An enthalpic basis of additivity in biphenyl hydroxamic acid ligands for stromelysin-1

Article abstract of DOI:10.1016/j.bmcl.2012.05.032

Fragment based drug discovery remains a successful tool for pharmaceutical lead discovery. Although based upon the principle of thermodynamic additivity, the underlying thermodynamic basis is poorly understood. A thermodynamic additivity analysis was performed using stromelysin-1 and a series of biphenyl hydroxamate ligands identified through fragment additivity. Our studies suggest that, in this instance, additivity arises from enthalpic effects, while interaction entropies are unfavorable; this thermodynamic behavior is masked by proton transfer. Evaluation of the changes in constant pressure heat capacities during binding suggest that solvent exclusion from the binding site does not account for the dramatic affinity enhancements observed.

Full text of DOI:10.1016/j.bmcl.2012.05.032

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6521–6524
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An enthalpic basis of additivity in biphenyl hydroxamic acid ligands
for stromelysin-1
⇑
Erin M. Wilfong, Yu Du, Eric J. Toone
Department of Chemistry, Duke University, LSRC B120, Box 90317, Durham, NC 27708, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Fragment based drug discovery remains a successful tool for pharmaceutical lead discovery. Although
based upon the principle of thermodynamic additivity, the underlying thermodynamic basis is poorly
understood. A thermodynamic additivity analysis was performed using stromelysin-1 and a series of
biphenyl hydroxamate ligands identified through fragment additivity. Our studies suggest that, in this
instance, additivity arises from enthalpic effects, while interaction entropies are unfavorable; this ther-
modynamic behavior is masked by proton transfer. Evaluation of the changes in constant pressure heat
capacities during binding suggest that solvent exclusion from the binding site does not account for the
dramatic affinity enhancements observed.
Received 29 October 2011
Revised 29 April 2012
Accepted 8 May 2012
Available online 30 May 2012
Keywords:
Additivity
Fragment based drug design
Stromelysin-1
Ó 2012 Elsevier Ltd. All rights reserved.
Matrix metalloproteinase-3
D
JAB
¼
D
JA þ
D
JB þ
DJi
In 1997, Fesik and co-workers first reported the use of fragment
based drug design (FBDD) to generate high affinity inhibitors for
stromelysin-1 (matrix metalloproteinase 3).1 Since then, FBDD
has taken its place among most successful approaches to lead com-
pound discovery. FBDD is based on the principle that proteins
binding sites can be parsed into multiple low affinity sub-sites.
The binding of small libraries (<200 compounds) of small ligands
(<150 Da) are evaluated using techniques that identify the binding
locus within the larger binding pocket; subsequent chemical link-
age of two or more fragments with affinities for adjacent pockets
yields high affinity ligands. Over a decade of use, FBDD had yielded
high affinity leads for over fifty proteins, at least some of which
have progressed to clinical trials.2
In this construct, DJi, is an interaction energy describing the
energetic consequences of physical linkage. Interaction energies
are most frequently considered in entropic terms, a trade-off be-
tween a ‘saving’ in losses in translational and rotational entropy
that arises through linkage, and in increase in losses in conforma-
tional degrees of freedom, as a flexible tether is constrained during
binding.
In contrast to this intellectually straightforward construct, the
thermodynamic origin of additivity has been investigated experi-
mentally in several systems, including multivalent carbohydrates
and metal chelates and found to be an enthalpic phenomena,4,5 that
is in the construct of Jencks both
DHi and TDSi are negative. On the
Despite its widespread utility, the thermodynamic principles
that underlie the success of FBDD are poorly understood. The no-
tion of additivity in ligand binding was first considered by Jencks.3
Although thermodynamic parameters are state functions and are
additive in and of themselves, there is no fundamental basis for
any simple relationship between the thermodynamic parameters
characterizing the interaction of an intact ligand and those of its
constituent fragments. Consider the binding of a ligand AB and of
its constituent fragments A and B. Any thermodynamic parameter,
J, describing the binding of AB is related to the corresponding
parameters for the binding of the constitutive fragments through
the relationship:
other hand, Borsi et al. recently conducted an additivity analysis
using matrix metalloproteinase-12 (MMP12) and determined that
additivity in that case was entropically driven and enthalpically
opposed.6 This finding contradicts Fesik’s original work which
determined an enthalpic origin of additivity in the context of FBDD,
a finding they attribute to proton transfer events that affect the
catalytic domain of stromelysin-1 (SCD) but not MMP12. Here,
we revisit the original Fesik ligand series making structural modi-
fications to ensure that proton transfer events are fully accounted
for in the additivity analysis.
In their original report, Fesik and coworkers analyzed a series of
linkage ligands for stromelysin-1 based on biphenol and hydroxa-
mic acid fragments (Fig. 1).1 While phenols are attractive from an
experimental perspective, primarily because of increased aqueous
solubility relative to corresponding ether, they are both hydrogen
bond donors and acceptors, while ethers act only as hydrogen bond
⇑
Corresponding author. Tel.: +1 919 681 3484; fax: +1 919 660 0000.
E-mail address: eric.toone@duke.edu (E.J. Toone).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.032

