An Integrated Biophysical Approach to Discovering Mechanisms of NDM-1\nInhibition for Several Thiol-Containing Drugs
Due to the rapid proliferation of antibiotic-resistant pathogenic bacteria, known as Carbapenem-Resistant Enterobacteriaceae (CRE), the efficacy of \xce\xb2-lactam antibiotics is threatened. \xce\xb2-Lactam antibiotics constitute over 50% of the available antibiotic arsenal. Recent efforts have been focused on developing inhibitors to these enzymes. In an effort to understand the mechanism of inhibition(s) of four FDA-approved thiol-containing drugs that were previously reported to be inhibitors of New Delhi Metallo-\xce\xb2-lactamase (NDM-1), various biochemical and spectroscopic techniques were used. Isothermal titration calorimetry demonstrated the binding affinity to NDM-1 corresponds to the reported IC50 values of the inhibitors. Equilibrium dialyses and metal analyses demonstrated that all of these inhibitors formed ternary complexes with ZnZn-NDM-1. Spectroscopic studies on CoCo-NDM-1, revealed two distinct binding modes for the thiol containing compounds. Our findings validate the need to further investigate the mechanism of inhibition of MBL inhibitors. Further research to identify inhibition capabilities beyond reported IC50 values is necessary for understanding the binding modes of these identified compounds and to provide the necessary foundation for developing clinically-relevant MBL inhibitors.
Introduction<!>Over-expression, purification, and characterization of NDM-1<!>Equilibrium dialysis<!>Isothermal titration calorimetry<!>UV-visible spectroscopy<!>NanoESI-mass spectrometry<!>NMR spectroscopy<!>EPR spectroscopy<!>Computational Modelling<!>Isothermal Titration Calorimetry (ITC)<!>Equilibrium Dialysis<!>UV-visible Spectroscopy<!>Electron Paramagnetic Resonance Spectroscopy<!>NMR Spectroscopy<!>Computational Docking Models/Results of Thiorphan with NDM-1<!>Summary and Conclusion
<p>Multidrug-resistant pathogenic bacteria, such as carbapenem-resistant
Enterobacteriaceae (CRE), have been classified as an "urgent threat,"
by the Centers for Disease Control and Prevention [1]. Gram-negative bacteria often contain several genes encoding
β-lactamases, which hydrolyze β-lactam antibiotics rendering them
ineffective. The significance of this problem lies in the fact that β-lactam
antibiotics constitute over 50% of the available antibiotic arsenal. The three most
clinically-relevant metallocarbapenemases are New Delhi metallo-β-lactamase
(NDM), Verona integrin encoded metallo-β-lactamase (VIM), and imipenemase
(IMP) [2]. These metallo-β-lactamases
(MBLs) use a dinuclear Zn(II)-containing active site to hydrolyze nearly all
β-lactam containing antibiotics. The substrate-binding abilities of these
enzymes reside in the positioning of the two Zn(II) ions in the active site [3]. Of the three MBLs, NDM is the most
widespread in U.S. patients, and NDM continues to evolve with twenty-eight variants
identified at the time of authoring this manuscript [2,4]. Given the need to address
MBLs in clinical strains, there have been numerous recent reports on new MBL
inhibitors. There is also over 30 years of prior investigations into
β-lactamase inhibition [5].
Unfortunately, there are no clinically-relevant MBL inhibitors yet reported [6]. The differences among the active sites, and
the catalytic mechanisms, of these clinically-important enzymes have severely
limited the development of common, selective inhibitors that do not strip metal from
the active site in vivo [7,8].</p><p>Recently, Klinger et al. screened FDA-approved drugs and
reported eleven potential MBL inhibitors containing thiol moieties [9]. Of the original eleven identified in their study:
L-captopril, DL-thiorphan, tiopronin, and 2,3-dimercaprol exhibited inhibition of
the three clinically-relevant MBLs (NDM, VIM and IMP). Dimercaprol is an approved
drug against toxic metal poisoning. DL-thiorphan is the active metabolite in the
antidiarrheal drug Racecadotril [9].
