Exploring the influence of the protein environment on metal-binding pharmacophores

Article abstract of DOI:10.1021/jm500984b

The binding of a series of metal-binding pharmacophores (MBPs) related to the ligand 1-hydroxypyridine-2-(1H)-thione (1,2-HOPTO) in the active site of human carbonic anhydrase II (hCAII) has been investigated. The presence and/or position of a single methyl substituent drastically alters inhibitor potency and can result in coordination modes not observed in small-molecule model complexes. It is shown that this unexpected binding mode is the result of a steric clash between the methyl group and a highly ordered water network in the active site that is further stabilized by the formation of a hydrogen bond and favorable hydrophobic contacts. The affinity of MBPs is dependent on a large number of factors including donor atom identity, orientation, electrostatics, and van der Waals interactions. These results suggest that metal coordination by metalloenzyme inhibitors is a malleable interaction and that it is thus more appropriate to consider the metal-binding motif of these inhibitors as a pharmacophore rather than a "chelator". The rational design of inhibitors targeting metalloenzymes will benefit greatly from a deeper understanding of the interplay between the variety of forces governing the binding of MBPs to active site metal ions.

Full text of DOI:10.1021/jm500984b

Article
pubs.acs.org/jmc
Open Access on 08/12/2015
Exploring the Influence of the Protein Environment on Metal-Binding
Pharmacophores
David P. Martin,† Patrick G. Blachly,† J. Andrew McCammon,†,‡,§ and Seth M. Cohen*,†
†Departments of Chemistry and Biochemistry, ‡Pharmacology, and §Howard Hughes Medical Institute, University of California, San
Diego, 9500 Gilman Drive, MC 0358, La Jolla, California 92093, United States
S
* Supporting Information
ABSTRACT: The binding of a series of metal-binding pharmacophores (MBPs)
related to the ligand 1-hydroxypyridine-2-(1H)-thione (1,2-HOPTO) in the
active site of human carbonic anhydrase II (hCAII) has been investigated. The
presence and/or position of a single methyl substituent drastically alters inhibitor
potency and can result in coordination modes not observed in small-molecule
model complexes. It is shown that this unexpected binding mode is the result of a
steric clash between the methyl group and a highly ordered water network in the active site that is further stabilized by the
formation of a hydrogen bond and favorable hydrophobic contacts. The affinity of MBPs is dependent on a large number of
factors including donor atom identity, orientation, electrostatics, and van der Waals interactions. These results suggest that metal
coordination by metalloenzyme inhibitors is a malleable interaction and that it is thus more appropriate to consider the metal-
binding motif of these inhibitors as a pharmacophore rather than a “chelator”. The rational design of inhibitors targeting
metalloenzymes will benefit greatly from a deeper understanding of the interplay between the variety of forces governing the
binding of MBPs to active site metal ions.
alternative metal-binding scaffolds, which have been evaluated
against a wide range of metalloprotein targets.10−16
INTRODUCTION
■
Metalloenzymes, those which require a metal ion cofactor to
function, are an emerging class of therapeutic targets.1,2
Inhibitors targeting carbonic anhydrases (CAs, Zn2+-depend-
ent),3 angiotensin converting enzyme (ACE, Zn2+-dependent),4
histone deacetylases (HDACs, Zn2+-dependent),5 5-lipoxyge-
nase (5-LO, Fe2+/3+-dependent),6 and HIV integrase (HIV1-IN,
Mg2+-dependent)7 are clinically approved for the treatment of
glaucoma, hypertension, cutaneous T-cell lymphoma, asthma,
and HIV infection, respectively. Furthermore, inhibitors
targeting these and other metalloenzymes are in various stages
of clinical trials for the treatment of a wide variety of ailments
including bacterial and viral infection, hypertension, cancer, and
inflammation. For the most part, metalloenzyme inhibitors
contain pharmacophores that bind directly to the catalytic
metal ion of the target.8 Given the extensive structural
optimization that goes into clinical candidates, it is surprising
how little diversity there is in the metal-binding pharmaco-
phores (MBPs) utilized in metalloenzyme inhibitor design;
thiols, carboxylic acids, phosphates, and hydroxamic acids have
historically dominated the chemical landscape for binding active
site metal ions. However, this has begun to change as illustrated
by the emergence of inhibitors targeting HIV1-IN that utilize
innovative hetrocyclic MBPs as well as HDAC inhibitors
containing the unconventional N-(2-aminophenyl)benzamide
MBP.9 The latter example is especially interesting because it is a
scaffold that would not be considered a strong metal-binding
motif but nonetheless strongly inhibits certain HDAC isoforms
by maximizing other interactions with the active site. In
addition, for nearly a decade, our laboratory has proposed
The utilization of novel MBPs has the potential to improve
both the target specificity and pharmacokinetic properties of
metalloenzyme inhibitors, thereby improving the clinical
success rate of this class of therapeutics. Numerous studies
have demonstrated that MBP optimization can aid in designing
selective and/or potent inhibitors for a variety of metal-
loenzyme targets.17−22 The use of structure-aided design can be
invaluable to this process, but due to the complexity of the
metal−inhibitor interaction, computational protocols to accu-
rately predict the binding mode and potency of MBPs have
been elusive.23−25 Consequently, in the absence of explicit
structural data, assumptions (e.g., bond lengths and coordina-
tion geometry) must be made about the MBP−metalloenzyme
interaction. In some cases, these parameters have been
estimated based on the structure of MBPs bound to small-
molecule model complexes, but these models do not account
for the constrained environment and nuanced features found in
a metalloenzyme active site.11,26−30 Consistent with the
aforementioned limitations, previous studies have shown that
the coordination modes of α-mercaptoketone (mono- vs
bidentate coordination), N-hydroxyurea (N-hydroxyl vs car-
bonyl monodentate coordination), and 3-hydroxy-(1H)-
pyridin-2-one (orientation of bidentate coordination) MBPs
can be changed based on relatively subtle changes in the
structure of the inhibitor backbone,31−33 an effect that would
not be recapitulated in model compounds.
