Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

Article abstract of DOI:10.1021/ja110571r

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site

Full text of DOI:10.1021/ja110571r

ARTICLE
pubs.acs.org/JACS
Fragment Growing Induces Conformational Changes in
Acetylcholine-Binding Protein: A Structural and Thermodynamic
Analysis
Ewald Edink,†,# Prakash Rucktooa,‡,# Kim Retra,† Atilla Akdemir,† Tariq Nahar,§ Obbe Zuiderveld,†
Renꢀe van Elk,§ Elwin Janssen,^ Pim van Nierop,§ Jacqueline van Muijlwijk-Koezen,† August B. Smit,§
Titia K. Sixma,‡ Rob Leurs,† and Iwan J. P. de Esch*,†
†Leiden/Amsterdam Center of Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences,
VU University Amsterdam, The Netherlands
‡Divison of Biochemistry, Netherlands Cancer Institute, The Netherlands
§Department of Molecular and Cellular Neurobiology, Center for Neurogenomics & Cognitive Research, VU University Amsterdam,
The Netherlands
^Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, The Netherlands
S Supporting Information
b
ABSTRACT: Optimization of fragment hits toward high-
affinity lead compounds is a crucial aspect of fragment-
based drug discovery (FBDD). In the current study, we
have successfully optimized a fragment by growing into a
ligand-inducible subpocket of the binding site of acetyl-
choline-binding protein (AChBP). This protein is a solu-
ble homologue of the ligand binding domain (LBD) of
Cys-loop receptors. The fragment optimization was moni-
tored with X-ray structures of ligand complexes and systematic
thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC).
Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles,
are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis
provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and
fragment-based discovery approaches.
’ INTRODUCTION
to develop new ligands that bind to acetylcholine-binding pro-
tein (AChBP). This water-soluble pentameric protein is widely
recognized as a structural homologue of the ligand binding
domain (LBD) of Cys-loop receptors.12,13 Currently, AChBPs
from different species such as Lymnaea stagnalis (Ls-AChBP),14-16
Aplysia californica (Ac-AChBP),17,18 and Bulinus truncatus (Bt-
AChBP)19 have been identified and crystallized, providing high-
resolution structural models of the extracellular domains of the
pentameric ligand-gated ion channels (LGICs) of the Cys-loop
family, such as nicotinic acetylcholine receptors (nAChRs), GA-
BAA-, serotonin 5-HT3-, and glycine receptors.13
In the past decade, fragment-based drug discovery (FBDD)
has become a well-established method in the field of drug
discovery.1-3 Compared to traditional high-throughput screen-
ing (HTS), FBDD is based on screening of smaller libraries of
compounds (typically containing 1000-5000 fragments) with
lower molecular weights (i.e., smaller than 300 Da).4 Fragment-
based screening results in a better coverage of chemical space and
usually higher hit rates than HTS.3,5 Identified fragment hits are
either optimized by linking or growing. In fragment linking, two
simultaneously binding fragments are connected via a chemical
linker. Unfortunately, the expected increase in affinity is often
compromised by perturbation of the binding modes of the
separate fragments or strain in the linker used to connect the
fragments.6,7 The preferred hit-optimization strategy in FBDD
has become fragment growing.1,8 In iterative cycles, additional
features are added to the hit fragment, leading to more-potent
compounds. Structural biology has been shown to be crucial to
guide the optimization of fragment hits toward novel and potent
drugs.1,3,9-11 In the current study, we employ FBDD approaches
In a recent study, tropine derivatives were identified as AChBP
ligands using an in silico screening protocol.18 A structural ana-
logue, the benzoate substituted nortropine fragment 1, exhibits
good ligand efficiency (LE)20 of 0.43 kcal mol-1 per heavy atom
3
for Ac-AChBP and is therefore considered to be a good starting
point for further optimization (Figure 1). In the current study, a
Received: November 24, 2010
Published: February 15, 2011
r
2011 American Chemical Society
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partially overlapping binding modes of hit fragment 1 and lobe-
line (2), we designed a fragment growing optimization study to
induce the tyrosine-flip and grow the fragment into the lobe-
line pocket (Figure 1). The ligand-induced opening of binding
site subpockets represents an interesting challenge and a poten-
tially rewarding drug discovery opportunity.23 Using X-ray ana-
lysis of cocrystal structures, molecular modeling, site-directed
mutagenesis, AChBP species differences, surface plasmon reso-
nance (SPR) biosensor analysis, and isothermal titration calo-
rimetry (ITC), we show that the thermodynamic signature of
ligand binding changes drastically when the lobeline pocket is
addressed. The obtained results illustrate that thermodynamic
analysis of ligand binding provides important information about
the binding mode and reveal the value of monitoring thermo-
dynamic aspects during fragment growing.