L-captopril is an approved angiotensin-converting enzyme (ACE) inhibitor for the
treatment of hypertension, and the crystal structure of L-captopril complexed with
NDM-1 was reported in 2012 [10]. Tiopronin is
used as a treatment for metal poisoning and cystinuria [9]. Klinger et al. previously reported a
crystal structure of NDM-1 complexed with tiopronin and proposed that the mode of
inhibition is similar to that of L-captopril [9]. However, it was not possible for them to unambiguously discriminate
all possible conformations of the ligand in the NDM-1 active site allowing for
several proposed orientations (PDB ID 5A5Z).</p><p>Previous crystallographic studies suggest that there are two distinct binding
modes for these thiol-containing compounds. Either the thiol sulfur bridges the two
Zn(II) ions (as it does in the structure of the NDM-1/L-captopril complex) or the
thiol sulfur coordinates terminally to only one Zn(II) ion (as in the structure of
the NDM-1/tiopronin complex). It is not clear how thiorphan or dimercaprol bind to
the metal center in the MBLs, and in the case of tiopronin, conflicting results
exist. Raczynska et al. reported a reanalysis of the reported
crystal structure of the tiopronin complex [11]. They successfully modelled and refined two water molecules in the
active site replacing the tiopronin ligand from the original coordinates, after
several failed attempts to model any ligand there, either tiopronin or such buffer
components. They concluded that the observed electron density reported by Klinger
et al. did not support modeling any ligand in the active site
(PDB ID 5NBK) [12]. As we show below,
spectroscopy provides valuable information to address this difference. We have been
developing a "toolbox" approach to biophysical characterization to
ascertain the mechanism of inhibition, through a series of solution-based analyses
[13], and the discrepancy in
interpretation of X-ray diffraction data is a perfect example to illustrate its
utility. The studies presented below were designed to determine how tiopronin,
thiorphan, and dimercaprol (Figure 1) inhibit
NDM-1, and whether they are indeed good lead candidates for further elaboration and
study. The work flow used herein can readily be used to examine any MBL inhibitor
and is easily transferrable to other types of systems.</p><!><p>Tag-free ZnZn-NDM-1 was prepared as described previously [15]. Briefly, over-expressed NDM-1 was purified with
a single-step HisTrap column, using TEV protease to remove the
His6-tag, as previously described [14]. The resulting NDM-1 exhibited Km and
kcat values (5 ± 1 μM and 4.2
s−1), using chromacef as substrate, which are similar to
previous reports [2]. ICP-AES confirmed
that the resulting NDM-1 binds 1.9 ± 0.1 eq of Zn(II), also consistent
with previous reports [2] [15] [16].
CoCo-NDM-1 was prepared by direct addition of 2 eq of Co(II) to apo-NDM-1, as we
have reported previously [2].</p><!><p>NDM-1 (final concentration 8 Î¼M and final volume of 5 mL) in 50 mM
HEPES, pH 7.5, was mixed with the compound of interest at concentrations of
0–128 μM. After incubation for 30 min, the solutions were dialyzed
versus 500 mL of metal-free 50 mM HEPES, pH 7.5, overnight using dialysis tubing
with MWCO of 6000–8000, Fisherbrand. The Zn(II) content in the resulting
NDM-1 samples was determined ICP-AES.</p><!><p>Isothermal titration calorimetry (ITC) experiments were carried out using
a Nano ITC System (TA Instruments-Waters LLC., USA) with a 500 Î¼L cell.
