Structure-Based Identification of Novel Ligands Targeting Multiple Sites within a Chemokine-G-Protein-Coupled-Receptor Interface

Article abstract of DOI:10.1021/acs.jmedchem.5b02042

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

Full text of DOI:10.1021/acs.jmedchem.5b02042

Article
pubs.acs.org/jmc
Structure-Based Identification of Novel Ligands Targeting Multiple
Sites within a Chemokine−G-Protein-Coupled-Receptor Interface
†
,⊥
‡,⊥
§
§
‡
Emmanuel W. Smith, Amanda M. Nevins, Zhen Qiao, Yan Liu, Anthony E. Getschman,
∥
∥
‡
§
,‡
Sai L. Vankayala, M. Trent Kemp, Francis C. Peterson, Rongshi Li, Brian F. Volkman,*
,†
and Yu Chen*
†Department of Molecular Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612, United
States
‡
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, United
States
§
Department of Pharmaceutical Sciences, Center for Drug Discovery, College of Pharmacy, and Cancer Genes and Molecular
Regulation Program, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 986805 Nebraska Medical
Center, Omaha, Nebraska 68198, United States
∥
Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, United States
*
S Supporting Information
ABSTRACT: CXCL12 is a human chemokine that recognizes
the CXCR4 receptor and is involved in immune responses and
metastatic cancer. Interactions between CXCL12 and CXCR4
are an important drug target but, like other elongated protein−
protein interfaces, present challenges for small molecule ligand
discovery due to the relatively shallow and featureless binding
surfaces. Calculations using an NMR complex structure
revealed a binding hot spot on CXCL12 that normally
interacts with the I4/I6 residues from CXCR4. Virtual
screening was performed against the NMR model, and
subsequent testing has verified the specific binding of multiple
docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine
residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that
protein−protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead
compounds for subsequent optimization by fragment based approaches.
INTRODUCTION
significant interest in investigating whether weak binders such
as fragments can be developed for such hot spots and
subsequently be used in lead optimization. In particular, with
the relative promiscuity displayed by fragments and the
conformational plasticity of many PPIs, there is uncertainty
about whether weak-binding ligands can interact with the
■
Protein−protein interactions (PPIs) are appealing yet challeng-
ing targets in drug discovery efforts to modulate protein
1
pathways in disease. In contrast to enzymatic active sites that
usually possess stable conformations and deep pockets, PPIs are
often highly dynamic structures presenting a collection of
shallow binding surfaces and are thereby frequently deemed
6
binding hot spots at PPIs with adequate specificity.
1
,2
The chemokine CXCL12 is involved in extensive protein−
protein interactions with the CXCR4 receptor and, like other
PPIs, presents an attractive yet difficult target for drug
“
undruggable”. However, studies have shown that PPIs are
not necessarily flat and that their binding is mediated by certain
grooves or hot spots, where specific localized interactions
provide most of the affinity. These binding hot spots are
primarily influenced by hydrophobic interactions and, to a
3
,4
7
discovery. Chemokines are small chemotactic cytokines that
bind and activate G-protein-coupled receptors (GPCRs), thus
guiding receptor-expressing cells to tissues of constitutive
2
lesser extent, electrostatic interactions and hydrogen bonds.
8
Additionally, reports have emerged where small molecules have5
successfully targeted PPIs by binding to such hot spots.
Therefore, drug discovery efforts against binding hot spots are
considered a viable strategy in targeting PPIs, especially
considering that a small molecule can occupy a surface of
chemokine expression. While this usually aids in the immune
response by attracting leukocytes that express chemokine
Special Issue: Computational Methods for Medicinal Chemistry
Received: December 31, 2015
2
2
2
3
00−1000 Å , while a hot-spot area is around 600 Å . With the
recent development of fragment-based approaches, there is
©
XXXX American Chemical Society
A
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Figure 1. Two-site/two-step binding of CXCL12 to CXCR4. The first step (1) is recognition of the core domain of CXCL12 by the flexible,
extracellular, N-terminal tail of CXCR4. The second step (2) is docking of the flexible, N-terminal tail of CXCL12 into CXCR4 leading to receptor
activation.
Figure 2. CXCL12−CXCR4 PPI and druggable hot spots. (A) Hot spots that mediate N-terminal CXCR4 peptide binding to monomeric CXCL12
(
L55C/I58C) (PDB code 2N55). To the left, tyrosines Y12 and Y21 of CXCR4 N-terminus recognize and bind to potential hot spots on the surface
of CXCL12. To the right, isoleucines I4 and I6 of the CXCR4 N-terminus bind to hot spots on the surface of CXCL12 (PDB code 2N55). (B)
FTMap analysis of potentially druggable hot spots on monomeric CXCL12 (L55C/I58C) (PDB code 2N55). Probe clusters were sorted based on
the number of probes in each cluster (cluster strength, CS). To the left, a large probe cluster, CS1 (green, 27 probes), was found at a site near the
Y21 binding site and a small probe cluster, CS6 (purple, 6 probes), overlaps with the Y12 binding site. To the right, five probe clusters, CS2 (cyan,
1
5 probes), CS3 (yellow, 14 probes), CS4 (green, 12 probes), CS5 (gray, 11 probes), and CS7 (blue, 5 probes), were found at sites overlapping with
the I4 and I6 binding site and surrounding area. (C) Hot spots that mediate sulfated N-terminal CXCR4 peptide binding to CXCL12 (PDB code
2
2
K05). Sulfotyrosines sY12 and sY21 of CXCR4 N-terminus recognize and bind to sulfotyrosine-binding sites on the surface of CXCL12 (PDB code
K05, only monomer A depicted). (D) FTMap analysis of potentially druggable hot spots on CXCL12 (PDB code 2K05, only monomer A used).
