X-ray structure of citrate bound to Src SH2 leads to a high-affinity, bone-targeted Src SH2 inhibitor

Full text of DOI:10.1021/jm0002681

660
J . Med. Chem. 2001, 44, 660-663
Letters
the interactions between pY and the residues of its
binding pocket are largely responsible for the peptide’s
binding affinity.8
X-r a y Str u ctu r e of Citr a te Bou n d to Sr c
SH2 Lea d s to a High -Affin ity,
Bon e-Ta r geted Sr c SH2 In h ibitor
Regine S. Bohacek,* David C. Dalgarno,
Marcos Hatada, Virginia A. J acobsen,
Berkley A. Lynch, Karina J . Macek, Taylor Merry,
Chester A. Metcalf III, Surinder S. Narula,
Tomi K. Sawyer, William C. Shakespeare,
Shelia M. Violette, and Manfred Weigele
ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street,
Cambridge, Massachusetts 02139-4234
Like pY in the Lck SH2/pYEEI and Src SH2/pYVPML
structures, citrate when bound to Src SH2 forms numer-
ous salt bridges and hydrogen bonds with side chain and
backbone protein atoms (Figure 1B). However, citrate
forms two a d d ition a l interactions not observed in the
SH2/peptide crystal structures: a salt bridge with Lys
206 and a hydrogen bond with the backbone NH of Thr
182. Thus, the pY-binding pocket offers hydrogen-
bonding opportunities in addition to those utilized by
the natural pY-containing ligands. Taking advantage
of this finding, we designed novel SH2 ligands that
incorporate the known interactions of pY as well as the
additional interactions revealed by the citrate structure.
This suggested the replacement of pY (1) by diphospho-
nomethylphenylalanine (dpmF, 2).
Received J une 22, 2000
In tr od u ction . Numerous studies provide compelling
evidence that the protein tyrosine kinase pp60c-Src (Src)
plays a crucial role in osteoclast-mediated bone resorp-
tion1,2 and, moreover, that the SH2 domain of Src
specifically regulates osteoclast activity.3,4
With the expectation that blocking the SH2 domain
of Src with high-affinity ligands might attenuate the
excessive signal transduction in bone resorption dis-
eases such as osteoporosis, we have been searching for
general ways to improve upon the phosphotyrosine-
containing peptides known to bind weakly to Src SH2.
To be useful, such ligands need to contain a nonhydro-
lyzable phosphotyrosine mimic incorporated into a non-
peptidic scaffold. Features that confer specificity for
bone would be an additional desirable property for the
use of these compounds as therapeutic agents in the
treatment of osteoporosis.
We were afforded a gratuitous starting point toward
this goal when one of us (M.H.) determined the crystal
structure of Src SH2 (in the absence of an inhibitor) at
1.5 Å resolution and found that the protein, when
crystallized from citrate buffer, contained a well-com-
plexed citrate ion in the phosphotyrosine-binding pocket.
We now describe how this Src SH2/citrate X-ray struc-
ture led us to design and synthesize a nonpeptidic SH2
ligand that contains a phosphotyrosine mimic with
bone-targeting properties.
To determine whether the SH2 domain of Src could
accommodate this novel amino acid in place of pY, dpmF
was flexibly docked into the pY pocket of the Src SH2/
citrate crystal structure using the conformational search-
ing/energy minimization procedure of FLO96.9,10 The
docking results indicate that, like the citrate, the two
phosphonates of dpmF can form an extensive network
of hydrogen bonds (shown in Figure 1C). One of the
phosphonates of dpmF occupies a position close to that
adopted by the phosphate of pYEEI and pYVPML and
forms the equivalent series of salt bridges. The second
phosphonate of dpmF forms hydrogen bonds with Ser
180 and the NH of Glu 181 which are also observed in
the Src SH2/pYVPML X-ray structure. However, this
second phosphonate also forms the a d d ition a l interac-
tions observed in the citrate structure.