6522
E. M. Wilfong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6521–6524
calorimetry buffer containing 50 mM buffer (Tris–HCl, HEPES or
MOPS), pH 7.5, 10 mM CaCl2, 1 lM ZnCl2. Protein concentrations
were determined by the method of Edelhoch.13
Table 1 summarizes the observed thermodynamics of binding.
Three percent DMSO was added to all solutions to facilitate com-
plete dissolution. The binding of compounds 1 and 4 was character-
ized by direct titration ITC; the weakly binding linkage ligands 2 and
3 and fragments 8–10 were characterized via displacement calorim-
etry.14 The hydrophobic fragments (compounds 5–7) presented
challenges due to poor solubility and only partial displacement
titrations were conducted.15 Binding thermodynamics of all ligands
were characterized in both Tris–HCl and HEPES; if differences in en-
thalpy were observed in the two buffers, a third titration in MOPS
was conducted to determine the extent of proton transfer by linear
Figure 1. Stromelysin-1 binding biphenol moiety in the S10 pocket and acetohy-
droxamic acid chelating catalytic zinc.
regression;
DHbind values reported in Table 1 are corrected for
proton transfer. All experiments were conducted in triplicate in
Tris–HCl and in duplicate for MOPS and HEPES buffers. The errors
acceptors. Additionally, no correction was made for proton transfer
events commonly observed for MMPs7 and certainly plausible for
electron deficient phenols.
reported
experiments. The errors for T
prorogation of errors; those errors for
D
G°,
D
H°, represent the standard deviation between
S and D i were determined via
CP were determined by
D
J
D
The conservation of atoms and functional groups is of para-
mount importance during linkage ligand design, as the tether itself
can interact—favorably or unfavorably—with the protein. Such is-
sues were considered when designing the ligand series depicted
in Figure 2. Linkage ligands 1–4, and acetohydroxamic acid 8 were
previously studied by Fesik. Potentially important differences
between phenols and phenyl ethers led to the inclusion of com-
pounds 5–7, which are ether equivalents to the phenols previously
studied by Fesik. To ensure conservation of atoms, hydroxamic acid
fragments 9 and 10 were extended to account for all atoms of the
linkage ligands.
regression. With the exception of compound 6, titrations were
conducted at 15, 25 and 37 °C to determine change in molar heat
capacity (
below room temperature, precluding
D
CP) accompanying binding. Compound 6 was insoluble
CP determination.
D
Proton transfer events are common among MMPs and previously
reported for stromelysin-1.7,16,17 The original characterization of the
biphenyl ligands did not include proton transfer events and was
conducted in Tris–HCl, a buffer which has an exceptionally large
ionization enthalpy
equation
(
D
Hion = ꢀ11.45 kcal molꢀ1). Using the
Synthesis of all ligands proceeded uneventfully.8 Linkage
ligands 1–4 were synthesized according to previously reported
procedures.9 Ligand fragments 5–7 were made by reacting the cor-
responding phenol with an iodoalkane.10 Hydroxamic acids 8–10
were made by reacting the corresponding ester with hydroxyl-
amine under methanolic conditions.11 Recombinant stromelysin-
D
Hbind
¼
D
Hobs þ nDHion
to account for proton transfer where n is the number of protons
transferred upon binding (0.21) and Hobs is the observed
D
enthalpy, our results are in good agreement with those of Fesik
(Supplemental materials). The difference in enthalpy for the
Hobs = ꢀ4.7 kcal molꢀ1 in Tris–HCl) and ether frag-
1
catalytic domain (SCD) was expressed in Escherichia coli
phenol
ment 5 (
(
D
D
according to previously described methods12 and dialyzed into
Hbind = ꢀ1.6 kcal molꢀ1) would correspond to an n of
Figure 2. Modified biphenyl series of ligands.