Probe clusters were sorted based on the number of probes in each cluster (cluster strength, CS). Five probe clusters, CS1 (cyan, 25 probes), CS4
yellow, 13 probes), CS5 (green, 9 probes), CS6 (blue, 5 probes), and CS8 (gray, 3 probes), were found at sites overlapping with the sY12 and sY21
(
binding sites and surrounding areas. Probe clusters CS2 (purple, 15 probes) and CS3 (yellow, 15 probes) were found at sites that would normally
interact with I4 and I6 in the monomeric CXCL12 complex.
1
1
receptors, cancer cells are known to highjack the same
mechanism by expressing chemokine receptors in order to9
metastasize to tissues where chemokine expression is high.
CXCL12 binds to CXCR4 in a two-step/two-site process
compete with the N-terminal domain of CXCL12. The first
step, however, represents a viable alternative target and a
prototypic example of PPIs, mediated by a binding surface area
covering a large portion of the chemokine. The flexible N-
terminus wraps more than halfway around the chemokine,
creating an interface between the CXCR4 N-terminus and
(Figure 1). First, the extracellular flexible N-terminal domain of
the receptor recognizes and binds to the surface of CXCL12,
and then CXCL12 “docks” its own flexible N-terminal domain
into the receptor resulting in activation and downstream
2
CXCL12 of 2093 Å and utilizing almost 25% of the chemokine
12
surface area. Because the binding energy is distributed across
a large interaction area, targeting this interface with small
molecule inhibitors is likely to be a major challenge.
Previous analyses have demonstrated potential binding hot
spots mediating the binding between the receptor N-terminus
10
signaling (Figure 1). Most drug discovery efforts for the
CXCL12/CXCR4 interaction have been focused on targeting
the second activation step by developing antagonists that would
bind in the deep hydrophobic pocket of the receptor and
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and CXCL12, which are facilitated by O-sulfation of tyrosine
residues (Y21 and Y12) on the N-terminal domain of CXCR4
which then recognize specific sites on the surface of CXCL12
(Figure 2A,B). FTMap analysis was also performed using
monomer A from the NMR complex structure of dimeric
CXCL12 bound to a triply sulfated (sY7, sY12, sY21), 38-
residue long (p38), N-terminal CXCR4 peptide (PDB code
12−14
and increase the affinity of the interaction.
Chemical
12
modifications and mutagenic analysis of the CXCR4 N-
terminus identified sulfated tyrosines sY21 and sY12 as the
residues providing the CXCL12 contacts with the greatest
2K05) (Figure 2C). Probe clusters were again ranked based
on the number of probes in each cluster (cluster strength (CS),
CS1−9), and five probe clusters were found at sites overlapping
with and in between the sY12 and sY21 binding sites, also
corresponding to the Y12 and Y21 binding sites in the
monomeric NMR structure (Figure 2C,D). Most probe clusters
occupy hot spots at the cleft between the sY12 and sY21
binding sites (CS1, CS5, CS8). One probe cluster (CS4)
occupies the sY21 binding site, and one (CS6) occupies the
sY12 binding site. These findings further support that the
(s)Y12/(s)Y21 recognition sites are potential binding hot
spots. In the dimer structure, however, the I4/I6 binding site is
masked by conformational changes in the protein as well as
intermolecular interactions across the dimer interface. Still, a
few probe clusters (CS2, CS3) were found in the area that
would usually interact with I4/I6 of CXCR4 (Figure 2D) but
not as many as were found in the constitutively monomeric
CXCL12 (L55C/I58C) NMR structure (Figure 2B), suggest-
ing that the constitutively monomeric CXCL12 (L55C/I58C)
NMR structure may be a better template for virtual screening
experiments against that site.
1
3,15
binding energy.
We have recently utilized virtual screening
10,16,17
to identify novel ligands targeting the sY21,
and sY12 (A.
E. Getschman et al., unpublished results) binding hot spots
separately, with binding affinities in the low micromolar range.
These studies have demonstrated that the sulfotyrosine-binding
sites on CXCL12 can indeed be utilized in targeting the initial
PPI of chemokine signaling.
The previous successes led us to question whether there were
other binding hot spots on CXCL12 and how they could be
18
identified and targeted. In this study we performed FTMap
analysis on the NMR structure of constitutively monomeric
CXCL12 (L55C/I58C) in complex with an unsulfated N-
terminal receptor peptide (PDB code 2N55) revealing a third
potential binding hot spot on CXCL12 that usually binds to the
I4/I6 residues of the receptor peptide. Then, through docking
to single and multiple chemokine conformations, novel small
molecule ligands have been uncovered that interact specifically
with the I4/I6 binding site. In addition, we present a compound
developed based on a previously discovered virtual screening
hit for the sY12 binding site, further confirming that specific
ligands can be engineered to target the different binding hot
spots of the PPI between CXCL12 and CXCR4. These results
demonstrate that CXCL12 can be targeted at multiple sites
with small molecule ligands and offer insights into computa-
tional approaches in targeting chemokine−GPCR interactions
as well as other PPIs.
To gain additional insight into the properties of the I4/I6
binding site compared to other hotspots and analyze its
recognition epitopes on the CXCR4 peptide, we also used
23,24
Schrodinger Prime MM-GBSA
to calculate the energetic
contribution of each residue side chain on the receptor N-
terminus to the PPI using the same constitutively monomeric
NMR CXCL12 (L55C/I58C) complex structure (PDB code
2N55) and also compare it to monomer A from dimeric
CXCL12 in complex with the sulfated CXCR4 peptide (PDB
code 2K05) (Figure S1 in Supporting Information). Whereas
the calculations demonstrated that I4 is the residue
contributing most to the binding of the nonsulfated N-
terminus of CXCR4 to CXCL12 (PDB code 2N55), the results
concerning other residues and binding hot spots correlated less
well with FTMap analysis and experimental data, as the
calculations deemphasized the contributions of some important
RESULTS
■
Computational Analysis Identifies Additional Binding
Hot Spots. Potentially druggable hot spots on protein surfaces
can be identified via computational solvent mapping pro-
1
9
20
grams, such as FTMap. These programs dock, in silico,
small organic probes onto the surface of a protein while
sampling a large number of conformations. Areas where
multiple probes cluster may be indicative of druggability. The
12,25
residues, such as Y21 (Supporting Information).
