Dynamic combinatorial mass spectrometry leads to metallo-β-lactamase inhibitors

Article abstract of DOI:10.1021/jm070866g

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-β-lactamase enabled the rapid identification of an inhibitor with a K_i of <1 μM. The study exemplifies the utility of protein-MS for screening dynamic mixtures of potential enzyme-inhibitors.

Full text of DOI:10.1021/jm070866g

684
J. Med. Chem. 2008, 51, 684–688
Brief Articles
Dynamic Combinatorial Mass Spectrometry Leads to Metallo-ꢀ-lactamase Inhibitors
Benoît M. R. Liénard,† Rebekka Hüting,† Patricia Lassaux,‡ Moreno Galleni,‡ Jean-Marie Frère,‡ and Christopher J. Schofield*,†
Chemistry Research Laboratory and OCISB, Mansfield Road, Oxford, OX1 3TA, United Kingdom, and Centre d’Ingénierie des Protéines,
UniVersité de Liège, Allé du 6 Août B6, Sart-Tilman 4000 Liège, Belgium
ReceiVed July 18, 2007
The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial
library of thiols/disulfides with the BcII metallo-ꢀ-lactamase enabled the rapid identification of an inhibitor
with a Ki of <1 µM. The study exemplifies the utility of protein-MS for screening dynamic mixtures of
potential enzyme–inhibitors.
Introduction
The application of dynamic combinatorial chemistry (DCC)1
to structure–activity relationship (SAR) studies is limited by
the lack of appropriate screening methodology. In some cases,
direct protein mass spectrometry provides a potential solution
to the problem. Poulsen has recently reported the use of
electrospray ionization Fourier transform ion cyclotron reso-
nance (ESI-FTICR) to identify carbonic anhydrase inhibitors
from a dynamic combinatorial library (DCL) of hydrazines and
aldehydes.2 We have applied a combined electrospray ionization
mass spectrometry (ESI-MS) and thiol-based DCL approach
under non-equilibrating conditions to identify inhibitors of the
BcII metallo-ꢀ-lactamase (MBL) from Bacillus cereus.3 Our
Dynamic Combinatorial Mass Spectrometry (DCMS) approach
employs a bifunctional dithiol-support ligand in which one thiol
is complexed to the active site zinc ions with another thiol-
enabling disulfide formation. ESI-MS was used to directly
analyze enzyme bound disulfides.
The MBL family are clinically important in resistance to
ꢀ-lactam antibiotics such as penicillins and carbapenems as they
catalyze hydrolysis of almost all the clinically used ꢀ-lactam
antibiotics.4 BcII is a subclass B1 MBL that requires two ZnII
ions in its active site for optimal catalytic activity. Among the
more potent reported inhibitors for the BcII MBL are the thiols
R-thiomandelic acid5 and D-captopril.6
Figure 1. Chemical structures of thiol inhibitors of the BcII MBL.
complex with BcII:Zn2 that was stable enough to be observed
by ESI-MS (Figure 3a).
The commercially available analogue of 1, meso-2,3-dimer-
captosuccinic acid (2, Figure 1), was then selected for DCMS
work, due to its potential ability to interact with BcII:Zn2 via
one SH group and support disulfide exchange using the other
thiol (Figures 2 and 3).
The results of the ESI-MS binding assays indicated that 2
binds to BcII:Zn2, as shown by a peak corresponding to BcII:
Zn2-2 (observed 25274 ( 3 Da; expected 25273 Da; Figure
3b). Because 3 (BcII:Zn2-3; observed 25239 ( 3 Da; expected
25239 Da; Figure 3c) but not 4 was observed to bind to BcII:
Zn2 (Figure 3d), and the dizinc site of the BcII:Zn2 has an
affinity for thiol groups,8,9 only one thiol group of thiol 2 is
likely to chelate by the active site ZnII ions. Together these
results indicated that 2 had potential to be used as a support
ligand (Figure 2).
