Structure-based discovery of a novel pentamidine-related inhibitor of the calcium-binding protein S100B

Article abstract of DOI:10.1021/ml300166s

Molecular dynamics simulations of the pentamidine-S100B complex, where two molecules of pentamidine bind per monomer of S100B, were performed in an effort to determine what properties would be desirable in a pentamidine-derived compound as an inhibitor fo

Full text of DOI:10.1021/ml300166s

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Discovery of a Novel Pentamidine-Related Inhibitor
of the Calcium-Binding Protein S100B
Laura E. McKnight,† E. Prabhu Raman,∥ Padmavani Bezawada,∥ Sucheta Kudrimoti,∥ Paul T. Wilder,†,‡
Kira G. Hartman,†,§ Raquel Godoy-Ruiz,† Eric A. Toth,†,‡ Andrew Coop,∥ Alexander D. MacKerell, Jr.,∥
and David J. Weber*,†,‡
‡
§
†Department of Biochemistry and Molecular Biology, Marlene and Stewart Greenbaum Cancer Center, and Graduate Program in
Molecular Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, United States
∥Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, United States
S
* Supporting Information
ABSTRACT: Molecular dynamics simulations of the pentamidine−S100B complex,
where two molecules of pentamidine bind per monomer of S100B, were performed in
an effort to determine what properties would be desirable in a pentamidine-derived
compound as an inhibitor for S100B. These simulations predicted that increasing the
linker length of the compound would allow a single molecule to span both pentamidine
binding sites on the protein. The resulting compound, SBi4211 (also known as
heptamidine), was synthesized, and experiments to study its inhibition of S100B were
performed. The 1.65 Å X-ray crystal structure was determined for Ca2+−S100B−
heptamdine and gives high-resolution information about key contacts that facilitate the
interaction between heptamidine and S100B. Additionally, NMR HSQC experiments
with both compounds show that heptamidine interacts with the same region of S100B as pentamidine. Heptamidine is able to
selectively kill melanoma cells with S100B over those without S100B, indicating that its binding to S100B has an inhibitory effect
and that this compound may be useful in designing higher affinity S100B inhibitors as a treatment for melanoma and other
S100B-related cancers.
KEYWORDS: structure-based discovery, pentamidine-related inhibitor, calcium-binding protein S100B
the S100B−p53 protein−protein interaction in an attempt to
he S100 family of proteins consists of over 20 family
Tmembers, which are so named for their solubility in 100% develop a compound that will restore p53 levels in patients with
ammonium sulfate.1 These proteins contain EF-hand domains
malignant melanoma or other cancers with elevated levels of
S100B. One such molecule, SBi4211 (also known as
heptamidine), was found to have significant activity against
melanoma cells in an S100B-dependent manner and was
predicted to bind in the same site as pentamidine using
molecular dynamics (MD) simulations. This compound was
further investigated using nuclear magnetic resonance (NMR),
and the structure of the S100B−heptamidine complex was
determined using X-ray crystallography.
MD simulations of the S100B dimer in complex with
pentamidine were performed to analyze the conformational
distribution of pentamidine when bound to the protein. In the
analysis, atoms of pentamidine were classified into four
functional group types: phenyl ring carbons, amidinium
hydrogen atoms, aliphatic chain carbons, and ether oxygen
atoms. Three-dimensional distributions for each atom type
were computed from the simulations.