We
1
8
therefore focused mainly on FTMap for our binding hot spot
analysis.
FTMap solvent mapping server was used to analyze the
druggability of putative hot spots on the surface of CXCL12. As
21
Novel Small Molecule Ligand Discovery against the
I4/I6 Binding Site Using Rigid Docking. On the basis of
FTMap analysis of constitutively monomeric CXCL12 (L55C/
I58C) (Figure 2B) and the energetic contribution calculations
for each residue of the N-terminal CXCR4 peptide (Figure S1),
the I4/I6 binding site of monomeric CXCL12 appeared to be a
promising binding hot spot for small molecule ligands. In order
to experimentally probe the druggability of this newly identified
candidate hot spot, we performed virtual screening experiments
against the NMR monomer structure using DOCK 3.5.54 and
CXCL12 exists as both monomer and dimer in vivo, two
structures were used for this analysis, corresponding to a
monomeric and dimeric state respectively (Figure 2). We first
focused on the NMR complex structure of constitutively
monomeric CXCL12 (L55C/I58C) bound to a nonsulfated,
4
2
0-residue long (p40), N-terminal CXCR4 peptide (PDB code
N55) (Figure 2A). Two mutations, L55C and I58C, were
used to engineer a disulfide bond locking the protein
conformation in the monomeric state and preventing
2
2
dimerization. Probe clusters were ranked based on the
26−28
number of probes in each cluster (cluster strength (CS), CS1−
the ZINC small molecule database.
Conformation 1 of the
7) and were found at several sites that represent potential
NMR ensemble was used as a rigid template. From the top
scoring compounds, 12 were screened experimentally using 2D
binding hot spots, including the binding site for Y12 (CS6) and
a cleft near the binding site for Y21 from the receptor (CS1)
1
15
H− N SOFAST-HMQC spectroscopy to test the binding of
(
Figure 2A,B). The highest density of probe clusters (five
potential ligands to WT-CXCL12, which existed as monomer
under the experimental conditions. Of the 12 compounds
experimentally analyzed in this screen, 3 showed binding to
clusters: CS2, CS3, CS4, CS5, and CS7) was found at and
around a site that recognizes two isoleucine residues, I4 and I6,
of the CXCR4 receptor, suggesting that this site might be more
druggable than the Y12 and Y21 binding sites in the monomer
2
6
WT-CXCL12 (1 (ZINC C04181455),
2 (ZINC
26
26
C40310216), and 3 (ZINC C16480049) ) (Figure S2),
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1
15
Figure 3. Novel small molecule ligand discovery against the I4/I6 binding site using rigid docking. (A) H− N HMQC overlay of titration of WT-
CXCL12 with 1 (inset) used for chemical shift mapping showing significant chemical shift perturbations for a subset of residues. (B) Quantification
1
15
of the change in total chemical shift ( H/ N Δδ chemical shift) for individual amino acid residues of CXCL12 with those experiencing the largest
shifts highlighted in green. (C) Fitted curves for selected residues used to calculate the affinity (K ) of 1 for WT-CXCL12. (D) Mapping of
d
significantly perturbed residues onto the NMR structure (conformation 1) of constitutively monomeric CXCL12 (L55C/I58C) (PDB code 2N55)
1
15
used to identify 1 in rigid virtual screening, corresponds to the predicted binding site for 1. (E) H− N HMQC overlay of titration of locked
dimeric CXCL12 (L36C/A65C) with 1 (inset) used for chemical shift mapping showing significant chemical shift perturbations for a subset of
1
15
residues. (F) Quantification of the change in total chemical shift ( H/ N Δδ chemical shift) for individual amino acid residues of LD-CXCL12
locked dimer) with those experiencing the largest shifts highlighted in green. (G) Mapping of significantly perturbed residues onto the NMR
(
structure (conformation 1) of dimeric CXCL12 (L36C/I65C) (PDB code 2K01). The two monomers are colored in gray and white with
corresponding perturbed residues colored in light green and dark green, respectively.
two of which (1 and 2) induced chemical shift perturbations to
residues associated with the targeted I4/I6 binding site, for a
final hit rate of 16.7% (Figure S2). In particular, 1 displayed
engineered residues at the dimer interface (L36C/A65C) link
both monomers together into the dimeric configuration
12
through a pair of symmetric intermolecular disulfide bonds.
concentration-dependent binding with the K estimated to be
The I4/I6 binding site is only present in the monomeric state
of CXCL12 and not accessible in the dimeric state of the
protein. In particular, it has been found that in the CXCL12
dimer, the α-helix constituting a major part of the I4/I6 binding
site adopts a different orientation relative to the rest of the
protein including but not limited to the β-sheet that is also part
d
1.2 ± 0.3 mM. The largest chemical shift perturbations were
observed for residues surrounding the I4/I6 binding site of
CXCL12 (Y7, C11, V18, R20, V23, H25, K27, V39, Q48, W57,
Y61, and L66) (Figure 3A−D, S4A). For 2, the concentration-
dependent binding was weaker with an estimated K of 1.7 ±
d
29,30
0
.7 mM.
of the I4/I6 binding site (Figure 2A,C).