’ RESULTS AND DISCUSSION
Chemistry. The designed compounds were prepared accord-
ing to the route depicted in Scheme 1. Acylation of tropine with
benzoyl chloride afforded tropine benzoate (7) in an excellent
yield.24 Subsequent demethylation using R-chloroethyl chloro-
formate gave the corresponding nortropinyl ester (1).25 The R-
hydroxylphenetyl extended fragment was synthesized by heating
1 with the (R)-enantiomer of phenyloxirane in the microwave.26
The enantiomeric excess (ee) of 3 was determined at 98% by
chiral HPLC. Reductive amination of phenylacetaldehyde with
nortropine benzoate 1 using sodium triacetoxyborohydride as
the reducing agent gave 4.27 Finally, treatment of 3 with iodo-
methane followed by recrystallization from chloroform resulted
in isolation of the endo-R-(R)-hydroxyphenetyl-substituted qua-
ternary ammonium derivative (6). The endo-configuration of the
R-(R)-hydroxyphenetyl substituent was confirmed by 2D NMR.
Structure-Based Design of a Novel ligand that Interacts
with the Lobeline Pocket. Fragment 1, which is a structural
analogue of hits that have been identified in an earlier study,18
exhibits good LE (0.43 kcal mol-1 per heavy atom) and was
3
cocrystallized with Ac-AChBP. A 3.65 Å resolution crystal struc-
ture was obtained, with good density for the ligand (part a of
Figure 2, and part a in both Figures S2 and S3 of the Supporting
Information) despite the low resolution. The ability to use 5-fold
restraints in crystallographic refinement allowed confident build-
ing ofthe compoundintoelectron density, thus enabling structure-
based optimization. The structure displays one Ac-AChBP pentamer
per asymmetric unit where the C-loops adopt a closed conformation
over all five binding sites. Fragment 1 is present at all five protomer-
protomer interfaces, with similar orientations. The ligand is stabilized
through hydrophobic interactions with Cys188, Cys189, Gln55,
Ile116, and Tyr91, and through a hydrogen bond between the proto-
nated amine atom of 1and the backbone carbonyl from Trp145 (part
a of Figures S1-S3 of the Supporting Information). When compared
with lobeline-bound Ac-AChBP (part b of Figure 1, PDB: 2BYS), the
fragment’s benzoate group is tilted ∼50ꢀ with respect to lobeline’s
1-phenylethanone moiety and stacked against the vicinal cys-
teines at the tip of loop C, placing the loop in a more open
conformation (2.7 Å when comparing Cys188 CR positions).
The structure further reveals that gatekeeper residue Tyr91 is in
the g-conformation, making the lobeline pocket inaccessible. From
the complex structures, we predicted that further optimization of
the fragment could be achieved by opening of and growing into the
lobeline pocket (Figure 1). To address this subpocket, the fragment
was merged with the R-hydroxyphenetyl group of lobeline (2),
Figure 1. Fragment optimization strategy. Surface representations of
the crystal structures of fragment 1-bound Ac-AChBP (a) and of lobeline-
bound Ac-AChBP (PDB 2BYS)16 (b). (a) In the fragment 1-Ac-AChBP
complex, Tyr91 (orange) is stabilized in the g- conformation through a
hydrogen bond with Ser144, rendering the lobeline pocket inaccessible.
(b) However, Tyr91 adopts a t conformation in the lobeline-Ac-AChBP
complex interacting with Tyr53 and Ser165 through hydrogen bonds,
thus leading to the opening of the lobeline pocket. (c) The superposition
of the fragment 1 and lobeline (2) molecules indicates that the fragment
may be grown into the lobeline pocket by decorating the tropine nitrogen
with the R-hydroxyphenetylmoietyoflobeline generating compound (3).
cocrystal complex of fragment 1 and Ac-AChBP was generated,
thereby enabling structure-based optimization. Comparison with
previously obtained cocrystal complexes reveals conformational
changes of the target protein upon ligand binding, mainly with
respect to loop C, which closes the binding site upon agonist
binding.15,17-19,21 A unique conformational change is observed
for AChBP while binding to lobeline, leading to the opening of a
subpocket that enables the binding of the R-hydroxyphenetyl
moiety of the ligand.17 More specifically, the subpocket, which
we will refer to as the lobeline pocket, becomes accessible after a
change in the rotameric state of Tyr91 (g- to t conformation,22
hereafter referred to as the tyrosine-flip). Considering the
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Scheme 1
resulting in 3 (Figure 1). Molecular docking using GOLD (version
4.0)28 suggests that the extended fragment 3 can adopt a bind-
ing mode in which the lobeline pocket is being addressed by the
R-hydroxyphenetyl moiety (Figure S4 of the Supporting Informa-
tion). Because these in silico results were promising, the designed
compound was synthesized and screened for AChBP affinity.