The DCMS assay was then applied to a set of thiols (Figure
4) in the presence of BcII. Strikingly, after 30 min of aerial
exposure, peaks corresponding to BcII:Zn2-disulfide complexes
were observed (Figure 3f). Selectivity in the disulfide recognition
by the BcII MBL was achieved since, for example, only
thiophenols 5a/b/c and 6a/b/c, and not carboxybenzylmercaptan
7 (Figure 4), were observed to react with 2 to form disulfide
complexes BcII:Zn2-(2∼5a/b/c) (observed 25,390 ( 2 Da;
predicted 25390 Da; Figure 3f; ∼ ) disulfide bond) and BcII:
Zn2-(2∼6a/b/c) (observed 25420 ( 2 Da; predicted 25419 Da;
Figure 3f). This selectivity is different to that observed when
using support ligand 8, which formed a disulfide with 7 and
not with 5 or 6 upon binding to BcII:Zn2 (Figure 3g).3
Because specific disulfides are not necessarily maintained
under the condition of the catalytic turnover assays, replacement
of the disulfide bond by stable linker analogues was then
Here we demonstrate that the DCMS method can be used to
identify further disulfides that bind to the BcII MBL and that
analogues of these disulfides are potent inhibitors. The dem-
onstration that the approach is not limited to a specific support
ligand, coupled to its speed, suggests that the technique may
be successfully applied to other targets.
Results and Discussion
Initially, we carried out ESI-MS analyses aimed at identifying
support ligands for DCMS screens. We found that mercapto-
propionic acid 1 (Figure 1), already known to inhibit the IMP-1
member of the MBL family (IC50 ) 170 µM),7 formed a
* To whom correspondence should be addressed. Tel.: 44 (0) 1865 275
625. Fax: 44 (0) 1865 275 674. E-mail: christopher.schofield@
chem.ox.ac.uk.
†
Chemistry Research Laboratory and OCISB.
Université de Liège.
‡
10.1021/jm070866g CCC: $40.75
2008 American Chemical Society
Published on Web 01/19/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 685
Support ligand 2 was incubated with 5, 6, and 10 to 17 in
the presence of BcII:Zn2 and the reaction was monitored by
ESI-MS. After 24 h of air exposure, three peaks A-C,
corresponding to BcII:Zn2-disulfide complexes with mass shifts
of 310, 336, and 343 Da relative to BcII:Zn2, were observed
(Figure 6b). Due to identities or similarities in mass between
some of the DCL ligands (e.g., 5 vs 12), it was not possible to
assign the binding partner of 2 using only the mass shifts. Peak
A (Figure 6b) could be assigned to either BcII:Zn2-(2∼5a/b/
c) or BcII:Zn2-(2∼12a/b/c), peak B (Figure 6b) could be
assigned to either BcII:Zn2-(2∼6a/b/c), BcII:Zn2-(2∼13) or
BcII:Zn2-(2∼14), and peak C (Figure 6b) could be assigned
to either BcII:Zn2-(2∼15) or BcII:Zn2-(2∼17). Knockout
experiments in which specific thiols were deleted were then
conducted. The results indicated that upon formation of the
mixed disulfide with support ligand 2, all of 5a-c (R ) NH2)
contributed to a similar extent to the formation of peak A,
whereas 12a-c (R ) OH) did not contribute. Peak B was
assigned to the BcII:Zn2-(2∼6a/b/c) (R ) CO2H) and BcII:
Zn2-(2∼13) (R ) B(OH)2) complexes and peak C was assigned
to the BcII:Zn2-(2∼15) (R ) NHAc) complex only. It was
notable that in the knockout experiments disulfides (2∼15)
displayed a significantly stronger binding to BcII:Zn2 compared
to the other disulfides, especially 2∼17, which was not observed
to bind to BcII:Zn2.
To test whether the functionalization pattern of the aromatic
ring of 9b obtained in the previous experiments could effectively
improve its inhibition against BcII, analogues of disulfides 2∼15
and 2∼17 (as a control) were synthesized (see Supporting
Information) and tested for inhibition of BcII. Despite remaining
relatively potent, BcII inhibitors 18 (Figure 6c, Ki ) 13.5 (
0.2 µM) and 19 (Figure 6c, Ki ) 8.4 ( 1 µM) did not improve
the original inhibitory activity of 9b. Interestingly, the relative
MS binding affinity of 18 for BcII:Zn2 was significantly stronger
than for 19 (see Supporting Information), consistent with the
DCMS results, despite having similar Ki values versus BcII.
