibitor
in the complex were calculated from a model adduct with
serine. Boron is not parametrized in Insight II so fixed standard
crystallographic B−C and B−O bond distances and a rigid
tetrahedral geometry at boron were assumed. The active site
was hydrated with a 15 Å sphere of water centered on Oγ of the
nucleophilic serine 64. The models were then subjected to 100
steps of steepest descent energy minimization followed by
short molecular dynamics runs (40 ps) to relax the active site
side chains and inhibitor. A typical snapshot from the molecular
dynamics runs was selected and subjected to 1000 steps of
steepest descent energy minimization, followed by 2000 steps
of conjugate gradients.
A similar procedure was followed to obtain a model of the
deacylation analogue adduct of 12 with the TEM-1 β-lactamase.
This was derived from the crystal structure of the complex of
15 with this enzyme [PDP entry 1NXY16]. Note, however, that
the thiophene ring of 15 has been retained in this model rather
than changed to phenyl. In the TEM active site, Glu166 was
neutral and Lys73 and Lys234 cationic. A generic model of a
tetrahedral acylation adduct of 12 with the TEM-1 enzyme was
derived directly from the crystal structure of its complex with
27 [PDB entry 1NY016]. Models of benzylpenicillin at the
CTX-M-9 and E. coli PBP4 active sites were built directly from
the published crystal structures [PDB entries 3HUO24 and
2EX8,25 respectively]. In each case, the acyl forms were
converted to tetrahedral intermediates by Insight modeling.
Streptomyces R61 DD-peptidases, and Bacillus subtilis PBP4a,
were generous gifts from Dr. J.-M. Frer
̀
e and Dr. P. Charlier of
the University of Liege, Liege, Belgium. The Escherichia coli
̀
̀
PBP5 DD-peptidase was a generous gift from Dr. R. A.
Nicholas of the University of North Carolina, Chapel Hill, NC.
The AmpC β-lactamase was provided by Dr. B. K. Shoichet of
the University of California at San Francisco, San Francisco,
CA. The class C P99 β-lactamase from Enterobacter cloacae, the
class A TEM-2 β-lactamase from E. coli W3310, and the class A
Staphylococcus aureus PC1 β-lactamase were purchased from the
Centre for Applied Microbiology and Research (Porton Down,
Wiltshire, UK). The class A SHV-1 enzyme was a gift from
Dr. Michiyoshi Nukaga of Jyosai International University, Japan.
Enzyme Kinetics Studies. DD-Peptidase Inhibition. a. In
Solution. Experiments designed to obtain equilibrium constants
of inhibition of the Actinomadura R39 DD-peptidase, B. subtilis
PBP4a, and E. coli PBP5 in solution by compounds 11 and 12
were performed as described previously17 from steady-state
competition experiments where N-(phenylacetyl)glycyl-D-thio-
lactic acid was employed as a spectrophotometric (245 nm, Δε =
2500 cm−1 M−1) substrate (0.5 mM). Enzyme concentrations
were between 0.1 and 0.2 μM. Initial rates of substrate
hydrolysis in the presence of a range of concentrations of 11
and 12 (0−1.0 mM) were obtained.
b. Membrane-Bound Enzymes. Equilibrium constants for
inhibition of E. coli DD-peptidases (PBPs) in membranes were
obtained as described previously, employing Bocillin Fl as a
fluorescent competitive β-lactam.18 Compounds 11 (0−1.0 mM)
and 12 (0−100 μM) were incubated with E. coli membrane
preparations for 1 h prior to addition of Bocillin Fl (20 μM).
β-Lactamase Inhibition. Equilibrium constants of inhibition
of the P99 and AmpC β-lactamases by compounds 11 and 12
(0−100 μM) were obtained from steady-state competition
experiments where cephalothin was employed as a spectro-
photometric (262 nm, Δε = 7660 cm−1 M−1) substrate (0.2 mM).
The reaction conditions were 20 mM MOPS buffer, pH 7.50,
25 °C, and enzyme concentrations of 2 nM, stabilized by 0.1%
bovine serum albumin in solution. Under these conditions, the
Km value of the substrate was 9.0 μM for the P99 enzyme19 and
13.8 μM for AmpC. No time-dependence of inhibition was
observed in the manual mixing time frame in these experiments.
Measurements of the initial rates, v, of cephalothin hydrolysis
catalyzed by the β-lactamase in the absence and presence of 11
or 12 were fitted to Scheme 3 by means of eq 1 (least-squares),
where v0 is the initial rate in the absence of inhibitor, to obtain
the Ki values. Attempts to obtain Ki values of 11 and 12 for the
class A TEM-2, SHV-1, and PC1 β-lactamases were conducted
similarly.
