Structure-based design of peptidomimetic antagonists of p56lck SH2 domain

Article abstract of DOI:10.1016/S0960-894X(02)00167-1

Starting from the tetrapeptide Ac-pYEEI-NHMe and using a structure-based approach, we have designed and synthesised a peptidomimetic ligand for p56^lck SH2 domain containing a conformationally restricted replacement for the two glutamate residue

Full text of DOI:10.1016/S0960-894X(02)00167-1

Bioorganic& Medicinal Chemistry Letters 12 (2002) 1365–1369
Structure-Based Design of Peptidomimetic Antagonists of
P56lck SH2 Domain
Christopher J. Hobbs,a,* Rino A. Bit,a Andrew D. Cansfield,a Bill Harris,a
b
Christopher H. Hill,a Katherine L. Hilyard,a Ian R. Kilford,a EricKitas,
Antonin Kroehn,a Peter Lovell,a,y David Pole,a Paul Rugman,a Brad S. Sherborne,a
Ian E. D. Smith,a David R. Vesey,a D. Lee Walmsley,a David Whittaker,a
Glyn Williams,a Fiona Wilson,a David Banner,b Allan Surgenora and Neera Borkakotia
aRoche Products Limited, 40 Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AY, UK
bF. Hoffmann-La Roche Ltd, Pharma Division, Preclinical Research, Basel, Switzerland
Received 16 August 2001; revised 18 February 2002; accepted 8 March 2002
Abstract—Starting from the tetrapeptide Ac-pYEEI-NHMe and using a structure-based approach, we have designed and synthe-
lck
sised a peptidomimeticligand for p56
SH2 domain containing a conformationally restricted replacement for the two glutamate
residues. We have explored replacments for the isoleucine residue in the pY+3 pocket and thus identified 1-(R)-amino-3-(S)-indane-
acetic acid as the most potent replacement. We also report the X-ray crystal structures of two of the antagonists. # 2002 Elsevier
Science Ltd. All rights reserved.
p56lck is a key non-receptor tyrosine kinase involved in
signal transduction through the T-cell receptor (TcR) in
T-helper (TH) cells. This signal is essential for the acti-
vation and proliferation of TH cells. p56lck consists of a
kinase domain which is responsible for phosphorylating
tyrosine-containing proteins, an SH3 domain which
binds proline-rich peptide motifs and an SH2 domain
which binds phosphotyrosine-containing proteins. As
part of our autoimmune disease programme, we are
interested in preventing TH cell proliferation by design-
ing p56lck SH2 domain antagonists.
our starting point for the design of p56lck SH2 domain
antagonists.2,3 The hamster middle-T antigen 1 is an
11-mer peptide and the structure revealed that of the 11
amino acids only four made hydrogen bond contact
with the SH2 domain. These contacts were bounded by
the backbone amide carbonyl C-terminal to the pY and
the backbone amide carbonyl C-terminal to the iso-
leucine at pY+3. A truncation analysis (Table 1) using
a series of peptides capped at the N-terminus with an
acetyl group and capped at the C-terminus with a
methylamide group indicated that the tetrapeptide 4,
which was only 5-fold less potent than the HmT anti-
gen, was the optimal length to be used as a lead for the
design of a p56lck SH2 antagonist. A crystal structure of
the related tetrapeptide with a carboxylic acid at the
C-terminus has now been reported4 and this binds in a
similar conformation to the corresponding part of the
middle-T antigen.
SH2 domains are modules of approximately 100 amino
acids. They bind phosphotyrosine containing sequences
with high affinity and specificity. The optimum binding
sequence for p56lck SH2 domain was determined to be
pTyr-Glu-Glu-Ile from
a
phosphopeptide library
screen.1 The crystal structure of p56lck SH2 domain
bound to a peptide (hamster middle-T antigen 1) con-
taining this sequence has been determined and this was
The design of a therapeutically useful compound which
could reach its target inside TH cells requires four prin-
cipal issues to be addressed: (1) the peptidic nature of
the compounds resulting in their instability to proteo-
lysis, (2) an improvement in the affinity of the com-
pound for the SH2 domain, (3) the propensity of the
phenylphosphate group to undergo hydrolysis, (4) the
*Corresponding author at present address: Ionix Pharmaceuticals Ltd,
10 Wellington Street, Cambridge CB1 1HW, UK. Tel.: +44-1223-306-
966; fax: +44-1223-306-977; e-mail: chobbs@ionixpharma.com
yPresent address: GlaxoSmithKline, New Frontiers Science Park,
Third Avenue, Marlow, Essex CM19 5AW, UK.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00167-1