Figure 2. The Dynamic Combinatorial Mass Spectrometry (DCMS)
approach. (a) The complex formed by binding of the thiol of the lead
compound 1 to the dizinc active site (BcII:Zn2-1 complex); (b) The
complex formed by binding of the dithiol support ligand 2 with one
thiol binding to the dizinc centre leaving one thiol free for disulfide
formation (BcII:Zn2-2); (c) The BcII:Zn2-2 complex in presence of
the dynamic thiol library; (d) Formation of a BcII:Zn2-disulfide
complex; Zn(II) ions are in green, BcII in grey (PDB ID: 1BVT),12
thiol groups in orange. Figure made using PyMol.
investigated (Figure 3h). ESI-MS studies had indicated that only
one of the two carboxylates of 2 was required for binding to
BcII:Zn2 (Figure 3a-d); we thus prepared a series of acylated
cysteine-based analogues 9a-f (Figure 1) as readily prepared
analogues of the disulfides observed to bind to BcII:Zn2.
Although imperfect disulfide analogues, the prepared compounds
were aimed at imitating the preferred linker length and stere-
ochemistry. Compounds 9a-f were synthesized in two steps
starting from L- and D-cystine (see Supporting Information) and
screened for inhibition versus BcII (Table 1).
The inhibition results revealed that the shortest amido-linker,
in agreement with the DCMS data, in combination with a
D-cysteine configuration, gave the most potent inhibition, with
a Ki value of 740 ( 20 nM for 9b, consistent with previously
reported data on the inhibition of BcII by structurally related
compounds.10 This also represents a significant increase in
inhibitory potency (about 170-fold) compared to the lead
inhibitor 2. Inhibition was observed to decrease significantly
with increasing linker length coupled to the L-configuration at
the cysteine CR, for example, 9c is 200-fold less potent than
9b. Furthermore, ESI-MS binding screens of 9a-f for the
binding to BcII:Zn2 were consistent with the solution inhibition
data (see Supporting Information).
Conclusions
Overall, our work demonstrates the efficiency of the DCMS
technique to rapidly generate inhibitors of clinically relevant
enzymes. When this method was used, the inhibitory activity
of the lead inhibitor 2 (Ki ) 125 µM) versus the BcII MBL
was improved by >150-fold, leading to the identification of
mercaptocarboxylate 9b with a Ki value of 740 nM for BcII,
60 times more potent than D-captopril. The work has revealed
limitations to the method in that the increased binding affinity,
by ESI-MS, of 18 relative to 9b was not reflected in increased
inhibitory potency. This difference may reflect the relative
efficiency with which hydrophobic and electrostatic interactions
are correlated in solution and under the ESI MS analysis
conditions.11 Alternatively, it may reflect the imperfect nature
of the disulfide analogues. Another serious limitation is that not
all proteins are amenable to nondenaturing MS analyses.
However, at least for amenable proteins, the speed with which
DCMS data can be generated means the technique is worthy of
further investigation.
Experimental Section
The initial DCMS screen had revealed that support ligand 2
selectively forms a disulphide with thiophenols to form BcII:
Zn2 complexes in the presence of aliphatic thiols and benzyl
mercaptans. In an attempt to improve the potency of inhibition
of 9b for BcII by optimizing the functionality of its aromatic
ring, a further set of thiols comprising exclusively thiophenol
derivatives was then employed in a DCMS screen (Figure 5).
DCL Preparation. Individual thiols of the library (2, 5, 6, and
10-17) were freshly prepared in DMSO at a final concentration
of 100 mM prior to each time-course in a glovebox ([O2] < 0.1
ppm). Each monothiol stock solution (5, 6, and 10-17) was then
diluted to 75 µM into the same mixture in 15 mM NH4OAc buffer.