RESULTS AND DISCUSSION
■
The syntheses of the boronic acids 11 and 12 are outlined in
Schemes 4 and 5, respectively. In these syntheses, we made use
of the recent discovery that stable α-boryl aldehydes can be
prepared when N-methyl or pinene iminodiacetic acid (MIDA
or PIDA) boronic acid protecting groups are employed.26,27
Our use of MIDA thus allowed synthesis of aldehydes 13 and
14 from which the thiazolidine rings of 11 and 12 could readily
be constructed. We also found that the MIDA protecting group
was conveniently removed when treated with penicillamine
in methanol during the last step of each synthesis, yielding 11
and 12 in unprotected form. High resolution mass spectral
characterization of 11 and 12 presented difficulties since free
boronic acids are generally not volatile or stable enough
under the conditions required.28 The mass spectrum of 11 as
Molecular Modeling. Simulations were performed on a SGI
workstation running the program Insight II, essentially as
6531
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
a
the pinacol ester, however, displayed a small peak at the
appropriate mass (m/z = 406.3), but even this represented a
metastable ion and was thus not suitable for accurate mass
measurement. For both 11 and 12 we were able to obtain
accurate mass measurements for M-BO2 cations from these
pinacol esters (see Supporting Information). The presence of the
boronic acid moiety in the parent boronic acids was indicated in
their 1H NMR spectra by the absence of a methylene hydrogen
resonance in the spectrum and the characteristic upfield shift of
the hydrogen α to the boryl group.29
Scheme 4. Synthesis of 11
These syntheses yielded, as expected, mixtures of diastero-
isomers that we did not attempt to separate since we could get
a good estimate of the activity of the likely most active
diastereoisomer from the activity of the mixture. The preparation
of 12 yielded a mixture of two diasteroisomers in an essentially
1
1:1 ratio as indicated by the H NMR spectrum. Presumably
these would have structures 12a and 12b (Scheme 6), formed
in the last step (j) of the synthesis. All precedent with β-lactam-
recognizing enzymes would suggest that 12a, which has the
stereochemistry of bicyclic β-lactams, would be the active
inhibitor. The situation with 11 was more complicated.
Epoxidation and rearrangement of the intermediate alkenes
(Scheme 4) led to a mixture of two enantiomers with a chiral
carbon α to the boronic acid group (Scheme 6, a/c and b/d) in
a 4:1 ratio, where the stereochemistry of (a), α to the boryl
group, is likely to be present in the major component.27
Subsequent formation of the thiazolidine ring (step d) would
a
Stereochemistry is discussed in the text. Reagents and conditions: (a)
Grubb’s II catalyst, CH2Cl2, reflux; (b) mCPBA, CH2Cl2, 0 °C; (c)
BF3.Et2O, CH2Cl2, −30 to 0 °C; (d) D-penicillamine, MeOH-H2O, rt.
a
Scheme 5. Synthesis of 12
a
Stereochemistry is discussed in the text. Reagents and conditions: (a) nBuLi, THF, −78 °C; (b) BnOH, nBuLi, THF, −78 °C; (c) CH2Cl2, nBuLi,
THF, −100 °C; (d) LHMDS, THF, −100 °C; (e) PhCH2COCl, CH2Cl2, −78 °C; (f) 1-methylpropylboronic acid, MeOH − H2O/hexane, 6 h;
(g) MIDA, DMF, 80 °C, 12 h; (h) H2, Pd on carbon, MeOH, 40 psi, 12 h; (i) DMP, AcOH − CH3CN; (j) D-penicillamine, MeOH.
6532
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
this rationale for the slow deacylation of β-lactams from DD-
peptidases were true, the tetrahedral boronate adduct 6 would
also be unstable, and thus 11 and 12 would not be DD-
peptidase inhibitors. Therefore, our results with these inhibitors
support the mechanism of action of β-lactams described above.
It is also reported that 15 does not inhibit Streptococcus
pneumoniae PBP1b.31 As would be anticipated from the above
discussion, analogues of 16, unsubstituted α to the boronic
acid, are more likely to be DD-peptidase inhibitors.32−36
The results of Table 1 show that the new boronic acids 11
and 12 at micromolar concentrations do inhibit class C
Scheme 6. Boronic Acid Stereochemistry
presumably, as with 12, subdivide these equally into a final
4:4:1:1 mixture, as observed by 1H NMR (Supporting
Information), where the likely most active 11a would represent
40% of the total. A minor component 11c might also have
some activity since this stereochemistry is found in carbapenem
antibiotics.30
The first important result was that neither 11 nor 12 in-
hibited representative DD-peptidases. In solution, no inhibition
of the low molecular mass DD-peptidases of Streptomyces R61
and Actinomadura R39, or of B. subtilis PBP4a was observed.