X-ray Structures Confirm Insertion into the Lobeline
Pocket. Extension of fragment 1 with an R-(R)-hydroxyphene-
tyl moiety yields 3 and resulted in a ∼50-fold increase in affinity
(pKi = 7.0 ( 0.1), although the LE drops slightly from 0.43 to
that observed in the Ac-AChBP-lobeline complex and is involved
in extensive hydrophobic interactions with Asp195, Tyr91, Lys141,
Gly143, and Thr89 (part b of Figure 2, and part b in Figures S1-S3 of
the Supporting Information). As a result, the experimentally deter-
mined binding mode corresponds very well to the predicted bind-
ing mode that was obtained by molecular docking (rmsd of 1.1 Å).
Nevertheless, the experimentally determined binding mode differs
from the predicted binding mode in that the hydroxyl group of 3is not
engaged in hydrogen bonding to the backbone carbonyl oxy-
gens of Ser144 or Trp145 but instead is involved in van der Waals
interactions with Tyr193 (Figure S4 of the Supporting Information).
Because the hydroxyl group of the optimized fragment 3 is not
involved in formation of hydrogen bonds with the binding site, it
was expected that its removal would lower the desolvation penal-
ty and thereby result in an increase in binding affinity. The nor-
hydroxyl derivative (4) was therefore synthesized and screened
for AChBP affinity. Indeed, 4 exhibited higher AChBP affinity
(pKi = 7.5 ( 0) than 3 and when compared with the starting frag-
ment 1, a 150-fold increase in binding affinity is observed (Figure 4).
To determine if 4 induces the tyrosine-flip and interacts with the
lobeline pocket, an additional cocrystal structure was generated.
The 4-Ac-AChBP structure was solved with a resolution of 3.30 Å
and shows an almost identical binding mode to 3 (rmsd of 0.6 Å),
exemplifying that a hydroxyl functionality is not required for
openingofandinsertionintothelobelinepocket(partc ofFigure2,
and part c of both Figures S2 and S3 of the Supporting Information).
Site-Directed Mutagenesis Study on Stabilization of the
Tyrosine-Flip. Visual inspection of the Ac-AChBP cocrystal
structures of the optimized fragments 3 and 4 and the structure
of the Ac-AChBP-lobeline complex by Hansen and co-workers17
shows that the side chains of two residues (Tyr53 and Ser165)
are likely to be involved in stabilization of the flipped state of
0.37 kcal mol-1 per heavy atom (part a of Figure 4). To confirm
3
successful insertion into the lobeline pocket, a crystal structure of
the optimized fragment (3) bound to Ac-AChBP was generated.
The 3.59 Å resolution crystal structure shows that fragment 1 was
successfully grown into the lobeline pocket (part b of Figure 2,
and part b in both Figures S2 and S3 of the Supporting Informa-
tion). The structure displays one pentamer in the asymmetric
unit with each of the protomer-protomer interfaces binding one
ligand molecule. The benzoate substituted nortropine moiety
adopts an orientation similar to 1, although displaced ∼1 Å deeper
into the binding site, hence bringing loop C into a conformation
intermediate between those observed for fragment 1 and lobeline
Ac-AChBP complexes. Interestingly, an ion, most likely a chloride,
could be refined in the position occupied by the lobeline 1-phe-
nylethanone moiety from the lobeline-Ac-AChBP complex. Ex-
tension of fragment 1 with an R-(R)-hydroxyphenetyl moiety
(resulting in 3), induces a change in rotameric state of Tyr91 (g- to
t-conformation) that opens the lobeline pocket. This tyrosine-flip
is stabilized by hydrogen bond formation between the phenolic
oxygen of Tyr91 and the side chain hydroxyl groups of Tyr53 and
Ser165. The R-(R)-hydroxyphenetyl moiety responsible for the
insertion into the lobeline pocket displays an orientation similar to
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Figure 2. Growing fragment 1 into or away from the lobeline pocket. Cartoon representations of the Ac-AChBP complexes with fragment 1
(a), optimized fragments 3 (b) and 4 (c) and quaternary ammonium derivative 6 (d). The SigmaA weighted 2Fo-Fc electron density map at a 1σ level,
carved 2 Å around the ligand is depicted as a mesh. The small sized fragment (1) does not interact with or induce an opening of the lobeline pocket and
maintains the gatekeeper Tyr91 (orange sticks) into a g- state stabilized by a hydrogen bond with the Ser144 carbonyl (a). Growing into the lobeline
pocket with 3 (b) or 4 (c) induces a change in rotameric state of Tyr91 (g- to t conformation), which opens the lobeline pocket. This tyrosine-flip is
stabilized by hydrogen bond formation with the side chains of Tyr53 and Ser165 (b, c). The green sphere in (b) corresponds to a chloride ion.