Design . Previous X-ray and NMR structural studies
provide detailed knowledge about the three-dimensional
structure of a number of different SH2 domains com-
plexed with peptidic, phosphotyrosine (pY)-containing
ligands. For example, the structure of the cognate
peptide, Ac-pTyr-Glu-Glu-Ile (pYEEI),5 bound to Lck
SH2 was determined at 1.0 Å resolution,6 and the
structure of the low-affinity peptide, pTyr-Val-Pro-Met-
Leu (pYVPML), bound to Src SH2 was determined at a
7
resolution of 1.5 Å. These structures show that pY
occupies a binding pocket composed of arginine, lysine,
serine, and threonine residues. The phosphate group
forms numerous hydrogen bonds and salt bridges with
these residues (see Figure 1A). Replacement of pY in
pYEEI with F abolishes binding affinity, suggesting that
To further improve the binding affinity as well as the
pharmacological potential of the projected SH2 ligands,
it was deemed essential to reduce the peptide character
of the portion linking the dpmF headgroup to the
hydrophobic C-terminal group present in peptide ligands
such as pYEEI. Lunney et al.11,12 had shown that a
* To whom correspondence should be addressed. Tel: 617-494-0400.
Fax: 617-494-8144. E-mail: regine@ariad.com.
10.1021/jm0002681 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/06/2001

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 661
F igu r e 2. Compound 4 docked into the binding site of Lck SH2.
The accessible surface of the binding site is displayed as a mesh,
colored to display chemical specificity: hydrophobic regions of the
binding site are shown in yellow, hydrogen bond donor regions in
red, and hydrogen bond acceptor regions in blue. For optimal van
der Waals contact, ligand atoms lie on or close to the mesh. Ligand
atoms forming hydrogen bonds with protein atoms penetrate the
mesh. Note the oxygen atoms of the dmpF moiety penetrating the
mesh as they from hydrogen bonds with atoms of the phosphoty-
rosine pocket. Generated using the Flo96 software.
unsatisfactory for modeling SH2 ligands. For example,
extensive docking studies did not succeed in correctly
fitting pYEEI into this binding site. Therefore, we used
the high-resolution (1.0 Å) structure of Lck SH2/pYEEI
for our modeling studies and conducted conformational
searching experiments to simulate the binding of com-
pound 4 to Lck SH2. Figure 2 shows the result of these
studies: the dpmF moiety forms the favorable interac-
tions described above; the benzene ring is positioned
above Tyr 181 (205 in Src)13 forming favorable stacking
interactions; the benzamide displaces two water mol-
ecules forming hydrogen bonds with the backbone NH
of Lys 182 (206 in Src) and the backbone carbonyl of Ile
193 (217 in Src); the ether oxygen forms an intramo-
lecular hydrogen bond with the NH of the benzamide
enabling the cyclohexyl side chain to form five hydro-
phobic contacts as it extends into the pY+3 pocket.
Because of the additional interactions formed by the
second phosphonate of dpmF, we predicted that 4 would
bind to Src SH2 with higher affinity than 3.
F igu r e 1. (A) Hydrogen bonds formed by phosphotyrosine
observed in the X-ray structure of pTyr-Val-Pro-Met-Leu bound
to the SH2 domain of Src. Hydrogen bonds are shown with dotted
lines. Atoms are colored as follows: oxygen, red; nitrogen, blue;
hydrogen, light blue; carbon atoms of the protein, purple; carbon
atoms of the ligand, white; phosphorus, green. (B) Hydrogen bonds
formed by citrate ion bound to Src SH2 from the Src SH2/citrate
X-ray structure. (C) Hydrogen bonds formed by dpmF with Src
SH2 predicted by molecular modeling studies.
Encouraged by these molecular modeling results, we
synthesized compound 4.
Syn th esis. The synthesis of compound 4 has been
optimized as outlined in Scheme 1. (Details of the
synthetic procedures are given in the Supporting Infor-
mation.) For comparison we also synthesized compound
5 (see Table 1).