and have no enzymatic activity but instead typically act as
calcium-activated switches in a manner similar to calmodulin.2,3
Through a Ca2+-induced conformational change, S100 proteins
bind and regulate numerous protein targets with a variety of
biological functions.4 One particular S100 protein, S100B, has
several targets, including the tumor suppressor protein p53.5−9
S100B protein levels are upregulated in multiple types of
cancer, including astrocytomas and glioblastomas, and levels of
S100B are used as a prognostic indicator for malignant
melanoma.10 It has been suggested that the S100B−p53
interaction may be involved in the progression of cancer; a
direct interaction between S100B and p53 has been
demonstrated in primary malignant melanoma cells, and
S100B is able to reduce levels of wild-type p53 and inhibit its
function.9,11,12 As further support for this idea, siRNA was used
to reduce levels of S100B, which resulted in restoration of p53
protein levels and functions including apoptosis and transcrip-
tional activation.13 In an attempt to recreate this phenomenon
in a clinically relevant situation, small molecule inhibitors have
been designed based on the previously discovered inhibition of
S100B by pentamidine, also known as SBi1.14−16 These
molecules, known as SBiX, are being tested for inhibition of
Figure 1,a,b shows the resulting 3D probabilities overlaid on
the S100B−pentamidine cocrystal conformation for the two
Received: June 26, 2012
Accepted: September 25, 2012
Published: September 25, 2012
© 2012 American Chemical Society
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Figure 1. (a and b) Binding sites of pentamidine on S100B onto which grid-based probability isocontour surfaces of pentamidine atoms are overlaid;
benzene carbons (purple), aliphatic chain carbons (green), amidinium hydrogen atoms (blue), and ether oxygen atoms (red, not visible clearly). (c)
Centers of high occupancy (>0.5%) amidinium hydrogen voxels in site A. (d) Distribution of distances between the high occupancy voxel centers
and that between amidinium hydrogen atoms of the two benzamidine groups in pentamidine, hexamidine, and heptamidine.
a
Table 1. Cellular Assay and ITC Data for Select SBiX Compounds
a
NB, no binding; KD listed is for the tight site; binding to the weaker site (KD = 40 5 μM) is described in detail elsewhere.14
1
crystal binding sites. The probability maps show that the
pentamidine molecules sample a diverse collection of
conformations and cover a wide region of the protein surface.
Consistent with the crystal structure in which two pentamidine
molecules in two conformations were observed, it is clear that
the benzamindine moieties are not located in defined binding
pockets. Rather, they are sampling a broad region on the
protein surface.
Because the range of conformations that pentamidine
sampled in the simulations was promising, additional analysis
was performed to better understand the spatial distributions of
the amidine moieties in pentamidine, based on the assumption
that these groups are essential for binding. To perform this, the
distribution of the distances between high probability voxels
sampled by amidinium hydrogen atoms was determined, and
those with an occupancy cutoff of 0.5% were used (Figure 1c).
The 3D distributions were then converted to a 1D distribution
of amidine H−H distances based on the distances between all
high occupancy voxels with respect to each other. The resulting
distribution (Figure 1d) shows a maximum at ∼18.5 Å. To
understand how this distance compares to the intrinsic
conformational flexibility of pentamidine alone, GBMV MD
simulations of pentamidine in the absence of protein were
performed. From these simulations, the 1D probability
distribution of the amidine H−H distances was obtained. As
shown in Figure 1d, the intrinsic conformational dynamics of
pentamidine yields distances between amidinium hydrogen
atoms ∼2 Å shorter than the optimal distance between high
probability voxels predicted from the pentamidine−S100B MD
simulations.
This observation indicated that increasing the linker length
between the amidine moieties in pentamidine would yield
intrinsic conformational properties better suited to optimize
ligand−protein interactions based on the pentamidine−S100B
simulations, thereby improving ligand affinity. To test this
hypothesis, we performed MD simulations on the pentamidine
derivatives heptamidine and hexamidine, which have a linker of
seven or six methylene units as compared to the 5-unit linker of
pentamidine, respectively. Figure 1d includes the distance
distribution obtained from these simulations, showing a peak at
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Figure 2. HSQC perturbations upon addition of compound to S100B. (a) HSQC spectrum of S100B with heptamidine (top, red) or pentamidine
(bottom, red) overlaid onto S100B control (black). Residues that experience significant perturbation are labeled. (b and c) Graphical representation
of the perturbation of chemical shifts experienced by S100B upon addition of heptamidine (b) or pentamidine (c). The red bar denotes twice the
average perturbation; values greater than this line are considered significant. The insets depict a surface representation of S100B bound to
heptamidine (b) or pentamidine (c); residues that are significantly perturbed or disappear completely upon addition of compound are colored red,
and atoms of the compound are colored yellow (carbon), blue (nitrogen), and red (oxygen). (d) The difference between perturbations of S100B
caused by pentamidine and heptamidine. The inset depicts residue perturbations that are not shared by the two compounds in yellow.