Quaternization of the tropine nitrogen of 3 with a methyl group, producing 6 locks the R-(R)-hydroxyphenetyl moiety in an endo configuration, unable
to induce the opening of the lobeline pocket (d).
gatekeeper residue Tyr91 by hydrogen bond formation with the
phenolic oxygen of Tyr91 (part b of Figure 1 and parts b and c of
Figure 2). Superposition of X-ray structures of Ac-AChBP (PDB:
2BYS)17 with its species variant Ls-AChBP (1UW6)16 shows that
the tyrosine-flip stabilizing residues, Tyr53 and Ser165, correspond
to a tryptophan and a tyrosine (Trp53 and Tyr164) respectively in
Ls-AChBP (Figure 3). The side chains of Trp53 and Tyr164 in Ls-
AChBP are positioned in such a way that these residues are not able
to stabilize the tyrosine-flip and hamper opening of the lobeline
pocket in Ls-AChBP (Figure 3). To study the importance of Tyr53
and Ser165 in stabilizing the tyrosine-flip in Ac-AChBP, site-
directed mutagenesis experiments were performed in which these
residues were substituted for their Ls-AChBP counterparts, a try-
ptophan and a tyrosine, respectively. Affinity measurements of Ac-
AChBP Y53W and S165Y mutants as well as wild-type Ac- and Ls-
AChBPs were performed with nicotine and acetylcholine, which
cannot interact with the lobeline pocket because of their small size,
and with optimized fragment 3 and lobeline, which have been
shown to interact with the lobeline pocket in Ac-AChBP from co-
crystal structures. The results summarized in Table 1, indicate that
Ser165 is essential for stabilizing the tyrosine-flip. Substitution of
Tyr53 with a nonstabilizing tryptophan decreases the affinity of 3
by 5-fold and generates a small increase in affinity for the three
other ligands. The switch from Ser165 to a nonstabilizing tyrosine
has a more dramatic effect. The affinity for lobeline and compound
3 decreases by >400- and 25-fold respectively, whereas the affinities
of acetylcholine and nicotine that do not interact with the lobeline
pocket are hardly affected. Strikingly, this single point mutation in
Ac-AChBP (S165Y) renders the affinity of 3 and lobeline very
similar to wild type Ls-AChBP. These results provide strong evi-
dence for a less accessible lobeline pocket in Ls-AChBP compared
to Ac-AChBP.
Thermodynamic Analysis of Fragment Optimization
Using SPR and ITC. The thermodynamic aspects of growing a
fragment into the ligand-induced lobeline pocket of Ac-AChBP
were studied by surface plasmon resonance (SPR) biosensor
analyses, using an assay developed by Geitmann et al.,29,30 at five
different temperatures (15, 20, 25, 30, and 35 ꢀC) and isothermal
titration calorimetry (ITC). SPR at different temperatures allows
dissection of binding affinity into the separate enthalpic and
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entropic contributions using van’t Hoff analysis31-33 (van’t
Hoff plots are depicted in Figure S5 of the Supporting Infor-
mation), whereas ITC analysis is directly related to enthalpic
contribution and free energy and entropy are derived through
titration curves34 (Figure S6 of the Supporting Information).
Our results on a large set of ligands show that these fundamen-
tally different techniques result in very comparable profiles
(Figure 4, Table 2). A major advantage of using SPR biosensor
analysis is that this method requires substantially lower amounts
of target protein compared to ITC.
Extending fragment 1 with an R-(R)-hydroxyl substituted
phenetyl moiety (leading to 3) results in a significant shift in
thermodynamic binding signature where a large increase in
favorable enthalpic contribution to the binding is observed for
the optimized fragment 3 (part c of Figure 4 and Table 2). An
even more enhanced increase in favorable enthalpic contribution
is observed when extending the fragment with a phenetyl moiety
(leading to 4). These favorable enthalpic contributions are com-
pensated to some extent by an unfavorable entropic contribution
to the binding for both optimized ligands. A rationale for the
difference in the enthalpic contributions to the binding between
the two optimized ligands can be derived from crystal structures
of the complexes of the two ligands to Ac-AChBP. These show
that the hydroxyl group of 3 displays an unsatisfied hydrogen
bond whereas the buried surface area is similar in both cases
(∼460 Å2 calculated using the PISA webserver35), possibly
causing the small difference in enthalpy.