Sr c SH 2 Bin d in g Assa y. A fluorescence polari-
zation-based competitive binding assay was used to
determine the IC50 of our compounds to the Src SH2
domain.14 Results are shown in Table 1.
benzamide, as in 3, can serve as such a linker. There-
fore, we modeled molecule 4 which exploits both the
dpmF and benzamide/cyclohexyl moieties.
Because the Src SH2/citrate structure contained no
ligand atoms in the binding site region beyond the
phosphate pocket, this structure was found to be

662 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Sch em e 1. Chemical Synthesis of 4
Letters
F igu r e 3. Conformation adopted by compound 4 upon binding
to Src SH2 predicted by molecular modeling in green, determined
by NMR in yellow, and determined by X-ray crystallography in
blue. Prepared by superimposition of binding site atoms.
measure the affinity of particular bone-targeting groups
to hydroxyapatite. Tighter binding compounds elute
later in the gradient. Retention time is expressed in
terms in K′, the number of void volumes required to
elute a particular compound.
Ta ble 1. Src SH2 Binding Properties
The hydroxyapatite chromatography results show
that neither Ac-pYEEI-NH2 nor compound 5 have
measurable affinity for hydroxyapatite (K′ < 0.1). On
the other hand, compound 4 has a similar affinity for
hydroxyapatite (K′ ) 3.7) as alendronate (K′ ) 3.6), a
bone-targeted bisphosphonate drug. These results sug-
gest that the diphosphonomethyl moiety of 4 is respon-
sible for its affinity to hydroxyapatite and that 4 will
bind to bone with affinity similar as alendronate.
Ra bbit Osteocla st Resor p tion . Because Src has
been implicated in the regulation of osteoclast functional
activity, we tested the ability of compound 4 to inhibit
osteoclast-mediated resorption of dentine. Compound 4
inhibited resorption of dentine with an IC50 of 2 µM.
This is a significant improvement over a recently
reported Src-modifying aldehyde compound (with an
IC50 of 43µM), also developed in these laboratories.4 To
rule out the possibility that the observed antiresorptive
activity of compound 4 was not due to nonspecific
association with the bone matrix, we synthesized an
analogue containing the dpmF moiety which possesses
no affinity for the Src SH2 domain. This molecule did
not demonstrate any antiresorptive activity.
Str u ctu r e Deter m in a tion . To confirm the predicted
binding mode of 4, the structure of its complex with Src
SH2 was determined by NMR and that of its complex
with the mutated Lck SH2 (S164C) by X-ray crystal-
lography. The experimentally determined binding con-
formations of 4 proved to be very similar to those
predicted by molecular modeling (Figure 3). NMR
experiments revealed 46 NOEs between compound 4
and the protein. These clearly indicate that the dmpF
of compound 4 occupies the pY pocket and that the
cyclohexyl moiety binds in the pY+3 pocket.
The X-ray structures of the Lck SH2/4 complex
reveals that the dpmF binds, as predicted, with the
phenyl ring in a position very similar to that of pY in
the pYEEI complex. The benzamide group is stacked
against the benzene ring of Tyr 181 (205 in Src). The
Comparison of compound 4 with compound 5 shows
that the incorporation of the second phosphonate group
in the dpmF headgroup resulted in a 22-fold increase
in binding affinity. Thus, the combination of the benz-
amide moiety with this high-affinity pY mimic resulted
in a compound with a 14-fold higher binding affinity
than that of the cognate peptide.
H yd r oxya p a t it e Bin d in g. The presence of the
diphosphonomethyl group in 4 suggests that the mol-
ecule might target to bone in vivo. The bisphosphonate
moiety has been shown to bind to bone and is present
in a number of drugs used clinically to treat osteoporo-
sis.15 To evaluate this in vitro, a bone-binding model was
developed. A microcrystalline hydroxyapatite adsorption
column with phosphate gradient elution was used to

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 663
ligands. Molecular modeling predicted that a geminal
bisphosphonate could form interactions similar to those
of citrate. Incorporation of this moiety into a nonpetidic
ligand for Src SH2 resulted in a compound which had
14 times greater binding affinity than the cognate
(natural) peptide, possessed bone-binding properties,
and inhibited osteoclast-mediated resorption of dentine.