∼18 Å, closest to the value of 18.5 Å obtained from the
pentamidine−S100B-based amidine H−H probability distribu-
tion. The data from the hexamidine probability distribution, on
the other hand, are not significantly different from that of
pentamidine. Thus, we concluded that heptamidine might bind
S100B more tightly than pentamidine because the conforma-
tions it frequently samples place the amidine moieties in a
position to make favorable contacts with S100B. We therefore
synthesized heptamidine to test its potency as an S100B
inhibitor. In Figure 3 in the Supporting Information, the
probability distributions that were obtained from the S100B−
pentamidine simulations in Figure 1a,b are overlaid with the
heptamidine crystal structure that we obtained, showing that
the heptamidine binding mode is largely consistent with our
predictions based on S100B−pentamidine simulations.
Heptamidine was synthesized as described in the Supporting
Information and assigned the database number SBi4211. This
compound, along with several similar compounds, was tested
for activity against the human melanoma cell line MALME-3M.
In this assay, cell death is assessed in both wild-type MALME-3
M cells, which have high levels of S100B and wild-type p53, and
in MALME-3 M cells, in which S100B has been reduced using a
stable siRNA knockdown. The effect of each compound on the
two lines is then compared to assess the specificity of
compounds against S100B. Additionally, KD values for
compound binding to S100B were determined using NMR
titrations. Table 1 summarizes the results from the cellular
assays and NMR, showing that heptamidine has better killing
activity in cells than pentamidine. This effect may be explained
by differential scanning calorimetry (DSC) results, which show
that heptamidine destabilizes S100B more than pentamidine
(Table 3 in the Supporting Information). Another possibility is
that both the tight and the weak sites of pentamidine need to
be at least partially occupied for pentamidine to have efficacy in
the cellular assays.
Interestingly, compound SBi4210 (hexamidine), which is
structurally related to pentamidine and heptamidine but has a
six-carbon linker, shows no activity in the cellular assay and no
binding by NMR. MD simulations predicted that this
compound would make less favorable contacts with S100B as
compared to heptamidine but would be comparable to
pentamidine. These results suggest that hexamidine may be
too long to take advantage of the interaction mode assumed by
pentamidine but too short to exploit the interactions that
stabilize the binding of heptamidine.
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Figure 3. High-resolution crystal structures of S100B bound to heptamidine and pentamidine. (a and b) Surface representation of the structure of
S100B bound to heptamidine (a) and pentamidine (b). Residues within 3 Å are colored yellow to highlight the binding pocket. Below, the electron
density maps calculated with the 2mFo − DFc coefficients (contoured at 1.0σ) for each compound are shown. The atoms of the compound are
colored orange (carbon), blue (nitrogen), and red (oxygen). (c and d) Cartoon representation of the structure of S100B bound to heptamidine (c)
and pentamidine (d) with helices visible. Compounds are colored as in a and b, and calcium ions are depicted as cyan spheres.
Heteronuclear single quantum coherence (HSQC) experi-
ments, which show peaks for backbone amides,17 were
performed on 15N-labeled S100B protein in the presence of
pentamidine or heptamidine. Perturbation of these signals from
those of the control is due to a change in the magnetic
environment and can indicate that compound is binding to this
region of the protein. The significantly perturbed residues for
both S100B−heptamidine and S100B−pentamidine HSQCs
are labeled in Figure 2a. Figure 2b shows all perturbations
caused by heptamidine, indicated both by bars and shading on
the protein surface in the inset, while Figure 2c shows the
perturbations caused by pentamidine. The similarities between
the two sets of perturbations indicate that pentamidine and
heptamidine occupy similar sites on S100B. In Figure 2d, the
difference in S100B−heptamidine perturbations from those of
S100B−pentamidine is mapped, highlighting regions that are
perturbed by pentamidine but not heptamidine.