All experiments were performed at 25 °C. Inhibitors were prepared by
diluting a stock solution (in 100 mM DMSO) with 20 mM HEPES, pH 7.5, containing
150 mM NaCl. Tag-free NDM-1 was also diluted in the same buffer. The ITC cell
was filled with 50 Î¼M NDM-1 (300 Î¼L), and the enzyme solutions
were titrated with 500 Î¼M L-captopril, DL-thiorphan, tiopronin, or
2,3-dimercaprol. The injection volume was 50 Î¼L, and the time between
injections was 210 s, allowing the inhibitor to bind. Kd values were
determined by fitting using NanoAnalyze (TA Instruments-Waters LLC, USA).</p><!><p>Aliquots from stock solutions (20 mM) of the thiol compounds were added
to 300 Î¼M CoCo-NDM-1 samples, and the samples were then incubated on ice
for 30 min. The UV-vis spectra of resulting samples between 250 and 800 nm were
collected on a PerkinElmer Lambda 750 UV/vis/NIR spectrometer. The blank
consisted of apo-NDM-1 (300 Î¼M) in 50 mM HEPES, pH 6.8, containing 150 mM
NaCl and 1 mM TCEP. Control spectra of an equimolar (600 Î¼M) mixture of
each inhibitor and aqueous Co(II) were recorded. The inhibitors were also
examined, in the absence of protein, as a function of pH. To obtain
pKa values, the absorptivity at Î»max was fitted
to Equation 1, a modified version
of the Henderson-Hasselbach equation, where c1 and c2
represent the limiting extinction coefficients of the high- and low-pH forms,
respectively [17].</p><!><p>ZnZn-NDM-1 (50 Î¼M) was buffer-exchanged, using three Bio-Rad spin
column wash tubes, into 100 mM ammonium acetate, pH 7.5. The eluted dizinc NDM-1
(50 Î¼M) was mixed with 500 Î¼M inhibitor and incubated for 10
minutes at room temperature. A Thermo Scientific LTQ Orbitrap XL™ hybrid
ion trap-orbitrap mass spectrometer equipped with a nano-electrospray ionization
(n-ESI) probe (Thermo Fisher Scientific, San Jose, CA, USA) with positive mode
protein detection was used to analyze the samples. The major parameters were
capillary temperature, 180 °C; sheath gas, 0; auxiliary gas, 0; sweep
gas, 0; and spray voltage, 1.1–1.9 kV; tube-lens, 150 V; capillary
voltage, 35 V. A full scan ranging from 1000 to 4000 (m/z) was used. The
resolution was set at 30,000. Automated gain control was set at 3 ×
104 in full scan, 1 × 104 in SIM, 1 ×
104 in MSn, and 3000 in zoom for ion trap, 3 ×
106 in full scan, 1 × 105 in SIM and 1 ×
105 in MSn for Fourier transform. The nESI source was equipped
with an offline unit (Cat # ES260), and the source was constructed based on
published work with modification. Briefly, a platinum wire (0.25 mm diameter)
was inserted in the center of the offline unit and in a pulled glass capillary
with a tip i.d. of about 1 Î¼m, produced in-house from a glass capillary
(i.d. 0.8 mm, o.d. 1.5 mm) using a micropipette puller (model P-87 Flaming/Brown
Micropipette Puller, Sutter Instrument Inc., USA). The sample solution (5
Î¼L) was loaded into the pulled glass capillary by an infusion syringe
(Thermo Scientific, USA). The platinum wire was inserted in the center of the
capillary. The position of the capillary tip was then adjusted to around 3 mm
away from the MS inlet.</p><!><p>Each NMR sample was buffered with 50 mM HEPES, pH 6.8, containing 150
mM NaCl and 10% D2O. Thiol inhibitors were dissolved in DMSO at high
concentration (50–100 mM) so they could be titrated in small (~5
Î¼L) aliquots. The inhibitor complex spectra presented here were obtained
from samples containing 2 molar equivalents of inhibitor per molar equivalent of
protein. Spectra were collected at 292 K on a Bruker ASX300 NMR spectrometer
operating at a frequency of 300.16 MHz. Spectra were collected using a frequency
switching method, applying a long, low power (270 ms) pulse centered at the
water frequency, followed by a high power 3 Î¼s pulse centered at 90 ppm
[18]. This method allows for
suppression of the water signal with enhancement of severely hyperfine-shifted
resonances. Spectra consisted of 30,000 transients of 16k data points over a 333