These conforma-
1
15
To further probe the specificity of 1, 2D H− N SOFAST-
tional changes in the dimer flatten the binding surface of the
I4/I6 binding site, making it significantly less druggable. A
specific ligand for the I4/I6 site in WT-CXCL12, as suggested
HMQC spectroscopy was carried out using the engineered
locked dimer of CXCL12 (L36C/A65C). For the locked dimer,
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Figure 4. Novel small molecule ligand discovery against the I4/I6 binding site using ensemble docking. (A) H− N HMQC overlay of titration of
WT-CXCL12 with 6 (inset) used for chemical shift mapping showing significant chemical shift perturbations for a subset of residues. (B)
Quantification of the change in chemical shift (Δδ chemical shift) for individual amino acid residues of CXCL12 with those experiencing the largest
shifts highlighted in green. (C) Fitted curves for selected residues used to calculate the affinity (K ) of 6 for WT-CXCL12. (D) Mapping of
d
significantly perturbed residues onto the NMR structure (conformation 13) of constitutively monomeric CXCL12 (L55C/I58C) (PDB code 2N55)
1
15
used to identify 6 in ensemble virtual screening, corresponds to the predicted binding site for 6. (E) H− N HMQC overlay of titration of locked
dimeric CXCL12 (L36C/A65C) with 6 (inset) used for chemical shift mapping showing significant chemical shift perturbations for a subset of
1
15
residues. (F) Quantification of the change in total chemical shift ( H/ N Δδ chemical shift) for individual amino acid residues of LD-CXCL12
locked dimer) with those experiencing the largest shifts highlighted in green. (G) Mapping of significantly perturbed residues onto the NMR
(
structure (conformation 1) of dimeric CXCL12 (L36C/I65C) (PDB code 2K01). The two monomers are colored in gray and white with
corresponding perturbed residues colored in light green and dark green, respectively.
by the NMR studies above, should only bind to the I4/I6 site in
the monomeric state but not in the CXCL12 dimer. During the
experiment with the locked dimer, 1 induced modest shift
perturbations (<0.5 ppm) for multiple residues (Y7, K24, K27,
I28, N30, I38, V39, A40, Q48) and larger (>0.5 ppm) shifts for
a small group (C11, T31, Q37, V49) (Figure 3E,F). The linear
in the C-terminal helix were not perturbed significantly by the
binding of 1 to the dimer, further suggesting that interactions
between 1 and this site occur uniquely with the monomeric
state of WT CXCL12 (Figure 3B,F).
Novel Small Molecule Ligand Discovery against the
I4/I6 Binding Site Using Ensemble Docking. In order to
more fully capture the flexibility of CXCL12, all 20
conformations of the NMR structure were used in an ensemble
docking experiment with each conformation serving as a rigid
template for virtual screening. The docking results of all 20
virtual screens were then combined, and compounds were
selected from the top-ranking list for experimental analysis.
(i.e., nonsaturable) concentration dependence of the largest
shifts (e.g., V49) was consistent with nonspecific binding. The
small perturbations of K24, K27, and I28 in the β1 strand and
I38, V39, and A40 in the β2 strand surround a cluster of
positively charged residues from both monomers (Figure 3G)
31
that bind other negatively charged molecules and may attract
through nonspecific electrostatic interactions. Importantly,
several residues in the I4/I6 binding site such as W57 and Y61
1
15
1
Twenty-four compounds were analyzed using 2D H− N
SOFAST-HMQC spectroscopy to test binding of the
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15
Figure 5. Successful targeting of the sY12 binding site of CXCL12. (A) H− N HMQC overlay of titration of WT-CXCL12 with 9 (inset) used for
chemical shift mapping showing significant chemical shift perturbations for a subset of residues. (B) Quantification of the change in chemical shift
(
Δδ chemical shift) for individual amino acid residues of CXCL12 with those experiencing the largest shifts highlighted in orange. (C) Fitted curves
for selected residues used to calculate the affinity (K ) of 9 for WT-CXCL12. (D) Mapping of significantly perturbed residues onto the NMR
d
structure (monomer A, conformation 1) of dimeric CXCL12 (PDB code 2K05) corresponds to the binding site for 9 as predicted by docking.
compounds to WT-CXCL12. From the 24 compounds
Development of a Specific Ligand Targeting the sY12
Binding Site of CXCL12 Monomer. It should be mentioned
that the binding site for 6 in the engineered dimer overlaps with
the sY12 binding pocket in the monomer, for which we have
also been developing ligands through virtual screening (A. E.
Getschman et al., unpublished results). Whereas those results
will be described in detail in a separate publication, it will be
interesting to compare binding to the sY12 site in the monomer
versus dimer for the current study. For this purpose we turned
to 2-((4-(carboxymethoxy)-6-phenyl-1,3,5-triazin-2-yl)thio)-
acetic acid (9), designed and synthesized (Scheme S1) based
on a novel inhibitor scaffold targeting sY12 binding site in the
monomer. 9 was found to bind to the WT sY12 binding site by
screened, five showed binding to WT-CXCL12 (Figure S3).
26
Among those five, 4 (ZINC C44978491) and 5 (ZINC
2
6
C69492022) were determined to bind nonspecifically (Figure
2
6
S3A,B). The rest, 6 (ZINC C12998741), 7 (ZINC
2
6
26
C15782120), and 8 (ZINC C07362052), induced chemical
shift perturbations to residues associated with the I4/I6 binding
site, resulting in a final hit rate of 12.5% (Figure S3C−E). In
particular, 6 induced concentration-dependent chemical shift
perturbations at a number of residues, the largest of which were
associated with the I4/I6 binding site of CXCL12 (A21, V23,
K24, H25, N33, V39, K43, N45, V49, W57, E60, K64, A65,
L66, and N67), and had an estimated K of 1.1 ± 0.3 mM
1
15
d
2
D H− N SOFAST-HMQC spectroscopy, with the largest
(
Figure 4A−D, S4B). 7 and 8 displayed weaker affinities with
chemical shift perturbations associated with residues surround-
ing the sY12 binding site (C11, K24, I28, V39, A40, and V49)
estimated K values of 4.1 ± 2 mM and 4.1 ± 1 mM,
d
respectively. The docking template that identified 6 corre-
sponded to conformation 13 of the NMR ensemble, similar to
and an estimated K of 200 ± 55 μM (Figure 5 and Figure
d
S4C). These results demonstrate that the sY12 binding site is
accessible in the monomer. 6 likely recognizes key structural
features unique to the dimer, such as residues across the
interface. The binding of 9 to the sY12 site also suggests that
specific interactions between small molecules and this site can
be recapitulated by designed compounds, thus paving the way
for future fragment-based lead optimization efforts.