Gel experiments also demonstrated that these compounds did
not inhibit E. coli PBP1a/1b, 2, 3, 4, and 5. These results con-
cretely support a current general proposal for the mechanism of
inhibition of DD-peptidases by β-lactams. Figure 1 shows the
Table 1. Enzyme Inhibition by Boronic Acids 11 and 12
a
Ki (μM)
enzyme
11
12
P99 β-lactamase
0.62 0.15
0.38 0.12
0.95 0.11
1.3 0.4
AmpC β-lactamase
TEM-2 β-lactamase
PCI β-lactamase
R39 DD-peptidase
b
b
NI
NI
b
b
NI
NI
c
c
NI
NI
a
The Ki values above are not corrected for the presence of
stereoisomers of 11 and 12 (see text). If it were assumed that the
most likely stereoisomer in each case was the only one with activity,
the values for 11 reported above would be multiplied by 0.4 and those
for 12 by 0.5 (see text) to obtain the Ki values of the active isomers.
b
c
NI, no inhibition observed at the concentration 1.0 mM. NI, no
inhibition observed at the concentration 0.10 mM.
β-lactamases, the P99 and AmpC enzymes. Notably, 11 is
comparably effective to 12, despite the absence in the former of
the amido side chain which is present in good substrates and
generally thought to be important for active site recognition
through hydrogen bonding.37 Apparently the hydrophobic side
chain of 11 is just as effective for inhibition as the amido
side chain of 12, which we can assume binds in the usual site
between the backbone carbonyl oxygen of residue 318 and the
amide side chain of the conserved Asn152.
Boronic acid 12 is similar in structure to 15, an inhibitor
described by Morandi et al.23 The latter compound is a 1 nM
inhibitor of the AmpC β-lactamase. A crystal structure of the
inhibitory complex has been published,20 from which the active
site diagram of Figure 2A has been taken. This shows the amide
side chain of 15 firmly hydrogen-bonded to the protein as
described above, and the boronate present as a tetrahedral
anion covalently bound to the nucleophilic serine of the active
site. One boronate oxygen is in the oxyanion hole (hydrogen-
bonded to backbone NH groups of Ser64 and Ala318), and the
other takes up the position of a leaving group or of the
deacylating water molecule, depending on whether the complex
is seen as an analogue of an acylation or deacylation tetrahedral
intermediate (see below).
Figure 1. Active site of the E. coli PBP4 DD-peptidase with
benzylpenicillin bound as a deacylation tetrahedral intermediate.
Modeled from the crystal structure of the acyl-enzyme.25
structure of a model of the putative tetrahedral intermediate for
deacylation of the covalent acyl-enzyme formed on reaction of
benzylpenicillin with E. coli PBP4. This structure was generated
directly from the acyl-enzyme structure.25 This structure is not
stable (and, consequently, deacylation is very slow) because of
unfavorable steric interactions between the hydroxyl group of
the water nucleophile and the adjacent carbon and nitrogen
atoms of the thiazolidine ring. These interactions are nicely seen
in a space-filling model (Supporting Information, Figure S1). If
Finally, the carboxyphenyl group is oriented with its plane
perpendicular to the chain formed by the amido side group, the
alkyl boronate, and the side chain of Ser64. In this position, the
carboxylate is directed above the β2-strand where it forms a
hydrogen bond with the amido side chain of Asn 289. This is
6533
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
a cephalosporin analog of 12, e.g., 17, which more closely
resembles 15, may be a better inhibitor of class C β-lactamases:
kcat values (deacylation rate constants) of cephalothin and
cephalexin are larger than that of benzylpenicillin for the P99
enzyme,28,29 suggesting that deacylation transition states of the
former may be better stabilized by the enzyme.
It might be noticed in passing that the interactions of the
inhibitor carboxylate group with the enzyme described above
hold the thiazolidine ring of 12 from sterically impeding
nucleophilic attack by water on the acyl-enzyme intermediate;
the thiazolidine nitrogen is held well away from both boronate
hydroxyl groups. This situation is in clear contrast to that seen
in DD-peptidases11 (see above) and presumably allows facile
hydrolysis of the acyl-enzyme by β-lactamases.
It might also be noted that Gln289 is not strictly conserved
in class C β-lactamases but is replaced by serine in Enterobacter
homologues such as the P99 β-lactamase. The hydroxyl of
serine in this position may also be able to form a hydrogen
bond with the carboxylate of a penicillin substrate and of an
inhibitor such as 15. Certainly 12 inhibits the P99 and AmpC
β-lactamases to very similar extents (Table 1).
Perhaps more striking than the results with class C enzymes
are those with class A. Neither the TEM-1, SHV-1, nor PCI
β-lactamases were inhibited by 11 or 12, even at 1 mM
concentrations. Given the fact that 12 should be the source of a
precise transition state analogue structure by reaction with
these enzymes (Schemes 1 and 2), much as with the class C
β-lactamases, the result is quite astonishing. In stark contrast,
15 is a powerful inhibitor of the TEM-2 β-lactamase; Ki = 64
nM.16 This profound and unexpected difference between 12
and 15 is discussed below.
First, it is necessary to recall that the geometry of the
acylation and deacylation transition states (and tetrahedral
intermediates, Scheme 2) is different in class A β-lactamases,
unlike the situation with the class C enzymes where the
acylation and deacylation tetrahedral intermediates have similar
structure (see above). This situation arises because, although
the hydrolytic water molecule is believed to attack the acyl-
enzyme intermediate from the solution side (Re face) in class C
β-lactamases, this attack comes from the protein side (Si face),
catalyzed by Glu 166, in class A β-lactamases.40,41 During
acylation, the leaving group is thought to depart from the Re
face of the acyl-enzyme in each case. These details are shown
diagrammatically in Scheme 7.