ppm spectral window (100 kHz, λaq ≈ 51 ms); signal
averaging took ~3 h per spectrum. The 1H NMR spectra of the
four inhibitors with Co(II), in 2.5-fold molar excess and in the absence of
protein, were recorded as controls.</p><!><p>EPR samples contained 1 mM CoCo-NDM-1, buffered with 50 mM HEPES, pH
7.5, containing 5 mM NaCl and ~10 % (v/v) glycerol, which served as a
glassing agent. Inhibitor complexes were prepared containing 2 molar equivalents
of inhibitor per molar equivalent of protein. Samples were loaded into EPR tubes
and degassed by repeated evacuation/purgation with N2 prior to data
collection. Spectra were collected on a Bruker EMX EPR spectrometer, equipped
with an ER4116-DM dual mode resonator (9.37 GHz, parallel; 9.62 GHz
perpendicular). The data were scaled so that the x-axes matched (perpendicular
mode field values were scaled by 9.37/9.62). Temperature control was
accomplished using an Oxford ESR900 cryostat and temperature controller. Other
spectral conditions: microwave power = 0.2 mW; field modulation = 10 G (100
kHz); receiver gain = 104; time constant/conversion time = 41 ms.</p><!><p>An initial model of NDM-1 was prepared from PDB ID 4exs [19] using the "prepack" module within
the Rosetta software suite. Initial three-dimensional coordinates for thiorphan
were generated using OpenBabel with the simplified molecular-input line-entry
system (SMILES) notation for thiorphan as input to generate a three-dimensional
structure-data file (SDF). A family of 512 conformations of thiorphan were
generated using OpenEye Omega version 3.1.2.2 [20] with the thiorphan SDF generated by OpenBabel [21]used as input. Rosetta ligand parameter files were
prepared from the Omega-generated thiorphan conformations using the automatic
RosettaLigand setup scripts packaged with Rosetta. Thiorphan was manually placed
in a starting pose near the NDM-1 active site prior to ligand docking. Docking
of thiorphan into NDM-1 was performed using Rosetta LigandDock [22,23].
Docking runs permitted side-chain and backbone flexibility with extra Chi1 and
aromatic-Chi2 rotamers and ligand translations up to ±5 Å along
the x, y, and z directions. A
total of 10,260 decoys were generated and scored. A representative pose,
selected as the lowest energy model of a cluster of 10 models based on the
overall Rosetta energy score, was selected using the Rosetta clustering app.</p><!><p>IC50 values for L-captopril,
D,L-thiorphan, tiopronin, and 2,3-dimercaprol binding to
NDM-1, previously reported by Klinger et al., [3] ranged from 1.3 to 84 Î¼M (Table 1; Figure
1). In an effort to expand on their findings, we examined the binding
affinities of the set of thiol-containing compounds by ITC. All four appeared to
form 1:1 complexes, and in general, the ITC-determined
KD values (see Table 1 and Supporting Information, Figure S1) were consistent with the
corresponding IC50, where tiopronin's was 5 to 10-fold larger
than the others. The data for thiorphan and captopril were sufficiently
well-fitted to extract thermodynamic parameters, and both gave enthalpies of
binding on the order of −30 kJ/mol (~ 7 kcal/mol). However, while
the data for captopril indicate a positive entropy of binding, presumably from
dehydration of the active site [24], the
data for thiorphan clearly indicate a negative entropy of binding, possibly due
to ordering of waters to solvate the thiorphan phenyl ring offsetting active
site dehydration [24]. This difference
may be due to the need to accommodate the hydrophobic pendant benzyl of
thiorphan, which is not present in captopril, and may represent a position to be
explored for further optimization.</p><!><p>To identify metal-stripping contributions, we incubated 8 Î¼M
ZnZn-NDM-1 with inhibitor concentrations ranging from 0–128 μM.
After incubation for approximately 30 minutes, the mixture was dialyzed for four
hours, to remove unbound Zn(II) and inhibitor. The resulting enzymes were
analyzed with ICP-AES to determine their remaining metal content (Figure 2) [14].
