Molecular recognition at the active site of catechol-omethyltransferase: Energetically favorable replacement of a water molecule imported by a bisubstrate inhibitor

Article abstract of DOI:10.1002/anie.200904410

The import business: The binding mode of highly potent bisubstrate inhibitors of catechol-O-methyltransferase (COMT) has been elucidated by X-ray crystal structures of ternary complexes with COMTand a Mg²⁺ ion (see picture). A single ligand-imp

Full text of DOI:10.1002/anie.200904410

Communications
DOI: 10.1002/anie.200904410
Inhibitors
Molecular Recognition at the Active Site of Catechol-O-
Methyltransferase: Energetically Favorable Replacement of a Water
Molecule Imported by a Bisubstrate Inhibitor**
Manuel Ellermann, Roland Jakob-Roetne, Christian Lerner, Edilio Borroni, Daniel Schlatter,
Doris Roth, Andreas Ehler, Markus Georg Rudolph,* and Franꢀois Diederich*
Biologically active catechols, such as l-DOPA and the
neurotransmitter dopamine, are inactivated by methylation.
This reaction is catalyzed by the enzyme catechol-O-methyl-
transferase (COMT) in the presence of S-adenosylmethionine
(SAM) and Mg2+ ions.[1] Small nitrocatechol-based inhibitors
of COMT find application in the treatment of Parkinson
disease by blocking unwanted methylation of the adminis-
tered l-DOPA, thereby enhancing dopamine levels in the
brain.[2,3] Recent studies have pointed towards additional
therapeutic applications of COMT inhibition in other disor-
ders of the central nervous system, such as schizophrenia[4]
and depression.[5]
The crystal structure of 1 in a ternary complex with
COMT and a Mg2+ ion shows that the adenine moiety forms
two hydrogen bonds, a moderately strong and a weak one
(d(N···O): 3.0 and 3.4 ꢀ, respectively), to a water molecule
We have developed a series of potent bisubstrate inhib-
itors for COMT which are competitive for both the catechol
and the SAM binding sites.[6] Based on the X-ray crystal
structure of ligand 1 (IC50 = 9 nm)[7a] in a ternary complex with
COMT and a Mg2+ ion (PDB code: 1JR4),[8] we started a
detailed exploration of the molecular recognition properties
of the entire active site of the enzyme.[9] Importantly, we
found that potentially hepatotoxic nitro groups, which are
mandatory in catechol-based monosubstrate inhibitors, are
not required for high-affinity bisubstrate inhibition.[10] We
substituted the nitro group in position 5 of 1 with appropriate
lipophilic residues, such as the 4-fluorophenyl ring in 2 (IC50 =
31 nm),[7] and found that the high, competitive inhibitory
potency was maintained. Computer modeling studies sug-
gested that the newly introduced lipophilic residue occupies a
hydrophobic cleft near the surface of the enzyme.[11] This
initial proposal is validated here experimentally by X-ray
crystallography.
[*] Dr. R. Jakob-Roetne, Dr. C. Lerner, Dr. E. Borroni, Dr. D. Schlatter,
Dr. D. Roth, A. Ehler, Dr. M. G. Rudolph
Pharma Division, Prꢁklinische Forschung
F. Hoffmann–La Roche AG, 4070 Basel (Switzerland)
E-mail: markus.rudolph@roche.com
M. Ellermann, Prof. Dr. F. Diederich
Laboratorium fꢂr Organische Chemie, ETH Zꢂrich
Wolfgang-Pauli-Strasse 10, 8093 Zurich (Switzerland)
Fax: (+41)44-632-1109
E-mail: diederich@org.chem.ethz.ch
[**] We thank F. Hoffmann–La Roche for generous support of this work,
and Dr. Manfred Kansy for valuable discussion. We are grateful to
Pia Warga and Valꢃrie Goetschy-Meyer for their advice concerning
the assay, and Jꢄrg Benz for his help in designing the crystallization
experiments.