Thus, although one would expect all boronic acid complexes
of class C enzymes to resemble 18 and 20 (i.e., have structure
21, the two forms of which are interconvertible by a small
single bond rotation), two possibilities, 24 and 26, exist for
class A β-lactamases. Indeed, examples of each kind have been
observed with class A β-lactamases in crystal structures of
boronate complexes.16 Note that 22 and 23 (or 24 and 26) are
not interconvertible by single bond rotations.
The crystal structure of the strongly inhibitory (64 nM)
complex between 15 and the TEM-1 β-lactamase represents a
mimic of a deacylation tetrahedral intermediate (Figure 3A),
where one boronate hydroxyl group is in hydrogen bond
contact with the deacylation catalyst Glu166.16 In this structure,
Figure 2. (A) Active site of the AmpC β-lactamase with the boronic
acid 15 bound, from the crystal structure.23 (B) An energy-minimized
model of 12 bound to the AmpC active site, derived directly from the
structure in A.
apparently an effective arrangement since the boronic acid 16,
lacking the carboxyphenyl group, has a Ki value of 0.57 μM.14
A model of the complex between 12 and the AmpC
β-lactamase was constructed based on the crystal structure with
15 as described in Materials and Methods. The initial structure
was subjected to a short molecular dynamics simulation to relax
active site interactions and a typical snapshot energy-minimized
(see Materials and Methods). This procedure led to the
structure of Figure 2B. This resembles the structure of the
complex of 15 described above but differs in the positioning
of the thiazolidine carboxylate vs the phenyl carboxylate of 15,
due to the nonplanar nature of the five- (vs six-) membered
thiazolidine ring and thus to the positioning of its substituents.
The short MD run suggested that the thiazolidine carboxylate
may prefer interaction with Arg349 over that with Asn 289. A
similar dynamics run on the complex with 15 suggested that it
too may be mobile in solution, with carboxylate access even to
the Arg204 side chain. At any event, it appeared that the
carboxylates of 12 and 15 may prefer somewhat different
environments with some complementary adjustment of the
active site structure. These effects presumably lead to the
weaker binding of 12 than 15 to the enzyme. It is possible that
6534
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
Scheme 7. Mechanism of Formation of Tetrahedral
a
Intermediates and Their Boronate Analogues
a
In this diagram, 18 and 20 represent the acylation and deacylation
tetrahedral intermediates in class C β-lactamase catalysis, and 22 and
23, respectively, represent these species for a class A enzyme. The
boronate analogues 21, 24, and 26 are shown below their respective
intermediates, and 19 is the central acyl-enzyme. The arc adjacent to
one oxygen ligand in each case represents the oxyanion hole, the
presence of which serves to distinguish the two boronate hydroxyl
groups.
the carboxylate group of the inhibitor forms a strong hydrogen
bond with the Thr235 hydroxyl group and, probably, with the
Arg244 side chain via a water molecule. An energy-minimized
model of a complex of 12 with this enzyme is shown in Figure 3B.
In this structure, the interactions with the active site are the
same as that of the complex with 15. Both structures seemed
stable during short MD runs. The experimental result, that 12
(or 11) is not an inhibitor, therefore remains a puzzle.
A possible way out of this dilemma may be found when the
mechanism of formation of deacylation analogue boronate
complexes is considered. Direct formation of these complexes
from the free boronic acids and enzyme might be difficult
since it would require prior or concerted displacement of
the deacylating water molecule from Glu166. An alternative,
perhaps more facile, mechanism would proceed by way of initial
direct formation of an acylation tetrahedral intermediate
analogue, 24. In an analogous fashion to the acylation/
deacylation sequence of a substrate (Scheme 7), consecutively a
neutral trigonal boronic acid intermediate 25 may be formed
from 24, followed by its attack by the hydrolytic water
molecule, presumably catalyzed by Glu166, to form the
deacylation tetrahedral intermediate analogue 26.
The crystal structure of the complex between the class A
TEM-1 β-lactamase with the boronic acid 27 resembles the
acylation tetrahedral intermediate 22 (i.e., it has the structure
24) while, as noted above, that with 15 resembles the
deacylation intermediate 23 (i.e., it has the structure 26).