Inhibitors that exhibit a metal-stripping mechanism are expected to show reduced
metal content, while inhibitors that form a stable ternary complex should show
little or no decrease in metal content [8]. EDTA was used as a control example of a metal-stripping inhibitor,
while L-captopril served as an example of a ternary
complex-forming inhibitor [25].As can be
seen in Figure 2, exposure to increasing
concentrations of EDTA led to proportionate reductions in NDM-1 metal content,
while none of the thiol-containing inhibitors (L-captopril, DL-thiorphan,
tiopronin, or 2,3-dimercaprol) appreciably altered the enzyme's zinc
content, even at 16-fold excess. These data, along with preceding studies,
[14] [26] suggests that all four compounds form ternary complexes with
NDM-1 in this concentration range (< 10 Î¼M).</p><!><p>To gain insight into the nature of the ternary complexes, we first
turned to UV-visible spectroscopy. The presence of the thiol in the inhibitor
structures provides a potential internal spectroscopic handle, but also a
potential complication due to background absorption. Therefore, we first
measured the optical spectrum of each inhibitor as a function of pH, shown in
Supporting
Information, Figure
S2. In each case, at low-pH, where the protonated thiol is expected
to be the dominant form, little (thiorphan) or no (tiopronin, dimercaprol, and
captopril) absorption can be observed. However, again in each case, the spectra
showed the onset of absorption with increasing pH. As perhaps could expected,
the alkyl thiolates give rise to very low intensity features
(λmax = 436 nm, ε < 2
M−1cm−1, for both), while thiorphan,
with a pendant phenyl group, gives rise to a more intense absorption. Both
thiorphan (ε > 240 M−1cm−1)
and dimercaprol (ε > 2,400
M−1cm−1) show a higher energy
Î»max, in the UV, below 250 nm. Dimercaprol appears to show
a second feature at longer wavelength (~ 260 nm), that levels off at
ε ~ 2400 M−1cm−1, but the data
are insufficient to claim the capture of multiple ionizations. Fitting the data
shows that the pKa values range from 8.4 (tiopronin) to 8.5
(captopril) to 9.6 for thiorphan. The ionization(s) observed for dimercaprol
indicate a minimum pKa of 8.5, with the longer wavelength better
fitted at 9.4. For even the lowest of these values (8.4), the inhibitor thiols
must be nearly fully protonated at pH 7.5. The relatively high pKa of
the thiorphan thiol may help explain the negative entropy of binding, as it has
a tenfold higher affinity for its thiol proton. Most importantly, the background
spectra show that the enzyme-bound spectra presented below are unaffected by any
contribution from unbound inhibitor.</p><p>We next examined the inhibitors' effect on the optical spectra of
Co(II)-substituted NDM-1. As previously reported, CoCo-NDM-1 exhibits an intense
peak at 330 nm, attributed to a Cys-S to Co(II) LMCT, and a group of four ligand
field transitions at 510, 549, 615, and 640 nm, with comparable contributions
from both metal ions (Figure 3).[27,2,28] The addition of 2 eq of
EDTA to CoCo-NDM-1 leads to disappearance of both the LMCT and ligand field
transitions, consistent with the loss of both Co(II) ions, while captopril,
which has been shown to bridge the metal ions via the thiol sulfur, leads to
increased LMCT intensity and a significant shift the ligand field bands.[27] As shown in Figure 3, the addition of 2 eq of thiorphan resulted
in increased LMCT intensity, with a shift of Î»max to 345 nm
(ε = 1,800 M−1 cm−1). Consistent with