Figure 1. Top: Bisubstrate inhibitors of COMT. cPr=cyclopropyl.
Bottom: Structure of the adenosine binding site of the ternary complex
of 1 with COMT and a Mg2+ ion as seen in the X-ray crystal structure.[8]
Distances are given in ꢀ (gray CCOMT, green Cligand, red O, blue N,
yellow S; PDB code: 1JR4).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904410.
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(H2O9, Figure 1). This water molecule is apparently imported
together with the ligand, as it does not undergo any hydrogen
bonding to the protein, but very likely does only to dynamic
bulk water.[8a] We reasoned that a replacement of this water
molecule by a hydrophobic residue of the ligand would result
in a favorable entropic and enthalpic gain, by freeing the
solvent molecule and establishing better interactions to the
protein.[12] The replacement of ordered water, as seen in
cocrystal structures, to enhance protein–ligand interactions is
currently a topic of intense interest in structure-based drug
design, both from experimental and theoretical viewpoints.[13]
The replacement of ordered, ligand-imported water mole-
cules, which do not interact with the protein[14] has, however,
not been previously addressed, despite such water being quite
abundant, as revealed by a preliminary search of the Protein
Data Bank (PDB; see the Supporting Information). Here, we
present the novel bisubstrate inhibitors 3–7 and show that
replacement of the ligand-imported water molecule seen in
the cocrystal structure of 1 (Figure 1) is indeed an energeti-
cally favorable process.
amine removed the phthalimide and acetyl protecting groups,
while at the same time substituting the chlorine atom of the
purine. The resulting allylic amine was coupled with the
protected catechol derivative 12 to yield 13, which was
deprotected to afford the targeted bisubstrate inhibitor 5. The
other inhibitors were accessible by the same route.
The biological activities of the novel bisubstrate inhibitors
were evaluated in a radiochemical assay (see the Supporting
Information);[6,18] the measured IC50 and calculated Ki values
are given in Table 1. The novel bisubstrate inhibitors which
have a substituent at the exocyclic N6 atom of the adenine
moiety display a similar potency to that of the previously
reported reference compound 2.[7] A Lineweaver–Burk plot
analysis[6] (see the Supporting Information) showed that all
the inhibitors showed a competitive inhibition mechanism
with respect to the SAM binding site.
The X-ray crystal structures of the four bisubstrate
inhibitors 3, 4, 5, and 7 in a ternary complex with COMT
and a Mg2+ ion (PDB codes: 3HVH (resolution: 1.30 ꢀ),
3HVI (1.20 ꢀ), 3HVJ (1.79 ꢀ), and 3HVK (1.30 ꢀ)) were
solved. The inhibitor binding mode is very similar in all four
structures, as illustrated by an overlay (see the Supporting
Information). Figure 2 depicts the X-ray crystal structure of
ethyladenine derivative 4 in a complex with COMT and a
Mg2+ ion.
The synthesis of the new bisubstrate inhibitors started
from alcohol 8 (as illustrated in Scheme 1 for propyl
derivative 5), which was transformed via 9[15] into the
triacetylated ribose derivative 10.[16] Nucleosidation with
6-chloropurine by a modified Vorbrꢁggen protocol gave
nucleoside 11 in excellent yield.[17] Treatment with propyl-
Scheme 1. Synthesis of bisubstrate inhibitor 5: a) phthalimide, DIAD, PPh3, THF, 208C, 20 h, 95%; b) 1. AcOH (80%), 708C, 15 h; 2. Ac2O,
pyridine, 4-(N,N-dimethylamino)pyridine, 208C, 16 h, 88%; c) BSA, Me3SiOSO2CF3, 6-chloropurine, toluene, 608C, 16 h, 84%; d) 1. propylamine,
MeOH, 208C, 6 days; 2. 12, HBTU, HOBt, iPr2NEt, DMF, 208C, 25%; e) trifluoroacetic acid (50%), THF, 08C, 1 h, 76%. TBS=tert-butyldimethyl-
silyl, DIAD=diisopropyl azodicarboxylate, BSA=N,O-bis(trimethylsilyl)acetamide, HBTU=O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluro-
nium hexafluorophosphate, HOBt=1H-benzo[d][1,2,3]triazol-1-ol.