4
and 5, and was unlike conformation 1 used in the original
rigid docking that identified 1 (Figure S3). The docking
templates that identified 7 and 8 corresponded to, respectively,
conformations 5 and 16 of the NMR ensemble (Figure S3).
This suggests that the compounds recognize slightly different
protein conformations and that multiple conformations of the
NMR structure can, and should, be used in virtual screening to
identify novel ligands, even though some conformations may
outperform others as docking template.
DISCUSSION
■
Multiple Binding Hot Spots for Targeting the CXCL12
PPI. Through a structure-based virtual screening strategy used
1
15
6
was also subjected to the binding analysis by 2D H− N
SOFAST-HMQC spectroscopy using the locked dimer.
Interestingly, whereas it exhibited some nonspecific interactions
with several binding surfaces on CXCL12, 6 appeared to bind
specifically to the β-sheet region of the positively charged dimer
in this study and together with the results from previous
10,16,17
studies,
we have identified novel small molecule ligands,
which despite their weak affinities (micromolar to millimolar)
are capable of binding to three distinct sites on the extensive
PPI of the CXCL12 chemokine (sY21, sY12, and I4/I6 binding
sites) with its receptor, CXCR4 (Figure 6). These results
suggest that these PPI binding hot spots possess enough unique
structural features, such as the shape of the pocket, the spatial
arrangement of polar functional groups for hydrogen bonding
interactions, significant hydrophobic surfaces, and a large
interface, with K of 74 ± 10 μM, suggesting that these binding
d
surfaces represent new hot spots in the dimer (Figure 4E−G).
Significantly, several residues in the I4/I6 binding site, such as
W57, were much less perturbed in the engineered dimer than
the WT, again indicating specific binding to the target site in
the WT monomer.
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earlier. The I4/I6 binding site may therefore play a role in the
different activities inherent to the CXCL12 monomeric and
dimeric states. The conformational variability of this site may
also partially explain the relatively low affinity of the ligands
targeting this site, due to the high entropic penalty imposed on
the protein upon ligand binding. Ligands targeting the I4/I6
site may provide valuable chemical probes to study CXCL12 by
shifting the conformation equilibrium toward the monomeric
state, whereas this unique binding pocket offers additional
protein surfaces to augment chemokine binding for small
molecules targeting multiple binding hot spots.
Our results also demonstrate the ability of computational
approaches in identifying PPI binding hot spots and uncovering
novel ligands using structural information from various
experimental sources including NMR. In particular, they
suggest that computational solvent mapping techniques such
as FTMap can be used to uncover multiple druggable binding
sites of PPIs. The successes of the virtual screening experiments
demonstrate that the distinct binding features of PPIs can be
recognized and utilized by computational docking programs to
select specific binders. However, whereas both FTMap and
binding energy analysis suggest the I4/I6 binding site to be the
most druggable, its suitability as a hot spot for small molecule
ligand binding may be affected by its overall conformational
instability as discussed above. In addition, although the relative
binding energy analysis offered insights into the importance of
I4 in the interactions between CXCR4 and CXL12, the
calculations may have been unable to accurately capture the
conformations of the small protein and its peptide ligand and
therefore the subsequent contributions of some residues to
binding.
Figure 6. Novel small molecule ligands targeting sY12, sY21, and I4/I6
binding sites on CXCL12. sY21 binding site inhibitor (yellow) was
retrieved from crystallographic complex structure (PDB code 4UAI)
and superimposed to monomeric CXCL12 (PDB code 2N55). 1
(
purple) and 9 (cyan) bind to the I4/I6 and Y12 binding sites,
respectively, as predicted by docking. A distance of 8.5 Å separates the
sY21 binding site inhibitor from 9, and a distance of 8.7 Å separates 1
from 9.
number of positively charged side chains, that can distinguish
between weak binders including fragments and are thus suitable
for fragment-based inhibitor discovery. Moreover, 9, located in
the sY12 binding pocket, is 8.5 and 8.7 Å away from the sY21
binding site inhibitor and 1 in the I4/I6 pocket, respectively
NMR Structure in Ligand Discovery against a Flexible
Target. In this study, we describe the use of both a rigid and an
ensemble virtual screening strategy to identify novel ligands
against a potentially transient binding pocket (i.e., the I4/I6
binding site) in an NMR structure. In our previous studies,
NMR and X-ray crystal structures were used to uncover the
(
Figure 6). The close proximity of these molecules to one
another suggests that a fragment-based linking or growing
strategy can be applied to develop ligands that can target
multiple hot spots and achieve higher binding affinities. In
particular, molecules such as 9 can provide a central scaffold
that extends from the sY12 pocket into the binding sites for
sY21 and I4/I6 from CXCR4.
17
ligands for the sY21, and sY12 binding sites, respectively (A.
E. Getschman et al., unpublished results). Because of the
structural ambiguity, in particular concerning side chain
conformations, NMR structures have been considered less
suitable for molecular docking experiments than crystal
Each of the three binding sites also plays a different role in
the function of CXCL12 and correspondingly has varied ligand-
binding characteristics that may contribute to the different
affinities and activities of the individual ligands targeting each
site. On the basis of previous mutagenesis and chemical
modification studies, the sY21 binding site appears to be one of
the main contributors to the binding affinity of the interactions
32
structures. However, similar to previous studies using an
33
NMR ensemble to target protein active sites, our results
demonstrate that NMR structures, including different con-
formations of the ensemble, can be successfully utilized for
identifying novel small molecule ligands targeting PPIs. Our
particular studies have benefitted from the fact that the NMR
structures being used are complexes of the target protein
1
2,13
with CXCR4.
Correspondingly, the ligands identified
against this site are the strongest binders among all three
series of novel CXCL12 ligands, with affinities in the low
1
0,16,17
micromolar range.