the relatively small KD from ITC (Table 1), the LMCT is nearly at its maximum with addition of the
first eq, increasing only 20% on addition of the second eq. Meanwhile, the
ligand field bands at 510 and 640 nm are reduced or eliminated when thiorphan
binds, while the central pair shift closer together (to 567 and 607 nm, ε
= 385 and 390 M−1 cm−1, respectively) and a
new band appears at 675 nm. Based on the similarity to the effect of captopril
on the spectrum of CoCo-NDM-1,[29] we
must infer that thiorphan binds in a similar fashion, with the thiol sulfur
occupying a bridging position between the two metal ions. In contrast, the
addition of tiopronin to CoCo-NDM-1 resulted in a more intense LMCT band,
without significantly affecting the ligand field transitions. Also consistent
with the ITC (Table 1), it is clear that
tiopronin does not saturate the active site as readily as thiorphan, as the LMCT
intensity increases more than 50% on addition of the second eq (from
~1,100 to 1,800 M−1cm−1, Fig. 3A). Meanwhile, the lack of any
significant effect on the ligand field region suggests it is terminally-bound
through the thiol sulfur, possibly replacing the terminal water molecule at the
Zn2 site.</p><p>Meanwhile, the addition of 2,3-dimercaprol to CoCo-NDM-1 results in
wholesale changes in the UV-vis spectra with strong absorption spanning 300 to
500 nm that largely obscures the ligand field transitions. However, closer
inspection of the ligand field region (see Figure
3B) shows that the longer wavelength ligand field transitions
disappear with the first addition of 2,3-dimercaprol, indicative of a
metal-stripping inhibitor at these concentrations. To gain further insight into
the interaction with 2,3-dimercaprol, we performed native mass spectrometry
(Figure S3). These
experiments, performed at similar concentrations (500 Î¼M vs. 300
Î¼M for the optical spectra), also showed evidence of metal stripping. On
exposure to 2,3-dimercaprol, the native-MS showed ZnZn-NDM-1 disappearing, with
the population shifting to the metal-free form. Importantly, we note that we
also observed a small portion of a binary complex between the metal-free enzyme
and dimercaprol but did not observe a ternary complex between the Zn-loaded
enzyme and the inhibitor. As 2,3-dimercaprol functions as a metal-stripping
agent at the necessarily high concentrations of the spectroscopic studies, we
will not present NMR and EPR data on dimercaprol-treated CoCo-NDM-1, which
largely showed reductions in signal intensity, without the observation of new
features.</p><!><p>EPR spectra for the CoCo-NDM-1/thiol complexes are shown in Figure 4. All of the spectra appear
quasi-axial in perpendicular mode (Figure
4A, B1⊥B0), with g⊥
~ 6.5 and g|| ~ 2.1. The width of the low-field,
perpendicular feature shows some modulation on addition of inhibitor. Tiopronin
causes the smallest perturbation, consistent with the UV-vis-derived suggestion
that it binds terminally to one metal ion. Somewhat surprisingly, given the
expectation that both thiorphan and captopril bind the NDM-1 active site in a
bridging fashion, captopril broadens the EPR signal, while thiorphan leads to
its sharpening. We believe this likely reflects differences in electron
relaxation rates, and may be a result of secondary interactions. All three
spectra are sufficiently altered to be considered indicative of binding to one
or both of the Co(II) ions, but a deeper analysis of the perpendicular mode
spectra is, at best, difficult [30].