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Table 1: Biological results obtained from a radiochemical assay.[a]
Gln120. The purine ring stacks with the side chain of Met91
(d(S···C5) = 3.6 ꢀ) and interacts in an edge-to-face arrange-
ment with the imidazole ring of His142 (d(CD2His···C6) =
3.8 ꢀ). The indole ring of Trp143, which shields the adenine
binding site from bulk solvent, adopts two different con-
formations in the complexes formed by 3–5, while only one
orientation is seen in the complex of 7 (see the Supporting
Information). Trp143 also displays an edge-to-face interaction
with the nucleobase (d(CD2Trp···C8) = 3.4 ꢀ in the complex of
4).
The cocrystal structure with bound 4 clearly shows that
the water molecule (H2O9), which binds to the adenine
moiety in the cocrystal structure of 1 (PDB code: 1JR4,
picture 1), has been replaced by the ethyl substituent.
Importantly, this replacement of the water molecule is
observed in all four cocrystal structures with ligands 3, 4, 5,
and 7 (see the Supporting Information).
A comparison of the thermodynamic quantities (Table 1)
at first sight suggests that the incorporation of the N6-alkyl
substituents and the concomitant increase in the lgP and
lgD values as well as the water displacement do not result in a
significant gain in the binding affinity: the IC50 and Ki values
for N6-alkylated 3–5 and 7 are only slightly lower than those
measured for the unsubstituted ligand 2 (Table 1).
Inhibitor
IC50[a] [nm]
Ki[b] [nm]
clgP[c]
lgD[d]
2 (R=H)[e]
3 (R=Me)
4 (R=Et)
5 (R=Pr)
6 (R=cPr)
31
12
21
9
41
15
7
3
5
2
9
3
1.12
1.96
2.48
3.01
2.31
1.19
1.64
2.14
2.00
3.15
2.57
1.58
7 (R=CH2CH2OH)
[a] IC50 =median inhibitory concentration. [b] Ki =inhibitory constant for
competitive inhibition of SAM. IC50 and Ki values were obtained with pre-
incubation as described in the Supporting Information. [c] Logarithmic
partition coefficient for octanol/water. [d] Logarithmic distribution
coefficient for octanol/water at pH 7.4. [e] Ref. [7]
However, the new ligands bind in the energetically
unfavorable s-trans conformation (Scheme 2). Previous
kinetic analyses of N6-methylated adenine systems revealed
Scheme 2. Conformational s-cis–s-trans equilibrium of N-alkylated
adenines.
Figure 2. Binding mode of bisubstrate inhibitor 4 with a Mg2+ ion in
the active site of COMT. The indole ring of Trp143 adopts two different
orientations (red O, blue N, yellow S, turquoise F, light green Mg,
green Cligand, gray Cprotein; PDB code: 3HVI). The numbering of the
residues and atoms correspond to those in the X-ray crystal structure.
activation free enthalpies (DG°) for the rotational barrier for
the s-cis–s-trans equilibration to be around 13 kcalmolꢀ1.[19–21]
This energy barrier allows observation of the conformational
1
isomer ratio by H NMR spectroscopy at low temperature
and slow exchange rates, with the Me resonance in the s-cis
conformer appearing around d = 3.0 ppm and the resonance
in the s-trans conformer around d = 3.5 ppm. In solvents of
different polarity, a strong preference for the s-cis isomer was
obtained, which was quantified as a DGs-trans!s-cis value of ꢀ1.5
to ꢀ1.9 kcalmolꢀ1. We recorded the spectra of 3 in perdeu-
terated 2,2,2-trifluoroethanol between 238 K and 310 K and
did not observe any signals for the s-trans isomer (see the
Supporting Information). A similar result was obtained for
measurements in D2O/(CD3)2SO between 277 K and 298 K.