The sY12 binding site is also involved
(
CXCL12) with a biological ligand (CXCR4 peptide). In
in the CXCR4 interaction but is equally important functionally
31
comparison, another virtual screening campaign using the apo
NMR structure of a different chemokine, CCL21, was
unsuccessful (unpublished data). Our analysis therefore
indicates that complex NMR structures contain enough
structural information for virtual screening. Additionally, it is
possible that certain conformations in the NMR ensemble may
be more suitable virtual screening templates than others
because of higher experimental accuracy or more druggable
features. However, systematic analysis is required to compare
the performance of various NMR conformations in virtual
screening and to develop methods to identify those that can
lead to higher hit rates.
for its association with heparin in the extracellular matrix.
Compounds targeting the sY12 binding site are less effective in
disrupting CXCL12−CXCR4 interactions when compared with
the ligands for the sY21 binding site but are instead able to
compete with heparin for CXCL12 binding (A. E. Getschman
et al., unpublished results). These molecules provide an
alternative opportunity to interfere with in vivo chemotaxis,
since heparin binding is used to protect CXCL12 from
proteolysis and to establish the chemokine gradient necessary
31
for adhesive migration. Meanwhile the focus of the current
study, the I4/I6 binding site, is only accessible in the
monomeric state and not in the dimeric state, as described
G
DOI: 10.1021/acs.jmedchem.5b02042
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and then visually inspected for complementarity. While certain
conformations in the NMR ensemble (i.e., conformation 18) were
not matched with any compounds in the top ranking results,
conformation 13 was the receptor template for the majority of the
top 2000 compounds (50% for the fragment subset and 77% for the
leadlike subset), possibly because of the particular arrangement of
positively charged functional groups that increased the positive
electrostatic potential in the binding pocket. Twenty-four compounds
were finally chosen for testing based on complementarity and purchase
availability. The compounds were 4, 5, 6, 7, 8, 2-(2-fluoro-4-
CONCLUSION
■
Our virtual screening approach has proven effective for
identifying ligands against multiple sites on the surface of a
small protein, CXCL12. Since each site also contributes to the
functional interaction with the CXCR4 receptor, their
proximity should enable fragment-based linking/growing/
merging strategies to develop potent and specific inhibitors of
CXCL12 by simultaneous targeting of multiple sites in future
studies. Our work also suggests similar computational
approaches can be applied to identify and target the PPI
binding hot spots of other proteins using fragment-based
methods.
2
6
sulfamoylphenoxy)acetic acid (ZINC C36948567),
2-
[
(
(carbamothioylamino)carbamoyl]cyclohexane-1-carboxylic acid
26
ZINC C19860584), N-(4-methylpyridin-2-yl)pyrrolidine-1-sulfona-
26
mide (ZINC C19909049), 4-[(4,6-dioxohexahydropyrimidin-2-
ylidene)amino]benzoic acid (ZINC C55134791), 2-(2,3-dihydro-1-
benzofuran-5-yl)-5-oxo-1-(pyridin-3-ylmethyl)pyrrolidine-3-carboxylic
2
6
EXPERIMENTAL SECTION
FTMap Analysis. FTMap analysis was performed using the
■
2
6
acid (ZINC C69779089), 2-(3-{[(cyclopropylcarbamoyl)methyl]-
18
sulfanyl}-5-(4-methylphenyl)-4H-1,2,4-triazol-4-yl)acetic acid (ZINC
FTMap computational map server. The constitutively monomeric
CXCL12 structure complexed to a nonsulfated N-terminal CXCR4
peptide (L55C/I58C) (PDB code 2N55) was uploaded into the
FTMap server and ran according to instructions provided (www.
2
6
C13035420), 4-methanesulfonamido-N-[(pyridin-3-yl)methyl]-
2
6
benzamide (ZINC C01063900), 2-(2-carbamimidamido-4-oxo-4,5-
dihydro-1,3-thiazol-5-yl)-N-(3-nitrophenyl)acetamide (ZINC
2
6
18
34
C20028245), 2-(2,3-dihydro-1-benzofuran-5-yl)-1-[(1-methylpyra-
ftmap.bu.edu). The results were visually inspected using PyMol.
zol-4-yl)methyl]-5-oxopyrrolidine-3-carboxylic acid (ZINC
Monomer A from dimeric CXCL12 structure complexed to a sulfated
N-terminal CXCR4 peptide (PDB code 2K05) was analyzed following
the same procedure.