However, previous studies on the di-Co(II) form of the related B1
metallo-α-lactamase, BcII, showed the spin coupling between the Co(II)
ions could readily be probed by parallel mode EPR (B1||B0)
[31].The parallel mode spectra of the
NDM-1-inhibitor complexes are compared to the resting enzyme in Figure 4B. All of the inhibitor complexes show the
same features as the resting enzyme, yet all show reduced signal intensity,
suggesting reduced spin coupling. This result is particularly apparent in the
negative feature near 800 G, and this feature has been correlated with coupling
strength in previous work [31]. While all
three inhibitors lead to reduced signal intensities, the level of attenuation at
800 G appears greater for thiorphan and captopril, consistent with the inhibitor
replacing the bridging hydroxyl with the thiol sulfur. These most likely are
facilitated by the thiol being protonated, which allows for protonation of the
bridging hydroxyl, allowing it to then leave as water. The lesser attenuation
seen in the tiopronin complex can be attributed to the addition of a second
electron-rich sulfur donor, which should, in turn, weaken the bonds to the
bridging group.</p><!><p>To gain more atomic level detail, we examined the 1H NMR
spectra of the thiol inhibitor complexes. The data, presented in Figure 5, are shown in three sections to allow them to
be scaled differentially; the as-collected spectra are presented in Figure S4. None of the
inhibitors alone form complexes with Co(II) free in solution (see Figure S5). The spectrum
for CoCo-NDM-1 (bottom trace in Figure 5)
has been previously reported and assigned, with solvent-exchangeable resonances
at 78, 73, and 65 ppm, assigned to the exchangeable NH protons of three
Co(II)-bound His residues at the Zn1 site. Meanwhile, peaks at 47 (aspartate
β-CH2) and 107 (histidine NH) ppm along with the doublet
near 170 ppm (cysteine β-CH2) were attributed to the Zn2 site
[27].</p><p>The addition of tiopronin to CoCo-NDM-1 leads to the appearance of two
new resonances to low field (> 140 ppm), consistent with coordination of
a second thiolate, and one other new resonance at 60 ppm, which may arise from
the terminal methyl of tiopronin. Aside from what appears to be a shift in the
cysteine dihedral, based on a change in the separation of their NMR lines, and
one fairly large change in the upfield region (~ 30 ppm, seen in all
three inhibitor complexes, discussed below), tiopronin causes remarkably little
else to change relative to the uncomplexed enzyme. This, again, is consistent
with terminal coordination of tiopronin through its thiol sulfur.</p><p>We have previously reported the 1H NMR spectrum of CoCo-NDM-1
in complex with captopril, though the data were not discussed [14]. Addition of captopril to CoCo-NDM-1 has profound
effects on signals attributed to both metal ions. These effects are evidenced by
~ 40 ppm chemical shift attenuation for two of the three Zn1 His ligands,
as well as new resonances above 170 ppm, a splitting of the symmetry-equivalent
aspartate β-CH2 (~ 47 ppm) and a sizable rearrangement
of the resonances upfield of the diamagnetic window (Î´ < 0), which
are commonly attributed to second sphere interactions with the Zn2 ion (see
Figure S6) [32]. As shown in Figures 5 and S6, thiorphan produces many similar
changes, including a set of new resonances above 170 ppm, indicative of
coordination of the thiol, and similar changes to the aspartate and histidine
resonances. Also present is one new broad resonance near 80 ppm which may come
from the benzylic carbon of thiorphan, or perhaps a subset of the phenyl
protons.</p><!><p>To further explore the potential binding mechanism of this set of
thiol-containing compounds as effective NDM-1 inhibitors, the computational
modelling of DL-thiorphan, tiopronin, and 2,3-dimercaprol were
attempted. The representative docking pose for DL-thiorphan
(Figure 6B) features the sulfur
positioned in a bridging position between the two active site zinc ions and the
thiorphan phenyl ring positioned to enable π-stacking with the side chain
imidazole ring of His122. Positioning of the acetyl-glycine substituent squarely
within the active site channel opens up opportunities for solvent-mediated
hydrogen bonds to surrounding residues; however, since the docking runs did not
include explicit solvent, these potential interactions were not observed.
Consistent with the EPR, NMR, and UV/visible spectroscopic results, the docking
pose for tiopronin (Figure 6C) features
binding to a single zinc ion, Zn2, through the tiopronin sulfur. Additional
interactions between NDM-1 and tiopronin include packing of the tiopronin methyl
group against the methylene groups of His189 and Cys208, and hydrogen bonds to
the side chain ε-amino of Lys211 and the backbone amide proton of Asn220,
each mediated by carbonyl oxygen atoms from tiopronin. Interestingly, the NDM-1
active site loops proximal to the pocket between His189 and Cys208 provide a
rich source of hydrogen bond donors that could interact with tiopronin. In
contrast with the binding modes observed for thiorphan and tiopronin, the
docking pose for dimercaprol (Figure 6D)
indicates interaction of two sulfur atoms with the active site zinc ions. One
sulfur occupies a bridging position between the two zinc ions while the second
sulfur coordinates to Zn2. Partly owing to the small size of dimercaprol,
additional interactions with NDM-1 are limited to a potential hydrogen bond
between the dimercaprol hydroxyl oxygen and the side chain amide of Asn220.</p><!><p>The sum of the methods utilized here have been developed by our labs
and others over a number of years, specifically for the study of MBLs. The
rationale for using such a wide array of approaches is summarized in Table 2. Each technique contributes
through a core strength, and each provides a unique piece of the puzzle.