E. M. Wilfong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6521–6524
6523
Table 1
conceptual underpinning for FBDD involves a ‘savings’ in transla-
tional and rotational entropy achieved by fragment tethering,
which effectively converts two particles to one, and diminishes
the loss in translational and rotational entropy by one particle.
Our results stand in direct opposition to this hypothesis. In all
cases, ligand linkage is associated with a significant enthalpic ben-
efit and large entropic penalty. Favorability of ligand linkage is
Thermodynamics of ligand binding for all compounds determined by isothermal
titration calorimetry, corrected for proton transfer events
G0
DCP
D
H0bind
TD
S0bind
Compound
D
1
2
3
4
5
6
7
8
9
10
ꢀ9.5 0.5
ꢀ6.4 0.1
ꢀ6.6 0.2
ꢀ10.5 0.2
ꢀ4.8 0.1
ꢀ5.3 0.1
ꢀ6.1 0.4
ꢀ2.4 0.3
ꢀ2.6 0.1
ꢀ2.7 0.1
ꢀ11.5 0.2
ꢀ8.0 0.4
ꢀ7.5 0.2
ꢀ9.2 0.4
ꢀ1.6 0.3
ꢀ0.8 1.0
ꢀ1.5 0.3
ꢀ2.3 0.2
ꢀ3.3 0.4
ꢀ2.2 0.1
ꢀ2.0 0.5
ꢀ1.6 0.4
ꢀ0.9 0.2
ꢀ0.8 0.4
3.2 0.3
4.5 1.0
4.6 0.4
0.1 0.3
ꢀ72
2
ꢀ60 11
ꢀ67 16
ꢀ59
5
determined solely by the magnitude of
DHi. Consistent with this
ꢀ48 60
notion is the greater variation in enthalpy rather than entropy.
One of two constructs is typically invoked to rationalize enthal-
pic additivity: hydrophobic effects20–22 and interfacial mobility/
protein contraction.21,23 Hydrophobic interactions have both an
enthalpic and entropic component. Van der Waals forces contrib-
ute to the association enthalpy as attractive forces between hydro-
phobic moieties are more favorable than the interactions of a
hydrophobic moiety and water. The classical hydrophobic effect
argues that entropic effects dominate hydrophobic interac-
tions,20–22,24–26 while a non-classical view hydrophobic effects fo-
cuses on enthalpic contributions.27–31
The enthalpic component of the hydrophobic effect arises pri-
marily from van der Waals forces, and the magnitude of this effect
trends with ligand surface area.32,33 Thus, linkage ligands with in-
creased tethers should show increased binding enthalpies, assum-
ing an increased surface area—that of the tether—is removed from
solvent during binding. If the enthalpy of binding decreases for a
ligand of equal or greater surface area, then the loss in enthalpy re-
sults from some other effect, for example, the loss of a hydrogen
bond, an unfavorable ligand conformation, or trapping of a lone
pair along the hydrophobic interface.21 Our experimental results
demonstrated a decreased enthalpy of binding upon tether elonga-
tion, while Fesik and co-workers previously reported that tether
elongation resulted in the loss of a hydrogen bond.1,9
nd
ꢀ41
6
ꢀ67 24
ꢀ0.7 0.4
ꢀ54
ꢀ59
8
6
0.5 0.1
D
D
G,
CP reported in cal molꢀ1 Kꢀ1
DHbind, TD .
S reported in kcal molꢀ1
.
0.27. 4-Cyanophenol has pKa of 7.95.18 Addition of a second phenyl
ring should further decrease the pKa making it plausible that the
phenol fragment is partially deprotonated at pH 7.5.
Using the data of Table 1, an additivity analysis was conducted
according to the method of Jencks (Table 2). In the cases of com-
pounds 2 and 3, two different combinations of ligand fragments
form the linkage ligands, and both combinations were analyzed.
Additionally, the linking coefficient, E, is also reported for each
linkage ligand and representative ligand fragment pairing. This
construct, recently described by Whittaker and coworkers de-
scribes the efficiency of fragment linkage. The linking coefficient,
E, is related to the dissociation constant KD such that
KADB ¼ KADKBDE
or the
DGi such that
Just as elongation of an alkyl tether should increase the enthal-
pic benefit of binding, the entropy of binding should also increase
with increasing ligand size. While desolvated ligand surface areas
were not calculated here, binding entropy should increase in a lin-