Combining the thermodynamic analysis with the identified
species differences, it was anticipated that complex formation
to Ls-AChBP (nonstabilized tyrosine-flip) of lobeline, and opti-
mized fragments 3 and 4 would be driven by less favorable en-
thalpy compared to Ac-AChBP (stabilized tyrosine-flip). Ther-
modynamic analysis using SPR biosensor analysis and ITC,
confirmed our hypothesis. Binding of ligands that interact with
the lobeline pocket in Ac-AChBP (3, 4 and lobeline) is char-
acterized by more favorable enthalpy when compared to Ls-
AChBP (parts c and d of Figure 4, Table 2). This is in contrast
with ligands that do not interact with the lobeline pocket. Their
binding to Ls-AChBP is driven by similar (nicotine) or more
(fragment 1) favorable enthalpy compared with binding to Ac-
AChBP.
In addition, these results show that growing fragment 1 into
the ligand-induced lobeline-pocket in Ac-AChBP renders the
fragment selective for Ac-AChBP over Ls-AChBP. A plausible
explanation for these observations that is in line with the site-
directed mutagenesis results, is a differential stabilization of the t-
conformation of gatekeeper residue between the AChBP species
variants. As such, the lobeline pocket is better accessible in Ac-
AChBP compared to Ls-AChBP and can be targeted as a species-
selectivity subpocket.
Ligand-Based Chemical Validation. Next to binding mode
characterization by considering site-directed mutagenesis and
species differences of the protein target, a ligand-based chemical
validation of the conclusions was pursued. The X-ray cocrystal
structure of the optimized fragment 3 shows that in order to
interact with the lobeline pocket the R-(R)-hydroxyphenetyl
moiety takes an exo-configuration with respect to the tropine
moiety, that is the substituent that is inserted into the lobeline
pocket is pointing toward the tropine ethylene bridge (part b of
Figure 2). Quaternization of the tropine nitrogen atom of 3 by
introduction of an additional methyl substituent, prevents pyr-
amidal inversion and locks the R-(R)-hydroxyphenetyl moiety in
the opposite endo-configuration, which is anticipated to prevent
this derivative from interacting with the lobeline pocket (chem-
ical synthesis and characterization is described in the Supporting
Information). Interestingly, pharmacological screening using
[3H]-epibatine displacement shows that upon quaternization
of the basic amine of 3 affording compound 6, the affinity for Ac-
AChBP is lowered 10-fold, whereas affinity for Ls-AChBP
increases 6-fold (part a of Figure 4). This minor modification,
that is addition of a single methyl substituent renders 3 from
8-fold Aplysia-selective to 8-fold selective for the Lymnaea-
AChBP species. The cocrystal structure of the quaternary
methylammonium derivative 6 with Ac-AChBP was solved to a
resolution of 3.25 Å and provides an explanation for the observed
change in AChBP species selectivity. Quaternary ammonium
Figure 3. AChBP species differences in stabilization of the tyrosine-flip.
The superposition of crystal structures of Ac-AChBP (in silicon) and Ls-
AChBP (in blue) suggests that Trp53 and Tyr164 in Ls-AChBP (repre-
sented as blue sticks) cannot stabilize the tyrosine-flip contrary to Tyr53
and Ser165 in Ac-AChBP (represented as silicon sticks).
Table 1. Investigation of AChBP Species Differences Using Site-Directed Mutagenesis
pKi ( SEMa
Ac-wt
Ac-Y53W
Ac-S165Y
Ls-wt
R-lobeline (2)
3
8.5 ( 0.1
7.0 ( 0.1
4.0 ( 0.1
5.6 ( 0.1
8.7 ( 0.1
6.3 ( 0.1
4.5 ( 0.1
6.3 ( 0.1
5.9 ( 0.1
5.6 ( 0.1
4.3 ( 0.1
5.5 ( 0.1
6.2 ( 0.1
6.2 ( 0.1
5.2 ( 0.1
6.4 ( 0.1
acetylcholine
nicotine (5)
a pKi values (SEM over 3 radioligand displacement experiments using [3H]-epibatidine.
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Figure 4. (a) Chemical structures and binding affinities for Ac-AChBP and Ls-AChBP as determined by [3H]-epibatidine displacement of the
compounds that were evaluated using SPR biosensor analysis and ITC. Ligand efficiency (LE) of each compound is depicted between brackets in
kcal mol-1 per heavy atom. (b) Representative sensorgrams of the compounds binding to Ac-AChBP at different concentrations at 25 ꢀC.