Received: July 1, 2014
Published: August 12, 2014
© 2014 American Chemical Society
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Because of many characteristics including its rigid structure,
stability, and ease of crystallization, human carbonic anhydrase
II (hCAII) is an excellent protein model system for examining
MBP−metalloenzyme interactions.34 The active site consists of
a His3-bound Zn2+ ion that sits in a relatively solvent-exposed
depression (Figure 1). Arylsulfonamide MBPs are used in CA
reported to be a moderate inhibitor of hCAII, binding in a
bidentate fashion to the active site Zn2+ ion (Supporting
Information, Figure S2), and derivatives with substituents on
the ring of the ligand can be readily synthesized.36 We find that
a methyl group appended to the 1,2-HOPTO scaffold in a
position analogous to that of TM results in the MBP (6-CH3-
1,2-HOPTO) adopting a similar monodentate coordination
mode. Furthermore, we show that the unexpected coordination
modes of these MBPs are likely the result of a significant
energetic penalty for disrupting the highly ordered water
network within the active site and are stabilized by favorable
interactions with the hydrophobic wall of the active site. The
relationship between metal coordination and other MBP−
metalloenzyme interactions is examined, showing that no single
interaction is dominant, but rather that the binding of these
molecules is influenced by a combination of a number of
interdependent forces. The results show that canonical metal
“chelators” used in metalloenzyme inhibitors are better
described as malleable pharmacophores, hence our suggestion
of the term “metal-binding pharmacophore”.
Figure 1. Structure of hCAII (PDB 3KS3). The active site Zn2+ ion
(shown in bronze) sits at the bottom of a cone-shaped depression
containing both hydrophobic and hydrophilic walls.
inhibitors and are prime examples of the effect that the active
site environment can have on enhancing MBP binding.
Although the sulfonamide MBP shows little activity against
other metalloprotein targets, it has very high activity against
CAs, which has been attributed to the MBP being optimally
positioned for hydrogen bonding with active site residues upon
metal binding (Figure 2A). Recently, we reported that in the
RESULTS
■
Methyl 1,2-HOPTO Derivatives. Previous studies have
shown that a single methyl substituent on the 3-hydroxy-(4H)-
pyran-4-thione MBP can alter its coordination mode in the
active site of hCAII.35 It was hypothesized that because the
steric restrictions of the active site force MBPs into nonideal
coordination, other ligand−protein interactions could influence
the coordination mode. To further understand these effects, a
series of methyl derivatives of the 1,2-HOPTO scaffold were
synthesized (Figure 3) and their inhibitory activity against
hCAII was investigated (Table 1). The previously reported
crystal structure of 1,2-HOPTO bound to hCAII shows that the
MBP coordinates the active site Zn2+ ion in a bidentate fashion,
resulting in a distorted trigonal bipyramidal geometry.36 The
sulfur atom of 1,2-HOPTO acts as an equatorial donor while
the oxygen atom acts as an axial donor, orienting the sulfur
toward the hydrophobic wall of the active site (see Supporting
Information, Figure S2). Derivatives of 1,2-HOPTO with a
methyl group positioned toward the hydrophobic wall (3-CH3-
1,2-HOPTO and 4-CH3-1,2-HOPTO) show over a 10-fold
increase in potency over unsubstituted 1,2-HOPTO (Table 1).
When the substituent is in a position that, based on the 1,2-
HOPTO crystal structure, would not be oriented toward either
active site wall (e.g., 5-CH3-1,2-HOPTO), inhibition is only
slightly improved. Importantly, when a methyl group is
positioned toward the hydrophilic side of the active site (6-
CH3-1,2-HOPTO), significantly weaker inhibition is observed
(Table 2) when compared to 1,2-HOPTO.
Figure 2. Diagram of MBP binding modes in the active site of hCAII.
(A) Aryl sulfonamides are positioned for hydrogen bonding upon
metal coordination. (B) 1-Hydroxypridine-2-(1H)-thione (1,2-
HOPTO, PDB 3M1K) acts as a bidentate ligand. (C) The MBP
allothiomaltol (PDB 4MLX) also shows bidentate coordination, but its
isomer thiomaltol (D, PDB 4MLT), which differs only in the position
of a methyl group, adopts monodentate coordination.
To understand the inhibition data reported in Table 2, the
crystal structures of 3-CH3-, 4-CH3-, and 5-CH3-1,2-HOPTO
bound in the active site of hCAII were determined. All of these
derivatives show bidentate coordination by the MBPs (Figure
4), identical to that of unsubstituted 1,2-HOPTO. The methyl
group of 3-CH3-1,2-HOPTO makes hydrophobic contacts with
the side chains of Val143, Leu198, and Val12 (see Supporting
Information, Figure S4), while the methyl group of 4-CH3-1,2-
HOPTO is positioned slightly further out of the active site,
interacting with Val121, Leu198, and Phe131 (see Supporting
Information, Figure S5). As predicted, the methyl group of 5-
CH3-1,2-HOPTO does not make any significant contacts (<4.5
Å) with the hydrophobic wall; the only active site residue in
active site of hCAII, 3-hydroxy-(4H)-pyran-4-thione derivatives
differing only in the presence or position of a single methyl
group show different binding modes (Figure 2B−D), including
unexpected monodentate coordination to the Zn2+ ion by
thiomaltol (TM, Figure 2D).35
To understand the origin of the unique coordination mode
adopted by TM in the active site of hCAII and, more generally,
gain greater insight into the influence of active site environ-
ments on MBP−metalloprotein interactions, this study
examines the binding of a series of related MBPs based on 1-
hydroxypyridine-2-(1H)-thione (1,2-HOPTO, Figure 2) to
hCAII. This MBP was chosen because it has been previously
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Figure 3. Structures and naming of MBPs.