The pH of the resulting mixture was then adjusted to pH 7.5 with
NH4OH using a HI 1290 Piccolo pH meter. Support ligand 2 was

686 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Articles
Figure 3. Deconvoluted ESI-MS spectra resulting from BcII:Zn2 (25089 ( 3 Da) incubated with (a) 1 (106 Da), (b) 2 (182 Da), (c) 3 (150 Da),
(d) 4 (118 Da), (e) 2 + DCL of thiol (Figure 4) after 1 min of aerial exposure, (f) 2 + DCL of thiol from Figure 4 after 30 min of aerial exposure,
(g) selectivity of the disulfide formation upon binding to BcII:Zn2 according to the type of support ligand used in the DCMS study (8 was used as
support ligand in previous studies using the set of thiols described in Figure 4);3 and (h) possible structural replacement of the disulfide linkage.
diluted to 100 µM in 15 mM NH4OAc buffer at pH 7.5. The
experimental samples were prepared by mixing the required
amounts of the monothiol mixture, 2 and BcII:Zn2 in NH4OAc
buffer pH 7.5 at a final concentrations of 15, 45, and 15 µM,
respectively. A total of 20 µL of this mixture was placed into a
96-well plate sealed with adhesive aluminum foil and taken out of
the oxygen-free environment to be analyzed. Samples were kept
in ice for the duration of the time-course (except during the time
required for measurement).
Soft Ionization Electrospray Mass Spectrometry. ESI-MS
analyses used a Q-TOF mass spectrometer (Q-TOFmicro Micro-
mass, Altrincham, U.K.) interfaced with a NanoMate chip-based
nano-ESI source (Advion Biosciences, Ithaca, NY, U.S.A.). The
time-course was started when the nanomate tip pierced the
aluminum seal covering the 96-well plate and introduced O2 into
the system. Samples were then infused to the Q-TOF through the
ESI chip (estimated flow rate ca. 100 nL/min). Typically a spraying
voltage of 1.72 ( 0.1 kV and a sample pressure of 0.25 psi were
applied. The instrument was equipped with a standard Z-spray
source block. Clusters of Na(n+1)In (1 mg/mL NaI in 100% methanol)
were used for calibration. Calibration and sample acquisitions were
performed in the positive ion mode in the range of m/z 500–5000.
Operating conditions for the mass spectrometer were sample cone
voltage (varied) between 50 to 200 V (only the data acquired at
sample cone voltage 50 V are shown in the Figures), source
temperature 40 °C, and acquisition and scan time were 30 and 1 s,
respectively. The pressure at the interface between the atmospheric
source and the high vacuum region was fixed at 6.6 mbar (measured
with the roughing pump Pirani gauge) by throttling the pumping
line using an Edwards Speedivalve to provide collisional cooling.
Kinetic Analyses. Solution of all tested inhibitors were prepared
as 10 mM DMSO solutions before dilution with 20 mM HEPES
buffer pH 7.0 containing 20 µg/mL BSA. The enzyme and inhibitor
were preincubated at room temperature before the substrate
(imipenem) was added. Substrate concentrations were varied
between 20 and 200 µM at a minimum of two inhibitor concentra-
tions and in its absence. Hydrolysis of imipenem was monitored
by following the variation in absorbance at 300 or 482 nm,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 687
Figure 4. Dynamic thiol library: first set (MW in Da).
Table 1. Ki Values vs the BcII MBL
cmpd
Ki (µM)
cmpd
Ki (µM)
2
125 ( 11
28 ( 0.5
0.74 ( 0.02
154 ( 5
9d
9e
9f
53 ( 1
44 ( 1.5
73 ( 1
9a
9b
9c
respectively, using a Uvikon 860 spectrophotometer connected to
a computer via a RS232 serial interface. Cells of 2 or 10 mm path
length were used depending on substrate concentration. The
experiments were performed at 30 °C and initial rate conditions
were used to study the inhibition with imipenem using the Hanes
linearization of the Henri-Michaelis equation and the KaleidaGraph
3.5 program.