Virtual Screening. Conformation 1 of the NMR ensemble of the
constitutively monomeric form of CXCL12 (L55C/I58C) (PDB code
2
6
C69492035), 2-(2-carbamimidamido-4-oxo-4,5-dihydro-1,3-thiazol-
26
5
-yl)-N-(4-nitrophenyl)acetamide (ZINC C20064260), 3-(4-me-
thoxyphenyl)-3-[(4-nitrophenyl)formamido]propanoic acid (ZINC
2
6
C00099591), 2-(2-carbamimidamido-4-oxo-4,5-dihydro-1,3-thiazol-
26
5
-yl)-N-(3-methoxyphenyl)acetamide (ZINC C13637710), 3-(6-
2
N55) complexed to the N-terminal CXCR4 peptide (p40) was used
methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamido)benzoic
for the rigid docking virtual screening experiment. Residues I4 and I6
from the CXCR4 peptide were used to specify the binding pocket and
generate the matching spheres. The spheres were chemically labeled
for matching based on ionization states and hydrogen bonding
properties of nearby protein residues. Both the fragment (575 530
compounds) and leadlike (4 552 896 compounds) subsets of the
ZINC small-molecule database (http://zinc.docking.org) were
docked into the binding pocket using DOCK 3.5.54
based on score. The top 1000 compounds were visually inspected for
complementarity, and 12 compounds were chosen for testing based on
complementarity and purchase availability. The compounds were 1, 2,
2
6
acid (ZINC C39361382), N-(3-cyanobenzenesulfonyl)-2-(2,5-
2
6
dimethylphenyl)acetamide (ZINC C69592086), N-({1-[2-(3,6-
dioxo-1,2,3,6-tetrahydropyridazin-1-yl)acetyl]piperidin-3-yl}methyl)-
2
6
methanesulfonamide (ZINC C48353628), 4-({3-[(2-methylprop-2-
en-1-yl)oxy]phenyl}sulfamoyl)-1H-pyrrole-2-carboxylic acid (ZINC
2
6
26
C66587652), 1-[4-(methanesulfonamido)-3-methylphenyl]-5-oxo-
27,28
26
and sorted
pyrrolidine-3-carboxylic acid (ZINC C65463160), 4-chloro-N-
[(furan-2-yl)methyl]-3-methanesulfonamidobenzamide (ZINC
2
6
C61341511), and 2-{8-[(4-fluorophenyl)amino]-1,3-dimethyl-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-7-yl}acetic acid (ZINC
2
6
3
, N-(1H-1,3-benzodiazol-2-yl)-2-{[(oxolan-2-yl)methyl]amino}-1,3-
thiazole-4-carboxamide (ZINC C44900490), (2S)-2-[3-(4-chloro-
H-indol-1-yl)propanamido]propanoic acid (ZINC C40310216), N-
C12443262). Purchased compounds were tested directly against
26
the protein without further purity analysis. The purity of 6 was
2
6
1
1
subsequently determined to be >90% by H NMR. Due to the lack of
{
4-[3-(3-cyanophenyl)prop-2-enoyl]phenyl}ethane-1-sulfonamide
material, we were unable to further purify this compound and retest it.
Because of the overall relatively low affinity of all the ligands against
the target binding site including 1 and the features of the binding site,
we deem it unlikely that the perturbations were caused by impurities of
6.
Synthesis of Compound 9. Aqueous sodium hydroxide (50% w/
v, 180 mg, 4.5 mmol) was added dropwise to a suspension of N-(4-
chlorobenzoyl)-N′-carbamoyl thiourea (10, 500 mg, 2.24 mmol) in
water (5 mL) as shown in Scheme S1. The reaction mixture was
stirred for 90 min at room temperature. The product was precipitated
by addition of glacial acetic acid (0.3 mL). The precipitate was filtered
2
6
(
ZINC C65565279), N-{4-[3-(3-hydroxyphenyl)prop-2-enoyl]-
26
phenyl}ethane-1-sulfonamide (ZINC C65566657), 1-(4-methylben-
zoyl)-N-(5-sulfanylidene-4,5-dihydro-1H-1,2,4-triazol-4-yl)piperidine-
26
3
-carboxamide (ZINC C28875196), 2-[(2E)-3-(4-methoxyphenyl)-
26
prop-2-enamido]propanoic acid (ZINC C03887304), 3-(2,5-dioxo-
2
6
1
-phenylimidazolidin-4-yl)propanoic acid (ZINC C06529806),
(
(
2S)-2-{[(3,4-dimethylphenyl)carbamoyl]amino}propanoic acid
26
ZINC C00534027), and (2E)-3-(4-oxo-3,4-dihydroquinazolin-2-
26
yl)prop-2-enoic acid (ZINC C17060315). Purchased compounds
were tested directly against the protein without further purity analysis.
The purity of 1 was subsequently determined to be >95% by H NMR.
1
and washed with distilled H O (3 × 5 mL) and then suspended in
2
For the ensemble virtual screening experiment, the same NMR
structure (PDB code 2N55) was used, and each conformation from
the ensemble was extracted and treated as a separate rigid docking
experiment. Residues I4 and I6 from the CXCR4 peptide were used to
specify the binding pocket in each conformation and generate the
matching spheres. The spheres were chemically labeled for matching
based on ionization states and hydrogen bonding properties of nearby
protein residues. Both the fragment and leadlike subsets of the ZINC
small-molecule database (http://zinc.docking.org) were docked into
the binding pocket of each conformation. The results from all
experiments were then combined and sorted by score. The overall
docking score for each compound against a particular receptor
conformation was used in the ranking of the combined results without
additional scaling or other modifications (such as considering the
internal energy of each receptor conformation). The top 2000
compounds were matched to their respective protein conformation
refluxing ethanol (5 mL) and filtered. 400 mg (87%) of the 6-phenyl-
4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (triazine, 11) was ob-
1
tained after the solid was dried under vacuum at 95 °C overnight. H
NMR (500 MHz, DMSO-d ) δ 13.19 (s, 1H), 12.78 (s, 1H), 8.09 (d, J
6
= 7.7 Hz, 2H), 7.68 (t, J = 6.8 Hz, 1H), 7.56 (t, J = 7.7 Hz, 2H).
Under an argon atmosphere and at room temperature, potassium
carbonate (828 mg, 6 mmol, 3 equiv) was added to a stirred solution
of 11 (410 mg, 2 mmol, 1 equiv) and ethyl bromoacetate (835 mg, 5
mmol, 2.5 equiv) in N,N-dimethylformamide (10 mL). The resulting
mixture was stirred at room temperature for 2 h. Water (20 mL) and
ethyl acetate (50 mL) were then added. The aqueous phase was
extracted three times with ethyl acetate (3 × 25 mL). The organic
phase was washed with brine three times (3 × 100 mL) and then dried
with anhydrous sodium sulfate and filtered. The filtrate was
concentrated and chromatographed on a silica gel column using
hexane and ethyl acetate as the eluent (4:1 to 1:1). Ethyl 2-((4-(2-
H
DOI: 10.1021/acs.jmedchem.5b02042
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
5
ethoxy-2-oxoethoxy)-6-phenyl-1,3,5-triazin-2-yl)thio)acetate (12, 560
mg, 74% yield) was obtained as colorless oil. H NMR (500 MHz,
were performed using 50 μM [U- N]-CXCL12WT with compound
concentrations ranging from 0 μM to 1600 μM prepared as described
1
1
15
CDCl ) δ 8.45 (d, J = 8.0 Hz, 2H), 7.58 (t, J = 7.3 Hz, 1H), 7.49 (t, J =
above and monitored using H− N SOFAST-heteronuclear multiple
3
7
.6 Hz, 2H), 4.99 (s, 2H), 4.26 (dq, J = 21.2, 7.1 Hz, 4H), 3.98 (s,
quantum coherence (HMQC) experiments, and chemical shift
36
2
H), 1.29 (td, J = 7.1, 2.3 Hz, 6H).
assignments were acquired from previously published sources.