ear manner as methylene units are added to the linker. Rather, we
observe remarkable consistency in both the entropies of binding
and the interaction entropies observed with the biphenyl linkage
ligands. No increase in binding entropy was observed with tether
elongation as predicted by a notion of hydrophobicity-driven
binding.
D
Gi ¼ ꢀRTlnE
Values less than 1 demonstrate a favorable thermodynamic
consequence of ligand linkage, values near 1 indicate linkage was
thermodynamically neutral, and values greater than 1 indicate a
negative thermodynamic consequence of fragment linkage.19
Several features of the Table 2 data are noteworthy. First, little
variation is observed in T
age of 4.7 1.0 kcal molꢀ1. The consistency of this value suggests
that Si is a property of the protein, rather the ligand. The variabil-
ity among Hi is larger than Si; the average interaction enthalpy
is 4.7 1.7 kcal molꢀ1. The discrepancy between the
Hi for com-
pounds 1/4 versus 2/3 has been previously attributed to the ability
of compounds 1 and 4 to hydrogen bond with Leu164. This hydro-
gen bond interaction is absent upon tether elongation.1
Examination of Table 2 reveals the counterintuitive result that
favorable additivity in ligand binding—that is a free energy of
binding greater than the sum of those for the constituent ligand
fragments—is enthalpic in origin. The most frequently cited
DSi across the entire series, with an aver-
D
D
D
Finally, hydrophobic hydration affects
ion. While changes in electrostatics or vibrational states of a pro-
tein during binding could, in principle, impact CP is the
CP,34
DCP in a predictable fash-
D
D
D
best available measurement of changes in solvent-exposed hydro-
phobic surface area and is therefore considered a hallmark of the
hydrophobic effect.35–37 The values of
DCP observed here offer no
evidence for major differences in the solvation behavior of the pro-
tein during binding. While there have been recent reports of Grb-2
SH2 domain exhibiting hydrophobic binding in the absence of
changes in
were as predicted by the hydrophobic effect. In this case, neither
H or T S nor CP behave in a manner consistent with the hydro-
phobic effect and we therefore reject this rationale for ligand
binding.
DCP, the enthalpic and entropic trends of ligand binding
D
D
D
Table 2
Additivity analysis of biphenyl ligands. Thermodynamic parameters reported in
kcal molꢀ1
Protein contraction and the interfacial mobility model arises
from a tightening of the ligand–protein interface that maxi-
mizes enthalpic intermolecular forces (ionic, van der Waals, and
dipole–dipole interactions), which vary inversely with
intermolecular distance.38–40 These favorable enthalpic interac-
tions are, in part, offset by entropic penalties that arise from
protein contraction with resultant rigidification. As ligand size
increases (as measured by the number of protein residues con-
tacted), the interfacial tightening required to maximize enthalpic
interactions requires too high an entropic penalty and enthalpy
Ligand
Ligand fragments
D
G°i
D
H°i
T
D
S°i
E
S10
Zn2+
1
2
5
5
6
5
6
7
8
9
8
10
9
ꢀ2.3 0.6
1.0 0.3
1.3 0.4
0.9 0.4
1.3 0.4
ꢀ2.0 0.7
ꢀ7.6 0.6
ꢀ3.1 0.6
ꢀ4.9 1.1
ꢀ3.7 0.4
ꢀ3.4 1.1
ꢀ5.4 0.5
ꢀ5.3 0.7
ꢀ4.1 0.6
ꢀ6.2 1.1
ꢀ4.6 0.4
ꢀ4.7 1.1
ꢀ3.4 0.8
0.021
5.354
8.857
4.527
8.857
0.035
3
4
8
D
D
G,
CP reported in cal molꢀ1 Kꢀ1
DHbind, TD .
S reported in kcal molꢀ1
.

6524
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DS among linkage ligands and the lack
of change in CP as they hydrophobic effect would not be expected
D
to contribute. Further, we have recently demonstrated that trends
in enthalpy–entropy compensation do appear to trend with pro-
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