3
Thermodynamic profiles for ligand binding to Ac-AChBP (c) and Ls-AChBP (d) were obtained using SPR biosensor analysis (dark bars ( SEM) and
ITC (light bars ( fitting errors) and are represented in bar charts. Shown are the changes that occur upon ligand binding in Gibbs energy (ΔGꢀ) (SPR,
dark blue; ITC, light blue), enthalpy (ΔHꢀ) (SPR, dark green; ITC, light green) and entropic contributions (-TΔSꢀ) (SPR, dark red; ITC, light red).
All thermodynamic parameters shown are in kcal mol-1. (e) Representative sensorgrams of the four compounds binding to Ls-AChBP at different
3
concentrations at 25 ꢀC.
derivative 6 was present in the five protomer-protomer inter-
faces with similar orientations, showing that the quaternized
ligand does not interact with the lobeline pocket. In this complex,
Tyr91 adopts a g- conformation with a χ2 value of -30ꢀ, and is
stabilized through a hydrogen bond between the phenolic
hydroxyl group and the carbonyl group from the Ser144
backbone (part d of Figure 2, part d of both Figures S2 and S3
of the Supporting Information). Such an orientation of the Tyr91
side chain is indicative of an inaccessible lobeline pocket.
Furthermore, in this complex Tyr91 adopts a different rotamer
state from the one observed in the Ac-AChBP- complex with
fragment 1 (χ2 = -85ꢀ) likely to accommodate the methyl group
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Table 2. Thermodynamic Parameters of Binding at 25 ꢀCa
compound
method
pKD
ΔGꢀ(kcal mol-1)
ΔHꢀ (kcal mol-1)
-TΔSꢀ (kcal mol-1)
3
3
3
Ac-AChBP
1
3
4
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
SPR
ITC
4.7 ( 0.1
5.9 ( 0.2
6.5 ( 0.1
7.4 ( 0.1
6.9 ( 0.1
7.3 ( 0.2
7.5 ( 0.1
8.3 ( 0.3
5.3 ( 0.1
6.1 ( 0.1
5.5 ( 0.1
6.0 ( 0.4
6.1 ( 0.1
6.7 ( 0.2
6.0 ( 0.1
6.6 ( 0.2
6.8 ( 0.1
7.2 ( 0.2
6.1 ( 0.1
6.6 ( 0.1
6.5 ( 0.1
7.3 ( 0.2
7.0 ( 0.1
7.2 ( 0.3
-6.5 ( 0.1
-8.0 ( 0.2
-8.8 ( 0.2
-10.1 ( 0.1
-9.3 ( 0.1
-9.9 ( 0.2
-10.2 ( 0.1
-11.3 ( 1.0
-7.3 ( 0.1
-8.3 ( 0.1
-7.5 ( 0.1
-8.2 ( 0.4
-8.4 ( 0.1
-9.1 ( 0.2
-8.2 ( 0.1
-9.0 ( 0.2
-9.3 ( 0.1
-9.8 ( 0.3
-8.4 ( 0.1
-9.0 ( 0.2
-8.9 ( 0.1
-10.0 ( 0.2
-9.6 ( 0.1
-9.8 ( 0.4
-2.1 ( 0.1
-4.5 ( 0.4
-14.2 ( 0.3
-12.3 ( 0.1
-15.0 ( 0.5
-16.0 ( 0.3
-8.4 ( 0.8
-13.7 ( 0.3
-11.1 ( 1.0
-12.5 ( 0.3
-4.8 ( 1.1
-3.8 ( 0.5
-7.5 ( 0.5
-12.3 ( 0.3
-5.5 ( 1.0
-7.0 ( 0.2
-11.1 ( 0.6
-10.9 ( 0.3
-5.4 ( 0.5
-7.4 ( 0.2
-11.0 ( 1.2
-14.5 ( 0.3
-8.6 ( 1.1
-11.0 ( 0.3
-4.5 ( 0.1
-3.5 ( 0.7
5.4 ( 0.5
2.3 ( 0.1
5.6 ( 0.6
6.1 ( 0.5
-1.9 ( 0.8
2.3 ( 1.4
3.8 ( 1.1
4.2 ( 0.2
-2.7 ( 1.2
-4 ( 1
R-lobeline (2)
(S)-nicotine (5)
6
Ls-AChBP
1
-0.9 ( 0.4
3.2 ( 0.5
-2.7 ( 1.0
-2.0 ( 0.3
1.8 ( 0.5
1.1 ( 0.5
-3.0 ( 0.5
-1.6 ( 0.5
1.3 ( 1.3
4.5 ( 0.5
-1.0 ( 1.1
1.2 ( 0.6
3
4
R-lobeline (2)
(S)-nicotine (5)
6
a SPR values are ( SEM over multiple experiments (n = 3-6). ITC values are ( fitting errors.