To examine binding in more detail, a high-resolution crystal
structure was solved for the complex of S100B bound to
heptamidine using molecular replacement methods. The final
asymmetric unit consists of 88 residues for S100B (Met0 to
Phe87), two calcium ions, and 89 water molecules. The
biologically significant model is a dimer comprised of the
asymmetric unit and a crystallographic symmetry mate. Nearly
all of the residues of S100B−Ca2+−heptamidine were in the
most favorable region of the Ramachandran plot (98.9%) with
the remaining residues in the additionally allowed region
(1.1%) (Table 4 in the Supporting Information). The resulting
structure, presented in Figure 3, reveals that one molecule of
heptamidine binds per monomer of S100B, as opposed to the
two molecules of pentamidine that bind each monomer in the
previously solved structure.14 This molecule of heptamidine
spans the two sites previously occupied by two molecules of
pentamidine (Figure 3a vs b), which nicely explains the NMR
chemical shift perturbations mapped in Figure 2. The global
fold of the protein was nearly identical to that of the S100B−
Ca2+−pentamidine X-ray structure reported previously,14 with
all of the Ca2+ ligands, ligand distances, helical angles, and EF-
hand angles found to be very similar. Specifically, each subunit
of S100B−Ca2+ contained four helices (helix 1, S1-G19; helix 2,
K28-L40; helix 3, E49-D61; and helix 4, Q70-F87) with the
dimer interface aligned as a symmetric X type four helix bundle
and two helix−loop−helix EF-hand calcium-binding domains
including an “S100 type” or “pseudo” EF-hand comprising
helices 1 and 2 and loop 1, and a “typical” EF-hand with 12
residues contributed by helices 3 and 4 and loop 3 (Figure
3c,d). Figure 5 in the Supporting Information provides a closer
view of the binding sites of heptamidine (a) and pentamidine
(b).
Heptamidine lies within close proximity of several residues of
S100B, and its amidinium groups are able to participate in
hydrogen bonds with Phe43 and His85 (Figure 4a in the
Supporting Information), while pentamidine does not.
Alternatively, the two molecules of pentamidine are positioned
to participate in π-stacking interactions with Phe88, Phe87, and
His42 (Figure 4b in the Supporting Information), and the
orientation of heptamidine precludes such interactions.
These differences, which result from the addition of only two
carbons in the linker region of the heptamidine, may explain
the different binding modes of heptamidine and pentamidine
and the number of molecules of each compound that binds per
dimer of S100B. In the future, these characteristics will be used
to engineer new S100B inhibitors (SBiXs) in an effort to
provide selective inhibition of S100B-dependent growth
properties and restore p53-dependent apoptosis in melanoma
cells at therapeutic concentrations.
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(12) Lin, J.; Yang, Q.; Wilder, P. T.; Carrier, F.; Weber, D. J. The
calcium-binding protein S100B down-regulates p53 and apoptosis in
malignant melanoma. J. Biol. Chem. 2010, 285, 27487−27498.
(13) Lin, J.; Yang, Q.; Yan, Z.; Markowitz, J.; Wilder, P. T.; Carrier,
F.; Weber, D. J. Inhibiting S100B restores p53 levels in primary
malignant melanoma cancer cells. J. Biol. Chem. 2004, 279, 34071−
34077.
(14) Charpentier, T. H.; Wilder, P. T.; Liriano, M. A.; Varney, K. M.;
Pozharski, E.; MacKerell, A. D., Jr.; Coop, A.; Toth, E. A.; Weber, D. J.
Divalent metal ion complexes of S100B in the absence and presence of
pentamidine. J. Mol. Biol. 2008, 382, 56−73.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for heptamidine synthesis, computational
methods, protein growth and purification, cellular assays, and
NMR and DSC as well as figures for additional MD
simulations, HSQC of 15N S100B, crystal and refinement
statistics, and a close view of the compound binding sites. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(15) Smith, J.; Stewart, B. J.; Glaysher, S.; Peregrin, K.; Knight, L. A.;
Weber, D. J.; Cree, I. A. The effect of pentamidine on melanoma ex
vivo. Anticancer Drugs 2010, 21, 181−185.
(16) Markowitz, J.; Chen, I.; Gitti, R.; Baldisseri, D. M.; Pan, Y.;
Udan, R.; Carrier, F.; MacKerell, A. D., Jr.; Weber, D. J. Identification
and characterization of small molecule inhibitors of the calcium-
dependent S100B-p53 tumor suppressor interaction. J. Med. Chem.
2004, 47, 5085−5093.
AUTHOR INFORMATION
Corresponding Author
*Tel: 410-706-4354. E-mail: dweber@som.umaryland.edu.
Author Contributions
All authors have given approval to the final version of this
manuscript.
■
(17) Bodenhausen, G. R.; Ruben, D. J. Natural abundance nitrogen-
15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett.
2002, 69, 185−189.
Funding
This work was supported by grants from the NIH (GM58888
and CA107331) to D.J.W.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
NMR, nuclear magnetic resonance; HSQC, heteronuclear
single quantum coherence; MD, molecular dynamics; LD50,
lethal dose for 50% of the population; DSC, differential
scanning calorimetry
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