Wang et al. have suggested that the preference for the
deacylation analogue structure 26 by 15 derives from the
presence of the pendant carboxylate group which interacts with
Arg244 in the complex, as seen in the crystal structure16
(Figure 3A). One might expect that the boronic acids, 11 and
12, bearing the thiazolidine carboxylate, would also form stable
complexes analogous to 26. Ke et al. have also discussed the
Figure 3. (A) Active site of the TEM-1 β-lactamase with the boronic
acid 15 bound, from the crystal structure.16 (B) An energy-minimized
model of 12 bound to the TEM-1 active site, derived directly from the
structure in A. Note, however, that the thiophene ring of 15 has been
retained in this model rather than changed to phenyl.
relative merits of acylation vs deacylation complexes in various
specific instances.42
We approached a structural model of acylation tetrahedral
intermediate complexes by way of the crystal structure of the
complex of 27 with the TEM-1 enzyme.16 An immediate
problem was that a bulky substituent α to the boronic acid, as
present in 11, 12, and 15, sterically interacts unfavorably with
Tyr105 (Figure 4), raising the likelihood of significant
conformational adjustment (Figure 4). Models of both 12
and 15 were unstable, both to energy minimization and to MD
simulation. In both cases, particularly that of 12, expansion of
the lower part of the active site, comprising Tyr105, Asn132,
and Glu166, occurred. In particular, Tyr 105 was “pushed
away” with motion of the Asp101 − Leu108 loop. This
distortion was more extreme with the bulkier (nonplanar)
thiazolidine of 12. Acylation tetrahedral intermediates thus
appeared likely to be less stable than the deacylation species
described above. This is certainly a reasonable explanation for
6535
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
Figure 5. Active site of the class A CTX-M-9 β-lactamase with
benzylpenicillin bound as an acylation tetrahedral intermediate.
Modeled from the crystal structure of the noncovalent complex of
penicillin with the Ser70Gly mutant.24
Figure 4. Active site of the TEM-1 β-lactamase with the boronic acid
27 bound, from the crystal structure.16 Also shown, in the form of an
added methyl group, is the general orientation of an L-α substituent, as
present in 11 and 12.
of deacylation tetrahedral species, 23 (Scheme 7). It is possible
that recently discovered cyclic borinates43,44 may better
approximate acylation complexes.
the two types of boronate structures obtained, the nature of the
complex depending on whether a bulky substituent α to the
boronic acid is present.
These considerations lead to another point and one that
provides direct evidence for the instability of acyclic boronate
adducts 24. Methyl penicilloate, 28, is known to be a substrate
of a Pseudomonas class C β-lactamase,45 and we have extended
this result to the P99 β-lactamase (see Supporting Informa-
tion). This compound is, however, neither a substrate nor a
covalent inhibitor of the class A BCI β-lactamase,46 and we have
extended this point by observations with the TEM-2 enzyme
(Supporting Information). These observations prove that while
the class C β-lactamase active site can significantly stabilize the
acylation tetrahedral intermediate 29 (a direct analogue of 18),
the class A active site cannot (stabilize the analogue of 22),
presumably for the reasons discussed above.
This problem of the instability of the intermediate 22 (and
thus the analogue 24) might also be sufficient to explain the
lack of inhibition by 11 and 12. The ability of 15 to act as
inhibitor but not 11 and 12 can be supposed to arise from the
greater bulk of the thiazolidine substituent of the latter than the
phenyl of the former (Figure S2, Supporting Information) and
thus the greater difficulty of the latter to achieve the acylation
analogue structure 24 required (Scheme 7) as a precursor of
the likely stable deacylation analogue structure 26. This is an
explanation in terms of unfavorable kinetics, but it should be
noted that no inhibition of the TEM-2 enzyme was observed
in 24 h.
In support of the rationalization above, it should be pointed
out here that acylation tetrahedral intermediate analogues
generated from acyclic boronic acids such as 11, 12, and 15 are
not likely to closely resemble the structure of a “real” acylation
tetrahedral intermediate 3 (Figure 5). This structure was
generated from the crystal structure of a noncovalent complex
between benzylpenicillin and a Ser70Ala mutant of the class A
CTX-M-9 β-lactamase.21 In this structure, the azetidine ring is
still intact, with the scissile C−N bond eclipsed by the C−C
bond on the opposite side of the ring. Such eclipsing will be lost
for steric reasons when the four-membered ring is opened, as
seen in the structures of Figure 3. In Figure 5, the carboxylate
substituent of the thiazolidine ring forms hydrogen bonds with
the side chains of Lys234 and Thr235. After rotation away of
the thiazolidine ring with opening of the four-membered ring,
this hydrogen-bond pattern is lost and replaced by the
interactions seen in Figure 3. Thus, acyclic boronates cannot
generate close acylation transition state analogue structures, at
least of bicyclic β-lactam substrates, hence the instability of the
boronate acylation complexes 24 of class A β-lactamases. On
the other hand, acyclic boronic acids can form direct analogues
SUMMARY AND CONCLUSIONS
■
Neither 11 nor 12 [or, most likely, 1531] inhibit DD-
peptidases, even at 0.1 mM concentrations, probably because
of unfavorable steric interactions at the active site of these
enzymes (Figure 1). Deacylation tetrahedral intermediates of
DD-peptidase catalysis are thought to be destabilized in the
same way.9,11 The results with 11 and 12 from experiments in
solution therefore strongly support the steric mechanism of
inhibition of DD-peptidases by β-lactams and thus the
mechanism of antibiotic action by these compounds. Previous
evidence for this mechanism largely rested on inspection of
crystal structures of inert complexes.