X-ray and NMR structures of the compound bound to
Src and Lck SH2 agreed with molecular modeling
predictions. These findings should provide a new para-
digm for the design of agents to treat osteoporosis.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Neel, B. G.; Birge, R. B.; Fajardo, J . E.; Chou, M. M.; Hanafusa,
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(11) Lunney, E. A.; Para, K. S.; Plummer, M. S.; Prasad, J . V. N. V.;
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(13) Protein residue numbers were obtained from Swiss-Prot
(www.expasy.ch) for human Src and human Lck. However,
unlike Swiss-Prot, we are taking the initiator Met as residue
number 1 rather than residue number 0. Where comparisons
between Lck and Src are relevant, residue numbers for Src are
given in parentheses.
F igu r e 4. Comparison between citrate and compound 4 from the
crystal structure of Src SH2/citrate and Lck SH2 (S164C)/4,
constructed by superimposition of the binding site atoms.
benzamide carbonyl oxygen is in a position occupied by
a water molecule in the pYEEI structure and forms a
hydrogen bond with the backbone NH of Lys 182 (206
in Src). There are also two water molecules in this
vicinity, and the temperature factors of these waters are
as low as the backbone chain of Lck SH2, indicating that
they are highly localized and held together by a number
of strong hydrogen bonds. There are two direct and four
water-mediated hydrogen bonds between the benzamide
group and the protein. The cyclohexyl group forms
extended contacts with the pY+3 pocket although it
does not extend as deeply into the pY+3 pocket as the
Ile side chain of pYEEI. An unexpected difference
between the Lck SH2/4 structure and that of previous
SH2/ligand complexes is the open conformation of the
BC loop. However, the crystal structure of a close
analogue of compound 4 displays the BC loop in a
position similar to that found in the Src SH2/citrate
structure, forming hydrogen bonds with the second
phosphate as predicted by our model.16 NMR results
also clearly indicate that the BC loop is in contact with
the dpmF group of compound 4. Thus, we conclude that
the open loop conformation observed in the X-ray
structure is due to crystal packing effects.
Another difference between the predicted ligand-
binding mode and the X-ray structure is the presence
in the X-ray structure of a water molecule between the
NH2 group of the benzamide and the carbonyl of Ile 193.
As no water molecules are present in our binding site
model, the NH2 group of the benzamide was predicted
to form a direct, weak hydrogen bond with the carbonyl
of Ile 193, instead of the water-mediated hydrogen bond
observed in the X-ray structure.
The superposition of complexes of Lck SH2 with
compound 4 predicted by molecular modeling and
determined by X-ray crystallography are shown in
Figure 3. The binding mode of compound 4 determined
by NMR has also been superimposed. Figure 4 shows a
close-up of the superposition of the X-ray structures of
the dpmF moiety from the Lck SH2/4 complex and the
Src SH2/citrate complex.
(14) Lynch, B. A.; Loiacono, K. A.; Tiong, C. L.; Adams, S. E.; A., M.
I. A fluoresence polarization based Src-SH2 binding assay. Anal.
Biochem. 1997, 247, 777-782.
(15) Geddes, A. D.; D′Souza, S. M.; Ebetino, F. H.; Ibbotson, K. J .
Bisphosphonates: Structure-activity relationships and thera-
peutic implications. Bone Miner. Res. 1994, 8, 265-306.
(16) Shakespeare, W.; Bohacek, R. S.; Azimioria, M. D.; Macek, K.
J .; Luke, G. P.; Hatada, M. H.; Lu, X.; Violette, S. M.; Bartlett,
C.; Sawyer, T. Structure-based design of novel bicyclic nonpep-
tide inhibitors of the Src SH2 domain. J . Med. Chem. 2000, 43,
3815-3819.
Con clu sion . A crystal structure of the citrate ion
bound to Src SH2 revealed that the phosphotyrosine
pocket of Src SH2 offers hydrogen-bonding opportunities
in addition to those displayed by natural pY-containing
J M0002681