Table 1. Ki Values (μM) of Methyl Derivatives of 1,2-HOPTO against hCAII
compd
Ki
compd
Ki
1,2-HOPTO
510 90
5-CH3-1,2-HOPTO
6-CH3-1,2-HOPTO
4,6-CH3-1,2-HOPTO
260 20
2300 200
950 100
3-CH3-1,2-HOPTO
4-CH3-1,2-HOPTO
48
38
5
5
Table 2. Ki Values (μM) of 1,2-HOPTO Derivatives with
Larger Substituents against hCAII
compd
Ki
compd
Ki
3-CF3-1,2-HOPTO
4-CF3-1,2-HOPTO
5-CF3-1,2-HOPTO
64
14
5
3
6-CF3-1,2-HOPTO
2,1-HIQTO
>10000
2300 200
950 100
270 30
1,2-HOQTO
Figure 5. Crystal structure of 6-CH3-1,2-HOPTO (A) bound to
hCAII shows monodentate coordination while that for 4,6-CH3-1,2-
HOPTO (B) shows that bidentate coordination is stabilized by the
presence of a methyl group in the 4-position. The |2Fo − Fc| map
(1.5σ) is shown in gray for the Zn2+ ion and protein residues while the
omit |Fo − Fc| map (3.5σ) is shown in red for the ligands. For clarity,
only the metal active site is shown.
Figure 4. Crystal structures of 3-CH3-1,2-HOPTO (A), 4-CH3-1,2-
HOPTO (B), and 5-CH3-1,2-HOPTO (C) bound to hCAII. All show
bidentate coordination similar to unsubstituted 1,2-HOPTO (Support-
ing Information, Figure S2). The |2Fo − Fc| map (1.5σ) is shown in
gray for the Zn2+ ion and protein residues while the omit |Fo − Fc| map
(4.0σ) is shown in red for the ligands. For clarity, only the metal active
site is shown.
Supporting Information, Figure S7). The change in coordina-
tion also places 6-CH3-1,2-HOPTO closer to the hydrophobic
wall so that the ring of the ligand makes contacts with Val121
and Val143 in addition to an improved interaction with Leu198
(∼0.2 Å closer) relative to unsubstituted 1,2-HOPTO (see
Supporting Information, Figure S7). Because a methyl group in
the 6-position appears to destabilize bidentate coordination,
4,6-CH3-1,2-HOPTO, which has methyl groups in the 4- and 6-
position, was synthesized. While this MBP shows more than a
20-fold decrease in affinity relative to 4-CH3-1,2-HOPTO, the
crystal structure of 4,6-CH3-1,2-HOPTO bound to hCAII
shows that bidentate coordination is maintained with the 6-
methyl group extending toward the hydrophilic region of the
active site (Figure 5).
close proximity to the methyl group is the side chain amide of
Gln92 (3.7 Å, see Supporting Information, Figure S6).
The crystal structure of 6-CH3-1,2-HOPTO bound in the
active site of hCAII reveals that its decreased inhibitory activity
is accompanied by a shift to monodentate coordination in the
active site of hCAII (Figure 5A). The Zn−S distance (2.3 Å) is
consistent with those in previously reported structures of
monodentate sulfur donors bound to active site Zn2+ ions.37
The hydroxyl group, which is 3.5 Å from the metal ion, remains
oriented toward the hydrophilic side of the active site, forming
a close interaction with the side chain of Thr200 (2.9 Å,
Larger Hydrophobic Substituents. The relatively
solvent-exposed methyl group of 5-CH3-1,2-HOPTO and the
monodentate coordination of 6-CH3-1,2-HOPTO when bound
to hCAII suggest that there is a significant penalty for swapping
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the position of the coordinating atoms of 1,2-HOPTO. That is,
rotation of the MBP by 180° so that the oxygen atom is on the
hydrophobic side of the active site and the sulfur atom is on the
hydrophilic side would position the methyl groups of these two
ligands to make favorable hydrophobic interactions similar to
those made by 3-CH3-1,2-HOPTO and 4-CH3-1,2-HOPTO.
However, this change in donor atom position is not observed.
We reasoned that if functional groups could be added to the
1,2-HOPTO scaffold that improve these hydrophobic inter-
actions, this barrier might be overcome for ligands with
substitutions in the 5- or 6-position. With this goal in mind, 1,2-
HOPTO ligands were synthesized with trifluoromethyl and aryl
groups appended (Figure 3). A slight decrease in potency is
observed for 3-CF3-1,2-HOPTO relative to its methyl analogue
(64 vs 48 μM), while the same substitution in the 4-position
leads to an improvement in potency (14 μM for 4-CF3-1,2-
HOPTO vs 38 μM for 4-CH3-1,2-HOPTO). The inhibitory
activity of 5-CF3-1,2-HOPTO is essentially unchanged from the
methyl analogue, while all activity is lost with a trifluoromethyl
group in the 6-position (6-CF3-1,2-HOPTO). Addition of a
second, fused aromatic ring to the 3/4-positions of the 1,2-
HOPTO scaffold also results in a significant increase in binding
affinity (2-hydroxyisoquinoline-1-(2H)-thione, 2,1-HIQTO, Ki
= 15 μM) but results in slightly diminished activity when
appended to the 5/6-positions (1-hydroxyquinoline-2-(1H)-
thione, 1,2-HOQTO, Ki = 730 μM).