The boronic acids 11 and 12 inhibit class C β-lactamases, at
micromolar concentrations, presumably by formation of
covalent tetrahedral adducts (Figure 2B) that resemble the
6536
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
(6) Beesley, T., Gascoyne, N., Knott-Hunziker, V., Petursson, S.,
Waley, S. G., Jaurin, B., and Grundstrom, T. (1983) The inhibition of
class C β-lactamases by boronic acids. Biochem. J. 209, 229−233.
(7) Pechenov, A., Stefanova, M. E., Nicholas, R. A., Peddi, S., and
Gutheil, W. G. (2003) Potential transition state analogue inhibitors for
the penicillin-binding proteins. Biochemistry 42, 579−588.
(8) Inglis, S. R., Zervosen, A., Woon, E. C. Y., Gerards, T., Teller, N.,
Fischer, D. S., Luxen, A., and Schofield, C. J. (2009) Synthesis and
evolution of 3-(dihydroxy-boryl) benzoic acids as DD-carboxypepti-
dase R39 inhibitors. J. Med. Chem. 52, 6097−6106.
(9) Kelly, J. A., Dideberg, O., Charlier, P., Wery, J. P., Libert, M.,
Moews, P. C., Knox, J. R., Duez, C., Fraipont, C., Joris, B., Dusart, J.
high energy tetrahedral intermediates of penicillin turnover.
Compounds 11 and 12 are not, however, as effective as
inhibitors as the phenyl analogue 15.23 It is possible that 17, a
cephalosporin analogue, closer in structure to 15, may be more
effective than 12. The new compounds do not inhibit class A
β-lactamases even at millimolar concentrations, in strong
contrast to 15, which, at nanomolar concentrations, forms 26,
a structural analogue of the deacylation tetrahedral intermediate
2316 (Scheme 7). A rationale for this surprising result is offered
in terms of a mechanism of formation of 26 (Scheme 7), which
requires the initial formation of an acylation tetrahedral
analogue 24, followed by that of a neutral trigonal boronic
acid intermediate 25, analogous to an acyl-enzyme, and finally
formation of 26 by intramolecular water attack on 25,
presumably catalyzed by Glu166. A model of the acylation
tetrahedral intermediate analogue from 12 suggests that this
species may be unstable on steric grounds, precluding progress
of 11 and 12 toward the deacylation analogue 26. An effective
transition state analogue inhibitor requires an energetically
accessible path to the inhibitory complex as well as transition
state mimicry in that complex.47
̀
M., Frere, J. M., and Ghuysen, J. M. (1986) On the origin of bacterial
resistance to penicillin: comparison of a β-lactamase and a penicillin
target. Science 231, 1429−1431.
(10) Ghuysen, J.-M. (1991) Serine β-lactamase and penicillin-binding
proteins. Annu. Rev. Microbiol. 45, 35−67.
(11) Pratt, R. F. (2002) Functional evolution of the serine β-
lactamase active site. J. Chem. Soc., Perkin Trans. 2, 851−861.
(12) Beta-Lactamases (2012) (Frere, J.-M., Ed.) Nova Science
Publishers, New York.
(13) Crompton, I. E., Cuthbert, B. K., Lowe, G., and Waley, S. G.
(1988) β-Lactamase inhibitors. The inhibition of serine β-lactamases
by specific boronic acids. Biochem. J. 251, 453−459.
(14) Caselli, E., Powers, R. A., Blasczcak, L. C., Wu, C. Y. E., Prati, F.,
and Shoichet, B. K. (2001) Energetic, structural, and antimicrobial
analyses of β-lactam side chain recognition by β-lactamases. Chem.
Biol. 8, 17−31.
(15) Ness, S., Martin, R., Kindler, A. M., Paetzel, M., Gold, M.,
Jensen, S. E., Jones, J. B., and Strynadka, N. C. J. (2000) Structure-
based design guides the improved efficacy and of deacylation transition
state analogue inhibitors of TEM-1 β-lactamase. Biochemistry 39,
5312−5321.
(16) Wang, X., Minasev, G., Blazquez, J., Caselli, E., Prati, F., and
Shoichet, B. K. (2003) Recognition and resistance in TEM β-
lactamase. Biochemistry 42, 8434−8444.
(17) Nemmara, V. V., Dzhekieva, L., Sarkar, K. S., Adediran, S. A.,
Duez, C., Nicholas, R. A., and Pratt, R. F. (2011) Substrate specificity
of low-molecular mass bacterial DD-peptidases. Biochemistry 50,
10091−10101.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic details for the preparation of compounds 11 and 12.
Figures S1and S2 illustrate steric effects of the thiazolidine ring.
This material is available free of charge via the Internet at
http://pubs.acs.org
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 860-685-2629; e-mail: rpratt@wesleyan.edu; fax:
860-685-2211.
Funding
This research was supported by National Institutes of Health
Grant AI-17986 (RFP) and by Wesleyan University.