N-Benzoyl-L-cysteine (9a). Compound 9a was prepared fol-
lowing a reported method.10 IR νmax (film) 3014 (b, OH), 2552
(m, SH), 1732 (s, CdO), 1639 (s, CdO), 1216 (s, C-O) cm-1
;
1H NMR (500 MHz, DMSO-d6) δ 12.85 (brs, 1H, COOH), 8.65
(d, J ) 8.0 Hz, 1H, NH), 7.91–7.90 (m, 2H, ArH), 7.59–7.55 (m,
1H, ArH), 7.51–7.49 (m, 2H, ArH), 4.55–4.51 (m, 1H, HR),
3.03–3.01 (m, 1H, Hꢀ), 2.92–2.88 (m, 1H, Hꢀ), 2.60 (brs, 1H, SH);
13C NMR (126 MHz, DMSO-d6) δ 172.3 (CdO), 167.0 (CdO),
134.3 (ArC), 132.0 (ArCH), 128.8 (2 × ArCH), 127.9 (2 × ArCH),
56.0 (CHR), 25.7 (CH2); HRMS (ESI-) (M - H)- calcd for
Figure 6. Deconvoluted ESI-MS spectra resulting from BcII:Zn2
incubated with support ligand 2 and the DCL of thiols (Figure 4) after
(a) 1 min and (b) 24 h of aerial exposure. Peaks A, B, and C correspond
to BcII:Zn2-disulfide quaternary complexes with a mass shift related
to BcII:Zn2 of 310, 336, and 343 Da, respectively; (c) derivatives of
9b designed according to the results of the second DCMS study (b).
C10H10NO3S-, 224.0381; found, 224.0676; [R]25 ) -15° (c )
D
0.54, CH3OH).
N-Benzoyl-D-cysteine (9b). Compound 9b was prepared fol-
lowing a reported method.10 All analytical data were identical to
that of 9a, with the exception of the optical rotation: [R]25D ) +19°
(c ) 0.48, CH3OH).
DMSO-d6) δ 12.90 (s, 1H), 8.43 (d, J ) 8.0 Hz, 1H), 7.33–7.19
(m, 5H), 4.40 (ddd, J ) 8.0 Hz, J ) 8.0 Hz, J ) 5.0 Hz, 1H), 3.52
(s, 2H), 2.87 (ddd, J ) 13.5 Hz, J ) 8.5 Hz, J ) 5.0 Hz, 1H),
2.80–2.73 (m, 1H), 2.39 (t, J ) 8.5 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6) δ 172.5, 171.1, 137.0, 129.9 ( × 2), 129.0 ( × 2), 127.2,
55.3, 42.8, 26.5; HRMS (ESI-) (M - H)-calcd for C11H12NO3S-,
N-Phenylacetyl-L-cysteine (9c). Compound 9c was prepared
following a reported method.10 Mp 96–98 °C; 1H NMR (400 MHz,
238.0538; found, 238.0532; [R]25 ) +2.8° (c ) 0.5, CH3OH).
D
N-Phenylacetyl-D-cysteine (9d). Compound 9d was prepared
following a reported method.10 All analytical data were identical
to that of 9c, with the exception of the optical rotation: [R]25
-1.6° (c ) 0.24, CH3OH).
)
D
N-Phenylpropionyl-L-cysteine (9e). Compound 9e was prepared
following a reported method.10 Mp 126–127 °C (white crystalline
solid); IR νmax (KBr disk) 3353 (s, OH), 1721 (s, CdO), 1617 (s,
1
Figure 5. Dynamic thiol library: second set (MW in Da).
CdO), 1224 (s, C-O) cm-1; H NMR (400 MHz, DMSO-d6) δ

688 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Articles
12.83 (brs, 1H, COOH), 8.20 (d, J ) 8.0 Hz, 1H, NH), 7.29–7.15
(m, 5H, ArH), 4.40 (ddd, J ) 8.0 Hz, J ) 7.5 Hz, J ) 4.5 Hz, 1H,
HR), 2.84–2.80 (m, 3H, CH2 + Hꢀ), 2.75–2.66 (m, 1H, Hꢀ),
2.52–2.41 (m, 2H, CH2), 2.29 (brs, 1H, SH); 13C NMR (101 MHz,
DMSO-d6) δ 172.6 (CdO), 172.4 (CdO), 142.1 (ArC), 129.14 (2
× ArCH), 129.12 (2 × ArCH), 126.8 (ArCH), 55.2 (CHR), 37.5
(CH2), 31.9 (CH2), 26.5 (CH2ꢀ); HRMS (ESI+) (M + Na)+ calcd
and additional ESI-MS experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
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for C12H15NNaO3S+, 276.0670; found, 276.0665; [R]25 ) -6°
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(c ) 0.62, CH3OH).