However, each technique has limitations, and the complete understanding of
the mechanism of an inhibitor requires multiple probes. We feel this
progression is important. Equilibrium dialysis can quickly identify
inhibitors that function via metal stripping, and, as was illustrated here,
because it is performed at a substantially lower concentration than the
spectroscopic studies, can validate potential hits that might otherwise be
overlooked. A potential limitation is that multiple rounds of dialysis may
lead to metal removal, generating a false positive with regards to metal
stripping. In addition, this technique, by itself, cannot determine whether
a ternary complex forms or not. Similarly, native mass spectrometry offers
an avenue to determine the presence of a ternary complex, although, in most
cases, it is not quantitative. ITC provides binding affinities and
thermodynamic parameters associated with binding, using moderate amounts of
enzyme and inhibitor. However as shown with dimercaprol in this study,
dissociation constants can be obtained, even for inhibitors that strip metal
from the MBL. UV/vis spectroscopic studies provide a rapid, albeit low
resolution method for identifying interactions between the inhibitor and the
active site metal ions. This technique, along with EPR and NMR discussed
below, requires the use of large quantities of non-native,
Co(II)-substituted MBL. EPR and NMR provide higher resolution data, with EPR
directly interrogating the active site metal ions and NMR probing their
interactions with the protons of functional groups that coordinate to the
active site metal ions (both constitutive and inhibitor-derived). Finally,
computational docking efforts offer an opportunity to propose specific
binding modes which can be evaluated for agreement with the biophysical data
from UV/Vis spectroscopy, EPR, and NMR, and potential positions that can
benefit from further elaboration.</p><p>The approach we employed was needed to investigate with accuracy the
mechanisms utilized by these thiol-containing compounds to inhibit MBLs. Our
structuring of this approach, beginning with simpler experiments utilizing
low enzyme and inhibitor concentrations prior to conducting more complex
spectroscopic experiments requiring higher quantities of protein and
inhibitor, offers an efficient route to identify those inhibitors that have
the potential for clinically relevancy. In contrast, conventional studies
examining enzymatic activities of MBLs, alone or in combination with
cell-based minimum inhibitory concentration assays, identify the capability
of compounds to inhibit MBL activities yet provide little understanding of
their inhibition mechanisms. Using our integrated biophysical approach, we
determined that DL-thiorphan exhibits a number of similarities to
L-captopril, including the mode of ternary complex formation through use of
a bridging sulfur between the two active site zinc ions, and thus likely
uses a similar inhibition mechanism. In contrast, the binding modes for
tiopronin and 2,3-dimercaprol are dissimilar to both thiorphan and
captopril. Our studies with tiopronin also match up well with a recent
reanalysis of the X-ray diffraction data for NDM-1 in complex with tiopronin
[11] that refutes the originally
proposed binding mode [26]. In
particular, this result shows that with two conflicting views of the
previously-reported tiopronin crystal structure, our toolbox of biochemical
and spectroscopic methods allowed us to confirm the most recent set of
findings [11]. We have suggested an
innovative approach that allows for a more thorough determination of the
mechanism of inhibition of MBL inhibitors using multiple techniques in
combination [13]. Although the four
inhibitors tested here were previously identified as potential
clinically-relevant inhibitors of MBLs, our data suggest additional research
with a broad toolbox of approaches is needed to identify novel MBL
inhibitors with potential for clinically-relevant MBL inhibition.</p>