(18) Dzhekieva, L., Kumar, I., and Pratt, R. F. (2012) Inhibition of
bacterial DD-peptidases (penicillin-binding proteins) in membranes
and in vivo by peptidoglycan-mimetic boronic acids. Biochemistry. 51,
2804−2811.
Notes
The authors declare no competing financial interest.
(19) Adediran, S. A., Cabaret, D., Pratt, R. F., and Wakselman, M.
(1999) A “cephalosporin-like” cyclic depsipeptide: Synthesis and
reaction with beta-lactam-recognizing enzymes. Bioorg. Med. Chem.
Lett. 9, 341−346.
ABBREVIATIONS
■
DMP, Dess−Martin periodinane; ESMS, electrospray ioniza-
tion mass spectroscopy; LHMDS, lithium hexamethyldisila-
zane; mCPBA, m-chloroperbenzoic acid; MIDA, methyl
iminodiacetic acid; MOPS, 3-morpholinopropanesulfonic acid;
NMR, nuclear magnetic resonance; PBP, penicillin-binding
protein; THF, tetrahydofuran; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide
(20) Kuzmic, P. (1996) Program DYNAFIT for the analysis of
enzyme kinetic data: application to HIV proteinase. Anal. Biochem.
237, 260−273.
(21) Nagarajan, R., and Pratt, R. F. (2004) Thermodynamic
evaluation of a covalently bonded transition state analogue inhibitor:
Inhibition of β-lactamases by phosphonates. Biochemistry 43, 9664−
9673.
REFERENCES
(22) Pelto, R. B., and Pratt, R. F. (2008) Kinetics and mechanisms of
inhibition of a serine β-lactamase by O-aryloxycarbonyl hydroxamates.
Biochemistry 47, 12037−12046.
■
(1) Robertson, J. G. (2005) Mechanistic basis of enzyme-targeted
drugs. Biochemistry 44, 5561−5571.
́
(23) Morandi, F., Caselli, E., Morandi, S., Focia, P. J., Blazquez, J.,
(2) Trippier, P. C., and McGuigan, C. (2010) Boronic acids in
medicinal chemistry: anticancer, antibacterial and antiviral applications.
MedChemComm 1, 183−198.
Shoichet, B. K., and Prati, F. (2003) Nanomolar inhibitors of AmpC β-
lactamase. J. Am. Chem. Soc. 125, 685−695.
(24) Leysenne, D., Delmas, J., Robin, F., Cougnoux, A., Gibold, L.,
and Bonnet, R. (2011) Noncovalent complexes of an inactive mutant
of CTX-M-9 with the substrate piperacillin and the corresponding
product. Antimicrob. Agents Chemother. 55, 5660−5665.
(3) Smoum, R., Rubinstein, A., Dembitsky, V. M., and Srebnik, M.
(2012) Boron containing compounds as protease inhibitors. Chem.
Rev. 112, 4156−4220.
(4) Drawz, S. M., Papp-Wallace, K. M., and Bonomo, R. A. (2014)
New β-lactamase inhibitors: a therapeutic renaissance in an MDR
world. Antimicrob. Agents Chemother. 58, 1835−1846.
(5) Kiener, P. A., and Waley, S. G. (1978) Reversible inhibitors of
penicillinases. Biochem. J. 169, 197−204.
(25) Kishida, H., Unzai, S., Roper, D. I., Lloyd, A., Park, S.-Y., and
Tame, J. R. H. (2006) Crystal structure of penicillin binding protein 4
(dacB) from Escherichia coli, both in the native form and covalently
linked to various antibiotics. Biochemistry 45, 783−792.
6537
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538

Biochemistry
Article
(26) He, Z., and Yudin, A. K. (2011) Amphoteric α-boryl aldehydes.
J. Am. Chem. Soc. 133, 13770−13773.
(27) Li, J., and Burke, M. D. (2011) Pinene-derived iminodiacetic
acid (PIDA): a powerful ligand for stereoselective synthesis and
iterative cross-coupling of C(sp3) boronate building blocks. J. Am.
Chem. Soc. 133, 13774−13777.
(28) Hall, D. G. (2005) Boronic Acids; Wiley-VCH, Germany.
(29) Rani, U., Karabacak, M., Tanriverdi, O., Kurt, M., and
Sundaragansan, N. (2012) The spectroscopic (FTIR, FT-RAMAN,
NMR AND UV), first order hyperpolarizability and HOMO-LUMO
analysis of methylboronic acid. Spectrochim. Acta, Part A 92, 67−77.
(30) Bryskier, A. (2005) in Antimicrobial Agents (Bryskier, A., Ed.)
Chapter 8, ASM Press, Washington, DC.
combined with a serine β-lactamase inhibitor, against anaerobic
bacteria. Antimicrob. Agents Chemother. 57, 2620−2630.
(45) Knott-Hunziker, V., Petursson, S., Waley, S. G., Jaurin, B., and
Grundstrom, T. (1982) The acyl-enzyme mechanism of β-lactamase
action. The evidence for class C β-lactamases. Biochem. J. 207, 315−
322.