The binding modes of 3-CF3-1,2-HOPTO and 4-CF3-1,2-
HOPTO to hCAII were determined and were found to be
similar to their corresponding methyl analogues (Supporting
Information Figures S8 and S9). The trifluoromethyl groups are
highly ordered in both structures, suggesting that their
interactions with the hydrophobic wall of the active site are
quite favorable. The crystal structure of 5-CF3-1,2-HOPTO
bound to hCAII shows two different binding modes, each with
∼50% occupancy (see Supporting Information, Figure S10). In
the first, the ligand is in fact “flipped” 180° and coordinated in a
bidentate fashion with the sulfur atom on the hydrophilic side
of the active site and the oxygen atom closer to the
hydrophobic wall. This switch in donor arrangement relative
to the other 1,2-HOPTO derivatives places the trifluoromethyl
group of 5-CF3-1,2-HOPTO in the same position as that of 4-
CF3-1,2-HOPTO (Figure 6). The S−Zn distance (2.2 Å) is
much shorter than the O−Zn distance (2.6 Å), and the
coordination geometry remains trigonal bipyramidal with the
oxygen atom as an axial donor. In the second binding mode,
there appears to be no metal coordination, as both the S−Zn
distance (3.4 Å) and O−Zn distance (4.3 Å) are too long for
the donors to have any significant interaction with the Zn2+ ion.
Instead of metal coordination, the oxygen atom sits in a
hydrophilic pocket lined by hydrogen bond donors including
the backbone amide nitrogen of Thr199 (3.0 Å) and the
backbone amide nitrogen (3.2 Å) and side chain (3.4 Å) of
Thr200 (see Supporting Information, Figure S10). The
position of this oxygen atom does not correspond to that of
a water molecule in the inhibitor-free structure of hCAII. In this
orientation, the ring of 5-CF3-1,2-HOPTO is positioned to
make contacts with Val121 (3.4−4.0 Å) and Leu198 (3.9 Å),
while the trifluoromethyl group interacts with Phe131 (3.4−4.0
Å).
The crystal structure of 2,1-HIQTO bound to hCAII shows
coordination similar to 1,2-HOPTO (see Supporting Informa-
tion, Figure S11). Consistent with the significant increase in
potency relative to the unsubstituted 1,2-HOPTO MBP, this
binding mode positions the fused aromatic ring for extensive
interactions with the hydrophobic wall of the active site
including Val 143 (3.9 Å), Val121 (3.5−4.0 Å), Leu198 (3.7−
4.0 Å), and Phe131 (4.0 Å). Attempts at soaking 1,2-HOQTO
into the active site of hCAII led to diffuse electron density that
suggested two binding modes similar to those of 5-CF3-1,2-
HOPTO; the complex could not be adequately modeled, likely
due to a combination of the low affinity and solubility of the
ligand as well as disordered binding (data not shown).
4-CH3-1,2-HOPTO Derivatives with Altered Donor
Sets. The divergent affinities and binding modes observed
for 6-CH3-1,2-HOPTO and 5-CF3-1,2-HOPTO indicate that
the coordinative ability of the MBP does not completely
dominate the strength and geometry of binding. To examine
this, MBP derivatives of 4-CH3-1,2-HOPTO with alternative
donor sets were synthesized (Figure 3). 2-Mercapto-4-
methylpyridine (4-CH3-2MPyr) contains the same sulfur
donor as 4-CH3-1,2-HOPTO but lacks the oxygen donor
and, as a result, is ∼200-fold less active against hCAII (Ki = 7.0
mM). The corresponding pyridine-N-oxide (4-CH3-PyrNO)
with the sulfur donor removed shows no measurable inhibition
of hCAII. The 1-hydroxypyridin-2-(1H)-one analogue (4-CH3-
1,2-HOPO), which has an oxygen donor instead of sulfur, is
more than 30-fold less potent than 4-CH3-1,2-HOPTO. These
results suggest that although the MBP interactions are
malleable, maintaining strong coordination to the metal ion is
important for good inhibitory activity.
Much like its thione analogue, 4-CH3-1,2-HOPO acts as a
bidentate ligand to the Zn2+ ion of hCAII (see Supporting
Information, Figure S12A), with the nitrogen-bound oxygen as
the axial donor (2.1 Å) and the carbonyl oxygen as the
equatorial donor (2.2 Å). This is the reverse of the binding
mode predicted by the TpPh,Me model complex in which the
carbonyl oxygen is the axial donor. The MBP is positioned such
that the methyl group forms contacts with the hydrophobic wall
of the active site similar to those made by 4-CH3-1,2-HOPTO.
A water molecule sits above the donor atoms, interacting with
the Zn2+-bound carbonyl oxygen of 4-CH3-1,2-HOPO (2.8 Å)
as well as both the backbone amide NH (2.9 Å) and side chain
(2.8 Å) of Thr199. This water molecule is not present in any of
the bidentate 1,2-HOPTO structures. The side chain of Thr200
is also rotated such that there is no interaction between it and
the N-hydroxyl group of 4-CH3-1,2-HOPO (4.5 Å).
Figure 6. Two views of the crystal structure of 5-CF3-1,2-HOPTO
(carbon atoms in magenta) bound to hCAII show “flipped”
coordination relative to 4-CF3-1,2-HOPTO (carbon atoms in
green), positioning the CF3 groups similarly in the active site. Analysis
of the Zn2+ coordination shows nearly ideal trigonal bipyramidal
geometry. Conserved water molecules in the hCAII active site are
shown as red spheres.
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The crystal structure of 4-CH3-2MPyr bound to hCAII
shows that, despite the lack of an oxygen donor, the ligand is
oriented in the active site in a very similar position to that of 4-
CH3-1,2-HOPTO, with the sulfur atom bound to the Zn2+ ion
(2.3 Å) and the methyl group interacting with the hydrophobic
wall (see Supporting Information, Figure S12B). The
endocyclic nitrogen atom does not interact with active site
protein residues but is in the proximity of a water molecule (2.8
Å) that in turn interacts with the side chains of both Thr199
(2.8 Å) and Thr200 (2.7 Å). This water molecule does not
appear in either inhibitor-free hCAII (PDB 3KS3) or in the
structures of other monodentate inhibitors bound in the active
site.