tethered to the amine of VUF11438. The quaternized nitrogen of
ligand 6 can make cation-π interactions with Trp145, Tyr91,
Tyr186, Tyr193, and Tyr53, whereas a hydrogen bond is made
between the hydroxyl group of the ligand and of Tyr53. The
benzoate moiety of 6 is oriented differently in the binding site in
an intermediate position relative to the benzoate of optimized frag-
ments 3 or 4 and the lobeline 1-phenylethanone moiety, where it
interacts via hydrophobic contacts with Ile116, Met114, and the
vicinal Cys188 and Cys 189 at the tip of loop C.
In line with these findings, is the thermodynamic data that
reveals that the favorable enthalpy of binding of compound 3 to
Ac-AChBP is dramatically reduced upon quaternization, leading
to ligand 6. However, the entropy of binding becomes more
favorable limiting the loss in affinity upon quaternization of 3 to
10-fold when binding to Ac-AChBP (parts a and c of Figure 4). In
the case of Ls-AChBP and in contrast with Ac-AChBP, quater-
nization of compound 3 affords a favorable change in enthalpy.
As a result, the quaternary ammonium derivative 6 binds with
more favorable enthalpy to Ls-AChBP compared to Ac-AChBP.
Similar to what is observed with ligands not interacting with the
lobeline pocket such as fragment 1, but not with ligands addres-
sing the lobeline pocket such as lobeline 2, 3, and 4. The more
favorable enthalpic contribution possibly arises from the differ-
ences in the complementary binding sites of Ls- and Ac-AChBP,
such as the replacement of Tyr53 by a tryptophan or of Ile116 by
a methionine. Such variations are likely to significantly alter the
interaction interface with the binding site and may be of influence
to the binding mode.
thermodynamic analysis and site-directed mutagenesis results,
provides evidence that the change in thermodynamic binding
signature upon fragment optimization is indicative of interactions
with the ligand-induced AChBP subpocket. Ligand 4 binds with
similar affinity to Ac-AChBP and Ls-AChBP. The affinity data
therefore does not provide any indication for a difference in
binding modes. However, the changes in binding enthalpy upon
extending fragment 1 with a phenetyl moiety (resulting in 4) are sub-
stantially different for Ac-AChBP (ΔΔHꢀ = -12.9 kcal mol-1
3
(SPR), -11.5 kcal mol-1 (ITC)) compared to Ls-AChBP
3
(ΔΔHꢀ -3.6 kcal mol-1 (SPR), þ1.4 kcal mol-1 (ITC)).
3
3
The significant favorable change in enthalpy for Ac-AChBP,
upon extending fragment VUF10663 with a phenetyl moiety is
likely to result from interactions with the ligand-induced AChBP
subpocket. The site-directed mutagenesis results strongly indi-
cate that the lobeline pocket is less accessible in Ls-AChBP com-
pared to Ac-AChBP. Therefore, we propose, that because of a
distinct binding mode in Ls-AChBP, in which the lobeline pocket
is not addressed, no significant favorable change in enthalpy,
upon extension of fragment 1 by a phenetyl moiety is observed.
In line with this proposal are the thermodynamic changes upon
growing fragment 1 into the quaternary ammonium derivative 4.
This chemical modification prevents the ligand from interacting
with the lobeline pocket in Ac-AChBP, and yields similar changes
in enthalpy for Ac-AChBP (ΔΔHꢀ -2.7 kcal mol-1 (SPR),
3
-1.1 kcal mol-1 (ITC)) and Ls-AChBP (ΔΔHꢀ -1.1 kcal mol-1
3
3
(SPR), þ1.3 kcal mol-1 (ITC)) (Figure 5). As such, these results
3
illustrate that more than affinity data alone, thermodynamic
analysis and focus on enthalpic and entropic contributions during
fragment optimization can provide valuable information on the
Integrating the X-ray data on the Ac-AChBP cocrystal com-
plexes of the optimized fragments 3 and 4 with the
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Journal of the American Chemical Society
ARTICLE
(pdb: 2W8E)18 shows that residue Tyr91 is in the g-conforma-
tion and functions as a gatekeeper making the lobeline pocket
inaccessible. It is therefore likely that, before complex formation,
the lobeline pocket is poorly solvated. A significant part of the
extensive favorable change in enthalpy may therefore result from
strong van der Waals interactions between the phenetyl moiety of 4
and the lobeline pocket that are not compensated by the solvent.