(46) Jones, M., Buckwell, S. C., Page, M. I., and Wrigglesworth, R.
(1989) A reversible inhibitor of β-lactamase I from Bacillus cereus.
Chem. Soc. Chem. Commun., 70−71.
(47) Moulin, A., Bell, J. H., Pratt, R. F., and Ringe, D. (2007)
Inhibition of chymotrypsin by a complex of ortho-vanadate and
benzohydroxamic acid: Structure of the inert complex and its
mechanistic interpretation. Biochemistry 46, 5982−5990.
(31) Contreras-Martel, C., Amoroso, A., Woon, E. C. Y., Zervosen,
A., Inglis, S., Martins, A., Verlaine, O., Rydzik, A. M., Job, V., Luxen, A.,
Joris, B., Schofield, C. J., and Dessen, A. (2011) Structure-guided
design of cell wall biosynthesis inhibitors that overcome β-lactam
resistance in Staphylococcus aureus (MRSA). ACS Chem. Biol. 6, 943−
951.
(32) Nicola, G., Peddi, S., Stefanova, M., Nicholas, R. A., Gutheil, W.
G., and Davies, C. (2005) Crystal structure of Escherichia coli
penicillin-binding protein 5 bound to a tripeptide boronic acid
inhibitor: A role for Ser 110 in deacylation. Biochemistry 44, 8207−
8217.
(33) Dzhekieva, L., Rocaboy, M., Kerff, F., Charlier, P., Sauvage, E.,
and Pratt, R. F. (2010) Crystal structure of a complex between the
Actinomadura R39 DD-peptidase and a peptidoglycan-mimetic
boronate inhibitor: Interpretation of a transition state analogue in
terms of catalytic mechanism. Biochemistry 49, 6411−6419.
(34) Zervosen, A., Herman, R., Kerff, F., Herman, A., Bouillez, A.,
̀
Prati, F., Pratt, R. F., Frere, J.-M., Joris, B., Luxen, A., Charlier, P., and
Sauvage, E. (2011) Unexpected tricovalent binding mode of boronic
acids within the active site of a penicillin-binding protein. J. Am. Chem.
Soc. 133, 10839−10848.
(35) Woon, E. C. Y., Zervosen, A., Sauvage, E., Simmons, K. J., Zivec,
M., Inglis, S. R., Fishwick, C. W. G., Gobec, S., Charlier, P., Luxen, A.,
and Schofield, C. J. (2011) Structure guided development of potent
reversibly binding penicillin binding protein inhibitors. ACS Med.
Chem. Lett. 2, 219−223.
(36) Zervosen, A., Sauvage, E., Frere, J.-M., Charlier, P., and Luxen,
̀
A. (2012) Development of new drugs for an old target - the penicillin
binding proteins. Molecules 17, 12478−12505.
(37) Beadle, B. M., Trehan, I., Focia, P. J., and Shoichet, B. K. (2002)
Structural milestones in the reaction pathway of an amide hydrolase:
substrate, acyl, and product complexes of a cephalothin with ampC β-
lactamase. Structure 10, 413−424.
̀
(38) Galleni, M., Amicosante, G., and Frere, J.-M. (1988) A survey of
the kinetic parameters of class C β-lactamases. Cephalosporins and
other β-lactam compounds. Biochem. J. 255, 123−129.
(39) Matagne, A., Misselyn-Bauduin, A.-M., Joris, B., Erpicum, J.,
̀
Granier, B., and Frere, J.-M. (1990) The diversity of catalytic
properties of class A β-lactamases. Biochem. J. 265, 131−146.
(40) Herzberg, O., and Moult, J. (1987) Bacterial resistance to β-
lactam antibiotics: crystal structure of β-lactamase from Staphylococcus
aureus PCI at 2.5 Å resolution. Science 236, 694−701.
(41) Herzberg, O., and Moult, J. (1991) Penicillin-binding and
degrading enzymes. Curr. Opin. Struct. Biol. 1, 946−953.
(42) Ke, W., Sampson, J. M., Ori, C., Prati, F., Drawz, S. M., Bethel,
C. R., Bonomo, R. A., and van den Akker, F. (2011) Novel insights
into the mode of inhibition of class A SHV-1 β-lactamases revealed by
boronic acid transition state inhibitors. Antimicrob. Agents Chemother.
55, 174−183.
(43) Hecker, S., Reddy, K., Totrov, M., Hirst, G., Sabet, M., Tarazi, Z.
Dudley, M. (2012) Discovery of RPX7009, a broad-spectrum β-
lactamase inhibitor with utility vs. class A serine carbapenemase,
abstract F-848. Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Francisco, CA.
(44) Goldstein, E. J., Citron, D. M., Tyrrell, K. L., and Merriam, C. V.
(2013) In vitro activity of biapenem plus RPX7009, a carbapenem
6538
dx.doi.org/10.1021/bi500970f | Biochemistry 2014, 53, 6530−6538