Although they are structurally similar to 4-CH3-2MPyr,
aromatic thiols such as thiophenol and 2-mercaptophenol (TP
and 2MP, Figure 3) have been previously reported to inhibit
hCAII with Ki values in the low micromolar range.38 To
determine whether the interactions perturbing 1,2-HOPTO
binding apply to other MBPs containing similar donor sets,
methyl derivatives of TP and 2MP analogous to 4-CH3-1,2-
HOPTO were obtained and their binding to hCAII
investigated. While 3-CH3-TP has similar inhibitory activity
to unsubstituted TP (Ki = 2.1 μM vs 3.5 μM), the 4-methyl
analogue of 2MP is significantly more potent (0.52 vs 3.1 μM).
Remarkably, the activity of 4-CH3-2MP is the same as
benzenesulfonamide (BSA, Ki = 0.49 μM),39 the parent MBP
of all FDA-approved CA inhibitors, illustrating the benefit that
a rational approach to MBP design that includes both metal
coordination and MBP−protein interactions can have.
The crystal structure of 3-CH3-TP to hCAII reveals
monodentate coordination to the Zn2+ ion similar to that
previously reported for 2MP (see Supporting Information,
Figure S13), and the binding mode is very similar to that of 6-
CH3-1,2-HOPTO. The methyl group does not interact with the
hydrophobic wall of the active site and is relatively solvent
exposed aside from a close contact (3.8 Å) with the side chain
of Gln92. However, the aromatic ring of the ligand makes
extensive contacts with Val121 (3.6−3.9 Å), Val143 (3.8 Å),
and Leu198 (3.8−4.0 Å). The binding of 4-CH3-2MP is
essentially the same, but the hydroxyl group extends toward the
hydrophobic wall, making additional contacts with Val143 and
Trp209 (see Supporting Information, Figure S14).
Table 3. Decomposition of Bonding Energies for 1,2-
HOPTO Derivatives Bound to the TpCZn Scaffold
substituent
−
4-CH3
4-CF3
a
c
“ideal”
ΔEelst
ΔEorb
−257.6
−101.1
95.0
−259.9
−103.3
96.4
−242.6
−99.0
91.1
d
e
ΔEsteric
f
ΔEtot
−263.7
−266.9
−250.5
b
“protein”
ΔEelst
ΔEorb
ΔEsteric
ΔEtot
−242.5
−97.1
84.4
−245.4
−99.1
86.1
−228.2
−95.5
81.1
−255.2
−258.3
−242.5
a
Results from EDAs performed on “ideal” geometries, corresponding
b
to freely optimized TpCZn(MBP) complexes. Results from EDAs
performed with the MBP in complex with TpCZn constrained to the
orientation observed in the crystal structure of 1,2-HOPTO bound to
c
hCAII. The electrostatic interaction energy between TpCZn and the
d
MBP. The orbital interaction energy between TpCZn and the MBP.
This term in the EDA implicitly accounts for charge transfer,
e
polarization, and electron bonding effects.69,71 The Pauli repulsion
f
energy associated with the bonding of TpCZn and the MBP. The total
bonding energy between TpCZn and the MBP: ΔEtot = ΔEelst + ΔEorb
69,71
+ ΔEsteric
.
Further details regarding the EDAs are included in the
Supporting Information.
CH3-1,2-HOPTO stems from both its more favorable electro-
static (ΔEelst = −259.9 kcal mol−1) and orbital (ΔEorb = −103.3
kcal mol−1) interactions with TpCZn. By contrast, the
TpCZn(4-CF3-1,2-HOPTO) complex has less stabilization
from electrostatic (ΔEelst = −242.6 kcal mol−1) and orbital
interaction (ΔEorb = −99.0 kcal mol−1) effects. This trend in
bonding energy observed for 1,2-HOPTO derivatives illustrates
the respective electron donating and electron withdrawing
effects of the methyl and fluoromethyl substituents. Indeed,
charge distributions obtained for TpCZn(4-CH3-1,2-HOPTO)
and TpCZn(4-CF3-1,2-HOPTO) complexes show the latter to
possess less negative charge on its S and O donor atoms (see
Supporting Information, Figure S16).
The ordering of 1,2-HOPTO bonding energies is similar
when the MBP orientation is constrained to its protein
geometry (Table 3, “Protein”), however, the strengths of the
individual interactions are significantly diminished when the
MBP orientation is distorted. For instance, the values of ΔEelst
and ΔEorb in the protein orientation are respectively shifted
15.1 and 4.2 kcal mol−1 more positive compared to their values
when the MBP geometry is freely optimized. Consequently, the
total bonding energies of TpCZn(MBP) complexes having
MBP orientations constrained to that of their hCAII complexes
are approximately 8 kcal mol−1 higher in energy (less favorable)
than geometries in which the MBP is freely optimized (Table
3).
While the aforementioned bonding energies suggest the
hCAII active site Zn2+ should preferentially bind, in order, 4-
CH3-1,2-HOPTO, 1,2-HOPTO, and 4-CF3-1,2-HOPTO, the
relative nonpolar binding free energies (ΔΔGnp, see Supporting
Information, Figure S17) obtained from thermodynamic
integration (TI) computations for these MBPs in complex
with hCAII oppose this energetic ordering. With respect to 4-
CF3-1,2-HOPTO, the values for ΔΔGnp of the 4-CH3-1,2-
HOPTO and 1,2-HOPTO MBPs are 0.8 and 1.8 kcal mol−1
higher in energy (see Supporting Information, Table S2).