1
.0 N aqueous LiOH solution (2 mL) was added to a solution of
Spectra were processed using in-house scripts, and chemical shift
diester 12 (100 mg, 0.27 mmol) in THF (8 mL) at 0 °C. The resulting
mixture was stirred for 1 h at the same temperature and evaporated to
dryness. The residue was acidified to pH 2 with aqueous hydrochloric
acid (1.0 N) to form a precipitate. The precipitate was purified by
preparative HPLC to give 2-((4-(carboxymethoxy)-6-phenyl-1,3,5-
tracking was performed using a combination of TitrView and CARA
2
2
1
15
software. The combined H/ N chemical shift perturbations were
2
2 0.5
calculated as ((5Δδ
the respective amide proton and nitrogen chemical shifts. Equilibrium
dissociation constants (K ) were determined using nonlinear fitting of
) + (ΔδNH) ) , where Δδ and ΔδNH represent
H
H
d
1
15
triazin-2-yl)thio)acetic acid (9, 25 mg, 29.4%) (Scheme S1). HPLC
the calculated H/ N chemical shift perturbations as a function of
compound concentration to a single-site quadratic equation (protein
concentration was held constant at 50 μM). For each compound, the
1
purity: 96.1%. H NMR (500 MHz, DMSO-d ) δ 8.36 (d, J = 7.4 Hz,
6
3
7
2
3
H), 7.66 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.7 Hz, 2H), 4.85 (s, 2H),
.97 (s, 2H). 13C NMR (100 MHz, DMSO-d ) δ 183.31 (s), 171.47
residues with the largest chemical shift perturbations were fitted
individually and the resulting K values and their respective errors were
d
averaged to produce the reported affinity and standard deviation.
6
(
(
s), 170.23 (s), 169.79 (s), 169.58 (s), 134.65 (s), 133.78 (s), 129.33
s), 129.14 (s), 64.95 (s), 33.86 (s).
Docking Pose Prediction of Compound 9. Monomer A of
conformation 1 in the NMR ensemble of the constitutively dimeric
form of CXCL12 (PDB code 2K05) complexed to the triply sulfated
N-terminal CXCR4 peptide (p38) was used to predict the binding
pose of 9 through docking. The sY12 residue from the CXCR4
peptide was used to specify the binding pocket and generate the
matching spheres. The spheres were chemically labeled for matching
based on ionization states and hydrogen bonding properties of nearby
protein residues. 9 was then docked into the matching spheres, and the
best pose was manually chosen based on complementarity.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b02042.
■
*
S
Binding energy analysis methods and results, ligand
discovery, and docking poses (PDF)
Purification and Sample Preparation of Recombinant
15
15
[
U- N]-CXCL12WT. Human [U- N]-CXCL12WT was purified as
AUTHOR INFORMATION
Corresponding Authors
B.F.V.: phone, (414) 955-8400; fax, (414) 955-6510; e-mail,
bvolkman@mcw.edu.
*Y.C.: phone, (813) 974-7809; fax, (813) 974-7357; e-mail,
ychen1@health.usf.edu
■
an N-terminal His6SUMO fusion protein in E. coli as described
17,35
previously.
Cells were grown in Terrific Broth and induced with 1
*
mM isopropyl β-D-1-thiogalactopyranoside (IPTG) prior to being
harvested. Cell pellets were lysed, and lysates were clarified by
centrifugation (12 000g for 20 min). The resolubilized inclusion body
pellets were then loaded onto Ni-NTA resin, and after 1 h proteins
were eluted with 6 M guanidinium chloride, 50 mM Na PO (pH 7.4),
3
4
Author Contributions
E.W.S. and A.M.N. contributed equally.
3
00 mM NaCl, 500 mM imidazole, 0.2% sodium azide, and 0.1% β-
⊥
mercaptoethanol. The eluate was pooled and refolded for 4 h before
cleavage of the His -SUMO fusion tag by Ulp1 protease overnight.
Author Contributions
6
The His -SUMO fusion tag and chemokine were separated using
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
6
cation-exchange chromatography (SP Sepharose Fast Flow resin GE
Healthcare UK Ltd.), and the eluate was subjected to reverse-phase
high-performance liquid chromatography as a final purification.
Proteins were frozen, lyophilized, and stored at −20 °C.
Automated Preparation of NMR Samples. Samples for NMR
analysis were prepared in groups of 8 (or fewer) compounds using a
Pal liquid handling robot configured for loading of 3 mm NMR sample
cells (Leap Technologies). For every 8 compounds, 33 mL of 50 μM
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is funded by Grant R01 GM097381 to B.F.V. and by
Grant CA173056 to R.L.
[U-15N]-CXCL12WT in a solution of 25 mM deuterated MES (pH =
6
.8), 10% (v/v) D O, and 0.02% (w/v) NaN was prepared in a glass
2
3
vial. Upon arrival and initial inventory each compound was brought up
to a concentration of 100 mM in deuterated DMSO and stored in
amber glass vials. For each compound an amount of 20 μL each of 80
mM and 7.5 mM stocks was prepared in plastic vials from the initial
ABBREVIATIONS USED
PPI, protein−protein interaction; CXCL12, CXC chemokine
ligand 12; CXCR4, CXC chemokine receptor 4
■
1
00 mM stock. Compounds were capped and arranged in a plastic rack
(
6 × 9) starting with the 7.5 mM stock, then the 80 mM stock for each
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