Besides providing insight into the thermodynamic aspects of
fragment growing, our investigations also reveal that growing the
fragment 1 into the lobeline pocket renders the fragment selec-
tive for Ac-AChBP, whereas introduction of an additional methyl
substituent preventing interactions with the lobeline pocket
reverses the AChBP species selectivity back to Lymnaea. Thus,
selectivity for Ac-AChBP over Ls-AChBP can be achieved by
addressing the lobeline pocket. The subtle differences in protein
conformational changes that induce the lobeline pocket may be
of interest in the design of subtype-selective ligands for human
nicotinic receptors as well. The gatekeeper tyrosine residue is
conserved among the human nAChR subtypes, whereas the
residue (Ser165 in Ac-AChBP) that stabilizes the open lobeline
pocket conformation is located in a highly variable region. As can
be seen from part a of Figure 4, lobeline exerts a 250-fold selec-
tivity for Ac-AChBP (accessible lobeline pocket) over Ls-AChBP
(inaccessible lobeline pocket) and ∼1000-fold selectivity for
R4β2 over R7 nAChRs.39,40 Differential stabilization of the rota-
meric states of the gatekeeper tyrosine residue may provide
an explanation for the observed nAChR subtype selectivity of
lobeline and the lobeline pocket may be targeted as a subtype-
selectivity pocket.
’ CONCLUSIONS
The work described here illustrates that fragment growing can
trigger ligand-induced conformational changes of the target
protein. The obtained results strongly indicate that the distinct
changes in thermodynamic binding signatures upon fragment
optimization between Ac-AChBP and Ls-AChBP result from a
difference in binding modes of the optimized fragments. We
conclude that, more than affinity data alone, dissection of binding
affinity into the separate enthalpic and entropic contributions
provides valuable information with regard to the binding mode of
a ligand. Furthermore, our studies show that thermodynamic
analysis, enabled by state-of-the-art technologies such as ITC
and SPR biosensor analysis, in our hands give comparable results.
Altogether, this study illustrates that, in combination with detailed
structural information (X-ray of cocrystals), thermodynamic data
provides crucial insights that enable efficient fragment optimization.
Figure 5. Thermodynamic analysis provides indication of successful
insertion into the lobeline pocket. Chemical structures and binding
modes as determined by X-ray analysis of Ac-AChBP cocrystal struc-
tures and the thermodynamic binding signatures for Ac-AChBP and Ls-
AChBP of optimized fragment 4 (a) fragment 1 (b) and quaternary
ammonium derivative 6 (c) as determined by SPR biosensor analysis
(dark bars ( SEM) and ITC (light bars ( fitting errors). Shown are the
changes that occur upon ligand binding in Gibbs energy (ΔGꢀ) (SPR,
dark blue; ITC, light blue), enthalpy (ΔHꢀ) (SPR, dark green; ITC, light
green) and entropic contributions (-TΔSꢀ) (SPR, dark red; ITC, light
red). All thermodynamic parameters shown are in kcal mol-1
.
3
binding mode of a ligand, thereby better guiding the rational
design process.
In the current study, we have grown a hit fragment into a
protein binding pocket that can be induced by triggering con-
formational changes of the protein. X-ray analysis confirmed the
successful design strategy. Thermodynamic binding analysis
showed that insertion into the lobeline pocket by extending frag-
ment 1 with a hydrophobic phenetyl moiety resulting in 4 affords
’ ASSOCIATED CONTENT
S
Supporting Information. Complete refs 10 and 11, full
b
experimental details, crystallographic data and refinement statis-
tics, van’t Hoff plots, and ITC titration curves. This material is
available free of charge via the Internet at http://pubs.acs.org.
The new AChBP cocrystal structures described in this paper
have been deposited in the Protein Data Bank (PDB codes
2Y54, 2Y56, 2Y57, and 2Y58).
a considerable favorable change in enthalpy of ∼-12 kcal
3
mol-1, that is partly compensated by an entropic penalty. This is
in contrast with the classical view on the hydrophobic effect, in
which increased burial of hydrophobic moieties in a hydrophobic
pocket results in favorable changes in entropy. Studies on major
mouse urinary protein suggest that enthalpy driven hydrophobic
association results from poor solvation of the binding site, prior
to complex formation.36-38 The apo-Ac-AChBP X-ray structure
’ AUTHOR INFORMATION
Corresponding Author
i.de.esch@few.vu.nl
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ARTICLE
Author Contributions
#These authors contributed equally.
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