N-Phenylpropionyl-D-cysteine (9f). Compound 9f was prepared
following a reported method.10 All analytical data were identical
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+6.4° (c ) 0.32, CH3OH).
)
D
N,N′-Bis(4-Acetamidobenzoyl)-D-cysteine (18). Compound 18
was prepared following a reported method.10 Mp 205–210 °C (white
solid); IR νmax (KBr disk) 3342 (s, OH), 2550 (m, SH), 1716 (s,
CdO), 1640 (s, CdO), 1593 (s, CdO); 1H NMR (500 MHz,
DMSO-d6) δ 12.84 (s, 1H, COOH), 10.17 (s, 1H, NH), 8.49 (d, J
) 8.5 Hz, 1H, NH), 7.84 (d, J ) 8.5 Hz, 2H, ArH), 7.66 (d, J )
8.5 Hz, 2H, ArH), 4.49 (ddd, J ) 9.0 Hz, J ) 8.0 Hz, J ) 4.5 Hz,
1H, HR), 2.96–3.01 (m, 1H, Hꢀ), 2.84–2.90 (m, 1H, Hꢀ), 2.56 (t, J
) 8.5 Hz, 1H, SH), 2.07 (s, 3H, CH3); 13C NMR (126 MHz,
DMSO-d6) δ 171.9 (CdO), 168.7 (CdO), 166.0 (CdO), 142.2
(ArC), 128.4 (2 × ArCH), 128.0 (ArC), 118.0 (2 × ArCH), 55.5
(CHR), 25.2 (CH2ꢀ), 24.1 (CH3); HRMS (ESI+) (M + Na)+ calcd
for C12H14N2NaO4S1+, 305.0572; found, 305.0566; [R]25D ) +23°
(c ) 0.08, CH3OH).
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B. L.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D.; Whitesides,
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N-(4-tert-Butylbenzoyl)-D-cysteine (19). Compound 19 was
prepared following a reported method.10 Mp 115–118 °C (white
crystalline solid); IR νmax (KBr disk) 3345 (b, OH), 2569 (m, SH),
1733 (s, CdO), 1638 (s, CdO) cm-1; 1H NMR (500 MHz, DMSO-
d6) δ 8.52 (d, J ) 8.5 Hz, 1H, NH), 7.82 (d, J ) 8.5 Hz, 2H,
ArH), 7.50 (d, J ) 8.5 Hz, 2H, ArH), 4.49 (ddd, J ) 9.0 Hz, J )
8.5 Hz, J ) 4.5 Hz, 1H, HR), 2.99 (J ) 13.5 Hz, J ) 9.0 Hz, 1H,
Hꢀ), 2.88 (J ) 13.5 Hz, J ) 9.0 Hz, 1H, Hꢀ), 1.31 (s, 9H, t-Bu);
13C NMR (126 MHz, DMSO-d6) δ 172.3 (COOH), 166.4 (CONH),
144.3 (ArC), 131.1 (ArC), 127.3 (2 × ArCH), 125.1 (2 × ArCH),
55.4 (CHR), 34.6 (Ct-Bu), 31.0 (3 × t-Bu), 25.3 (CH2ꢀ); HRMS
(ESI+) (M + Na)+ calcd for C14H19NNaO3S1+, 304.0978; found,
304.0978; [R]25 ) +23.3° (c ) 0.42, CH3OH).
D
Acknowledgment. We thank the Biotechnology and Bio-
logical Sciences Research Council and the E.U. (Contract No.
HPRN-CT-2002-00264) for support of B.M.R.L. and P.L.
(12) Carfi, A.; Duee, E.; Galleni, M.; Frere, J. M.; Dideberg, O. 1.85 A
resolution structure of the zinc (II) beta-lactamase from Bacillus cereus.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54 (Pt 3), 313–23.
Supporting Information Available: Experimental details cor-
responding to the synthesis of the compounds described in this paper
JM070866G