These relative energies estimate the nonpolar interactions
between the different MBPs and the hydrophobic wall in the
Energetic Contributions to MBP Binding Affinities. To
assess the electronic effects of methyl and trifluoromethyl
substituents at the 4-position of 1,2-HOPTO, energy
decomposition analyses (EDAs) were performed and compared
with experimental binding affinities (Table 3). All EDA
computations are performed on two different geometries of
the TpCZn(MBP) complex: (a) a freely optimized TpCZn-
(MBP) complex, where the binding orientation of the MBP is
ideal (denoted “ideal” in Table 3), and (b) a constrained
TpCZn(MBP) complex, where the angle between the plane of
the MBP and the plane comprising the pyrazole nitrogens that
coordinate Zn2+ (ϕ) is set to ∼130° (see Supporting
Information, Figure S3), roughly mimicking the conformation
from the crystal structure of 1,2-HOPTO bound to hCAII
(denoted “protein” in Table 3).
Considering first the freely optimized TpCZn(MBP)
complexes (optimized geometries shown in the Supporting
Information, Figure S15), the 4-CH3-1,2-HOPTO MBP is
observed to interact more favorably with TpCZn (ΔETot
=
−266.9) than either 1,2-HOPTO (−263.7) or 4-CF3-1,2-
HOPTO (−250.5). The more negative bonding energy of 4-
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hCAII active site, indicating that the 4-CF3 substituent forms
the strongest interaction. It should be noted that these values
are intended to describe the individual forces driving MBP
binding and are not meant to be quantitative descriptions of the
full binding affinity of the MBPs to hCAII.
not “flip” in the active site to form similar contacts (Figure 8).
This suggests that there is a significant penalty for changing the
DISCUSSION
■
The results described above demonstrate that relatively minor
changes in MBP structure can have a drastic effect on both the
affinity and binding mode in a metalloenzyme active site. A
wide variety of forces play a part including ligand electrostatics,
steric restrictions of the active site, and interactions between the
MBP and the active site environment. By examining the
binding of 4,6-(CH3)2-1,2-HOPTO, which maintains bidentate
coordination due to stabilization by the methyl group in the 4-
position, the origin of monodentate binding by 6-CH3-1,2-
HOPTO in the active site of hCAII could be determined.
When the MBP adopts bidentate coordination, the methyl
group in the 6-position extends toward the hydrophilic side of
the active site, disrupting the highly ordered water network
(Figure 7). Two water molecules (W2 and W3) are displaced
Figure 8. Schematic of the two bidentate conformations available for
1,2-HOPTO. (A) The binding mode observed for unsubstituted 1,2-
HOPTO. In addition to binding the metal ion, interactions between
the Zn2+-bound oxygen atom and the hydrophilic active site
environment are observed. (B) When the ligand is “flipped” 180°, as
with 5-CF3-1,2-HOPTO, the interactions with the hydrophilic
environment are weakened and the anionic oxygen atom is positioned
near the hydrophobic wall of the active site.
positions of the O and S donor atoms, which likely originates
from several features, including (1) Coordination preferences:
small-molecule model complexes of His3Zn active sites
coordinated by bidentate (O,S)-donor ligands exclusively
show the sulfur and oxygen atoms as the equatorial and axial
donors, respectively.11,29,30,40−42 Geometry optimizations of
TpC(1,2-HOPTO) complexes indicate having the O-donor
coordinate axially is only slightly preferred (ΔE < 0.7 kcal
mol−1), thus indicating there is a small penalty for donor-
swapping in the absence of a protein environment. (2) Active
site electrostatics: If the donors were to swap, the greater
anionic charge on the oxygen would be positioned closer to the
hydrophobic side of the active site in hCAII, which would be
accompanied by an energetic penalty. (3) Hydrogen bonding:
The interaction between the active site water/hydrogen
bonding network and the Zn2+-bound donor atom would be
significantly weakened with a sulfur atom instead of oxygen
atom (Figure 8B, shown in red).43
Unlike its methyl analogue, 5-CF3-1,2-HOPTO does, in fact,
adopt a “flipped” coordination mode (Figure 8B) in the active
site of hCAII. The primary reason for this is likely the greatly
improved vdW interaction between the trifluoromethyl group
and the hydrophobic wall compared to CH3. Indeed, the
nonpolar contributions of having different hydrophobic groups
attached to the 4-position of 1,2-HOPTO are quantified by
thermodynamic integration (TI) computations performed on a
classical representation of the hCAII(MBP) complexes and
indicate that the 4-CF3 group provides 0.8 kcal mol−1
stabilization over the 4-CH3 group which, in turn, is favored
by 1.0 kcal mol−1 over unsubstituted 1,2-HOPTO (see
Supporting Information, Table S2). Despite a likely weakening
of metal coordination in 4-CF3-1,2-HOPTO compared to 4-
CH3-1,2-HOPTO (due to the electron-withdrawing nature of
the trifluoromethyl group), these improved interactions yield
excellent activity for the trifluoromethyl derivative. In the case
of 3-CF3-1,2-HOPTO, the vdW contacts are not improved
enough to compensate for the loss in metal binding affinity,
resulting in lower inhibition compared to its methyl analogue.