The accuracy of the NMR integration is about 5%, and thus a
minimum isomeric ratio of 95:5 (s-cis/s-trans) can be
expected. This results in a minimum DGs-trans!s-cis value of
ꢀ1.8 kcalmolꢀ1 (DG = ꢀRTlnK; K = 95/5) at 310 K (the
temperature at which the assay was conducted), which is in
good agreement with the previous studies.[19–21] Thus, around
As expected, the adenosine moiety occupies the SAM
binding site, while the catechol moiety coordinates to the
Mg2+ ion. The 4-fluorophenyl substituent on the catechol ring
occupies the hydrophobic cleft near the surface of the protein,
as predicted by modeling studies,[10] and undergoes several
van der Waals interactions (C···C contacts below 4.0 ꢀ with
Pro174, Val173, Leu189, and Trp38) with the protein (Figure 2
and the Supporting Information). These apolar contacts,
together with the binding-induced entropically favorable
desolvation of the cleft, contribute strongly to the high
affinity of the bisubstrate inhibitors that lack a nitro group on
the catechol ring.
The adenine ring of 4 forms a hydrogen bond (d(N1···N) =
3.0 ꢀ) to the backbone NH atom of Ser119 and a second,
weak hydrogen bond (d(N6···O) = 3.6 ꢀ) to the side chain of
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1.8 kcalmolꢀ1 of binding free enthalpy has to be invested in
forcing the N-alkylated ligand into the s-trans conformer in
the ternary complex with COMT.
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Nevertheless, non-alkylated 2 and N-alkylated 3–7 all
form complexes of similar stabilities. The energy costs of the
unfavorable conformation must be compensated by the
energetic gains resulting from the replacement of water by
the N6-alkyl substituent in the bound s-trans form, which
therefore provides a gain in binding free enthalpy of at least
DDG = ꢀ1.8 kcalmolꢀ1. Since the alkyl substituents point into
a highly polar region near the surface, there are few additional
van der Waals contacts with the protein (see the Supporting
Information). Partitioning into this highly polar environment
is energetically not very favorable: although the lgD value
strongly increases upon changing from 3 (Me) to 5 (Pr)
(Table 1), no additional binding free enthalpy is gained.
Furthermore, hydroxyethyl derivative 7, with a low lgD value
similar to that of 2, has a comparable binding affinity.
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ca. ꢀ1.8 kcalmolꢀ1 originates from the displacement of the
water molecule.[22] This displacement occurs as a result of the
change in the N-alkyl substituent from the s-cis conformation
in the free ligand, which presumably maintains adenine
solvation as shown in Figure 1,[23] into the s-trans form in
COMT.
In summary, we have described highly potent bisubstrate
inhibitors of COMT and validated their predicted complex-
ation mode by four X-ray cocrystal structures. The N6-
alkylated inhibitors in the unfavorable s-trans conformation
bind with similar strength as the corresponding non-alkylated
derivative. Our analysis shows that the displacement of the
water molecule in the s-trans conformation of the bound
ligand contributes at least 1.8 kcalmolꢀ1 to the binding
affinity of the ligand to the protein, thereby compensating
the similar energetic costs resulting from the unfavorable
s-trans ligand conformation. The study clearly suggests that
replacement of ligand-imported water molecules using struc-
ture-based design is worthwhile, in particular since a prelimi-
nary search of the PDB shows that such ligand-imported
water molecules, which do not interact with the protein
through hydrogen bonds, are frequently observed.
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Received: August 6, 2009
Published online: October 30, 2009
Keywords: drug discovery · enzymes · inhibitors ·
.
medicinal chemistry · structure–activity relationships
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