In addition, the trifluoromethyl derivatives show diminished
interactions with Thr200, most likely due to the electron-
withdrawing nature of the trifluoromethyl group. The O−O
distance for this interaction increases significantly for both CF3
Figure 7. Comparison of the crystal structures of 4-CH3-1,2-HOPTO
(A) and 4,6-(CH3)2-1,2-HOPTO (B) bound to hCAII reveals
disruption of the highly ordered active site water network. (C) The
two MBPs essentially overlay, suggesting that the water network
disruption is responsible for the ∼25-fold decrease in potency for 4,6-
(CH3)2-1,2-HOPTO compared to 4-CH3-1,2-HOPTO. (D) The
monodentate binding mode of 6-CH3-1,2-HOPTO is stabilized by a
hydrogen bond with Thr200 and hydrophobic interactions with
Val143, Val121, and Gln92.
by >0.8 Å as a result of the methyl group, breaking a hydrogen
bond between them (O−O distances of 4.3 and 2.8 Å in the
4,6-(CH3)2-1,2-HOPTO and 4-CH3-1,2-HOPTO structures,
respectively). A third water molecule (W1) is displaced by ∼0.2
Å, resulting in a longer interaction with the Zn2+-bound oxygen
atom (O−O distances of 2.9 and 3.1 Å in the 4-CH3-1,2-
HOPTO and 4,6-(CH3)2-1,2-HOPTO complexes, respec-
tively). We conclude that in order to avoid these unfavorable
interactions, 6-CH3-1,2-HOPTO adopts monodentate coordi-
nation in the active site of hCAII.
Given the favorable interactions formed between the methyl
groups of 3-CH3-1,2-HOPTO and the hydrophobic wall of the
active site, it is surprising that 5- and 6-CH3-1,2-HOPTO do
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derivatives relative to their methyl analogues (4.0 Å vs 3.0 and
3.7 Å vs 2.9 Å for 3-CF3-1,2-HOPTO and 4-CF3-1,2-HOPTO,
respectively), mostly due to a change in the position of the side
chain of Thr200 rather than a change in the position of the
MBP. The observation of a “flipped” coordination mode for 5-
CF3-1,2-HOPTO is likely a result of both the improved vdW
interactions (stabilizing the “flipped” conformation, Figure 8B)
as well as decreased anionic character on the Zn2+-bound
oxygen atom (destabilizing the “normal” conformation, Figure
8A).
case of anthrax lethal factor, both MBPs show weak affinity
(IC50 ∼5 mM),46 while with matrix metalloproteinases11 and
hCAII, the 1,2-HOPTO MBP is significantly more potent
(∼50-fold) than 1,2-HOPO.
CONCLUSION
■
Both the coordination mode and affinity of metal-binding
pharmacophores (MBPs) can be significantly influenced by
other interactions within the active site environment. Depend-
ing on its position, a peripheral methyl substituent on the 1,2-
HOPTO scaffold can cause either a greater than 10-fold
increase in potency against hCAII (4-CH3-1,2-HOPTO) or a 5-
fold decrease in potency accompanied by a shift to
monodentate coordination to the active site Zn2+ ion (6-
CH3-1,2-HOPTO). Despite greatly improved interactions with
the surrounding active site, 4-CF3-1,2-HOPTO only shows a
slight increase in potency relative to its methyl analogue due to
a weakening of MBP-metal binding. While the inhibitory
activity of a MBP is unquestionably influenced by the strength
of metal coordination, our findings indicate that the
interactions formed between the MBP and the surrounding
active site environment upon binding are equally important.
Furthermore, the affinities of MBPs for active site metal ions
are much closer to those observed for traditional pharmaco-
phore−protein interactions than to those of known metal
chelators (Figure 9).47−51 It is thus more appropriate to
consider the metal-binding portion of metalloprotein inhibitors
as pharmacophores rather than chelators. By recognizing the
pharmacophore nature of these functional groups, we can use
appropriate design principles to optimize MBPs, not only for
metal coordination but also for other interactions with the
target active site, in a rational and effective manner.
MPy-4CH3, which binds in the same conformation as 4-CH3-
1,2-HOPTO, but makes no interactions through the endocyclic
nitrogen, is 250-fold less potent. This suggests that the
interactions between the anionic oxygen and both the Zn2+
ion and the hydrophilic active site environment make a
significant contribution to the affinity of 1,2-HOPTO.
However, it is important to note that the pKa of MPy-4CH3
is significantly higher than that of 1,2-HOPTO and it is
therefore likely to bind as a neutral species. In this respect, the
low binding affinity of MPy-4CH3 also underscores the
importance of ligand electrostatics. Molecules with sulfur as
an anionic donor atom (thiophenols) are very potent, while
analogous compounds with less anionic character on the sulfur
(1,2-HOPTO derivatives) show lower affinity; those with little
to no anionic character on the sulfur (2-mercaptopyridines)
show an even greater decrease in binding. While this result
would seem to indicate that the sulfur donor plays a limited role
in the binding of 1,2-HOPTO derivatives to hCAII, exclusion
of a second donor atom entirely (PyNO-4CH3) results in a
molecule with no observable activity, and replacing the sulfur
with oxygen (4-CH3-1,2-HOPO) results in a greater than 30-
fold decrease in binding affinity. Further demonstrating the
intricacies of metal coordination in the hCAII active site,
aliphatic thiols such as 2-mercaptoethanol have been shown to
lack inhibitory activity, underscoring the importance of the
aromatic ring system.44
METHODS
■
Synthesis. MBPs were synthesized using a modified version of
previously reported procedures.52,53 Details can be found in the
Supporting Information.
The diminished inhibition for 1,2-HOPO relative to that of
the 1,2-HOPTO also supports the case for considering these
molecular scaffolds as pharmacophores and not chelators. The
Zn2+ affinity in free solution is comparable for 1,2-HOPO and
1,2-HOPTO (Kd of 5−10 μM, Figure 9),45 but their affinities to
Zn2+ ion active sites in metalloproteins is highly variable. In the
hCAII Activity Assay. hCAII was expressed and purified as
previously reported,54 and a detailed procedure can be found in the
Supporting Information. Assays were performed in 50 mM HEPES,
pH 8.0, containing Na2SO4 to an ionic strength of 100 mM. Enzyme
(100 nM final concentration) was incubated with varying concen-
trations of inhibitor for 10 min at room temperature before the
addition of substrate (p-nitrophenyl acetate, final concentration
between 0.05 and 10 mM). The reaction was monitored by the
increase in absorbance at 405 nm. Initial reaction rates vs substrate
concentration were plotted for three concentrations of inhibitor and
the curves simultaneously fit for Ki using GraphPad Prism.
Representative examples of curve fitting can be found in the
Supporting Information (Figure S1).
hCAII Crystallization. Crystals of hCAII were obtained by the
sitting-drop or hanging-drop vapor diffusion method. The protein
solution consisted of 20 mg/mL hCAII and 1 mM p-chloromercur-
ibenzoic acid in 50 mM Tris-SO4, pH 8.0. The precipitant solution
contained 2.7−3.0 M (NH4)2SO4 in 50 mM Tris-SO4 pH 8.15. Drops
consisted of 3 μL of protein solution plus 2.5−4.0 μL of precipitant
solution and were equilibrated at 18 °C against 750 μL of precipitant
solution. Crystals roughly 0.3 × 0.3 × 0.3 mm in size appeared after 2
days to 3 weeks depending on the drop size and precipitant
concentration. Once formed, crystals were transferred to 15 μL of
soak solutions containing MBP (at saturation, generally ∼1 mM), 1.5
M sodium citrate, 50 mM HEPES, pH 8.15, 5% glycerol, and 1−5%
DMSO. Soak solutions of 3-CH3-TP and 4-CH3-2MP also contained 2
mM tris(carboxyethyl)phosphine (TCEP) as a reducing agent.
Crystals were taken directly from the soak solutions for data collection.
Figure 9. Binding affinities of MBPs such as 1,2-HOPTO and
benzenesulfonamide (BSA) are much closer to traditional pharmaco-
phores than to conventional metal chelators. Examples of such
chelators include ethylenediaminetetraacetic acid (EDTA) and
deferoxamine (DFO). The affinity of MBPs for metal ions are also
well below those of metalloproteins (e.g., zinc fingers, hCAII, etc.) and
regulatory proteins such as metallothioneins and transferrin, indicative
of the pharamcophore nature of these functional groups.
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Crystal Structure Determination. X-ray diffraction studies on
hCAII crystals were carried out at 100 K with a Bruker D8 Smart 6000
CCD detector and utilizing Cu Kα radiation (λ = 1.5478 Å) from a
Bruker-Nonius FR-591 rotating anode generator. The data were
integrated and scaled using the Bruker APEX software suite. All
crystals belonged to the monoclinic space group P21. The data were
phased by molecular replacement using a previously reported hCAII
structure (PDB 3KS355) with water molecules removed. Models were
built by alternating refinement using REFMAC556 and manual
visualization and model building in Coot.57 Ligand topologies were
generated using the PRODRG server.58 The structures contain p-
mercuribenzoic acid bound to Cys206. Complete data collection and
refinement statistics can be found in the Supporting Information
(Table S1).
ASSOCIATED CONTENT
* Supporting Information
Full experimental details for ligand synthesis, protein expression
and purification, crystal structure collection details and
refinement statistics, and complete computational procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
Accession Codes
Coordinate and structure factor files for all hCAII structures
have been deposited with the Protein Data Bank: 4Q7P, 4Q7S,
4Q7V, 4Q7W, 4Q81, 4Q83, 4Q87, 4Q8X, 4Q8Y, 4Q8Z, 4Q90,
4Q9Y, 4Q99.
Density Functional Computations. To assess MBP binding
modes in the absence of a protein environment, geometry
optimizations were performed utilizing a tris(5-methylpyrazolyl)-
methane scaffold, referred to here as TpCZn (see Supporting
Information, Figure S3). This is a modification of the TpPh,MeZn
(TpPh,Me = hydrotris(5,3-methylphenylpyrazolyl)borate) model com-
plex commonly used experimentally to study MBP coordina-
tion.11,26−30 In addition to omitting the phenyl groups of the TpPh,Me
ligand to eliminate steric bulk, the TpCZn system provides a rigid
scaffold with the correct net charge (q = +2) for modeling the hCAII
His3Zn center in a computationally efficient manner.
AUTHOR INFORMATION
Corresponding Author
*Phone: 858-822-5596. Fax: 858-822-5598. E-mail: scohen@
ucsd.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Geometry optimizations are performed with Gaussian 09,59 using
Becke’s three-parameter hybrid method with the Lee, Yang, and Parr
correlation functional (B3LYP)60−63 and the 6-311++G(2d,2p) basis
set. This level of theory has previously been used to successfully
recapitulate geometric parameters of model active sites for Zn2+
metalloproteins64 as well as free energies of water−chloride exchange
in zinc chloride complexes.65 Further, implicit solvation is employed in
all computations using the conductor-like polarizable continuum
model (CPCM) with ε = 10,66−68 consistent with the crystallization
environment previously used to structurally characterize TpPh,MeZn-
(MBP) complexes.35 Where indicated, energy decomposition
analyses69−71 were performed on the optimized geometries of
TpCZn(MBP) complexes using the Amsterdam Density Functional
2009 suite of programs71,72 to enable assessments of electrostatic,
steric (Pauli repulsion), and orbital (which accounts for charge
transfer, polarization, and electron pair bonding effects) contributions
to the bond energy between TpCZn and the different MBPs.
Additional details and explanations can be found in the Supporting
Information.
J.A.M. acknowledges support from the National Institutes of
Health (NIH GM31749), National Science Foundation (MCB-
1020765), Howard Hughes Medical Institute, National
Biomedical Computation Resource, and NSF supercomputer
centers. P.G.B. acknowledges support from the National
Institutes of Health Molecular Biophysics Training Grant
(2T32GM008326-21). S.M.C. acknowledges support from the
National Institutes of Health (R01 GM098435). D.P.M. is
supported by a SMART scholarship from the Office of the
Secretary of Defense−Test and Evaluation (N00244-09-1-
0081).
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