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C. J. Hobbs et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1365–1369
Table 1. Truncation analysis on hamster middle-T antigen
Compd
Amino acid sequence
IC50 (mM)a
1
2
3
4
5
EPQpYEEIPIYL
AcpYENHMe
AcpYEENHMe
AcpYEEINHMe
AcpYEEIPNHMe
0.36
350
17
2.0
1.6
aCompounds were tested using a scintillation proximity assay.5
low likelihood of cell penetration due to the charged
nature of the phosphotyrosine containing peptides. In
this paper, we describe the identification of an
uncharged replacement for the doubly charged Glu-Glu
sequence and the preparation of compounds containing
potent replacements for the isoleucine substituent in the
pY+3 pocket.6
Figure 1. Conformational restriction of the Glu-Glu linker with
9-aminopyridazinodiazepine.
A search was carried out of internal angles in
N–C(C¼O)–N–C–C¼O substructures found in the
Cambridge crystallographic database, which identified
the 9-aminopyridazinodiazepine carboxamide 67 as a
potential Glu-Glu mimetic. This substructure is shown
overlaid on the tetrapeptide in Figure 2. It can be seen
that both of the b carbon atoms of the glutamic acid
side-chains are overlaid by ring carbons of the 9-amino-
pyridazinodiazepine. Synthesis of the tetrapeptide
mimetic containing the bicycle 7 confirmed it was a
suitable replacement for the Glu-Glu linker since 7 was
essentially equipotent with the tetrapeptide (Fig. 1). We
obtained co-crystals of 7 bound to lck SH2 domain
(Fig. 3).8 This structure shows that the tyrosine phos-
phate adopts a similar conformation to that of the pep-
tide structure reported by Eck2, and indeed the
hydrogen bond contacts for the acetamide and the
phosphotyrosine side-chain correspond to those
observed in the Eck structure. However, the 9-amino-
pyridazinodiazepine is considerably displaced from the
peptide backbone in Eck’s structure and as a result the
isoleucine does not penetrate as deeply into the pY+3
pocket. 1H NMR experiments showed that the side-
chain of isoleucine was relatively mobile in the pY+3
pocket. The bicyclic ring system makes a number of
contacts with the SH2 domain. There is a hydrogen
bond from the side-chain NH of Lys 57 to the C6 car-
bonyl of the 9-aminopyridazinodiazepine. The C8 and
C9 carbons of the 9-aminopyridazinodiazepine make
Van der Waals contacts with the aromatic ring of tyr-
osine 59, which may explain why the isoleucine does not
penetrate the pY+3 pocket very deeply, or it may be
due to crystal packing effects. There are water mediated
hydrogen bonds from the carbonyl of the acetamide to
the backbone NH of histidine 58 and from the C10
carbonyl of the 9-aminopyridazinodiazepine to the
backbone NH of lysine 60. More detailed analysis of
this structure will be published shortly.
Figure 2. Molecular modelling showing the the 9-aminopyr-
idazinodiazepine 6 (magenta) overlaid on the peptide backbone of the
hmT antigen (multicolour) from Eck’s structure.2
Figure 3. Comparison of the crystal structures of lck SH2/iso-
leucinemethylamide 7 (multicolour) and lck SH2/adamantylmethyl-
amide 31 (magenta).
molecule close to the base of the pocket. Our initial
strategy was to more completely occupy the pocket by
incorporating replacements for isoleucine methylamide
into the structure.
We subsequently turned our attention towards improv-
ing the activity of the lead series by finding improved
ligands for the pY+3 pocket. The pocket is bounded
predominantly by hydrophobicresidues with a tyrosine
located at the bottom of the pocket and also a water
The hmT antigen was prepared by Fmoc-SPPS on a
Milligen 9050 peptide synthesiser using the solid-phase

C. J. Hobbs et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1365–1369
1367
Target compounds were prepared as described in
Scheme 1. The dibenzylphosphotyrosine 8 was coupled
to the 9-aminopyridazinodiazepine 9 using a mixed
anhydride procedure to give products 10. The tert-
butylester was removed using TFA and the resulting
acid was coupled to the appropriate amine using the
mixed anhydride procedure. Deprotection of the di-
benzyl phosphate and tert-butyl or benzyl esters present
on the amine was carried out with either TFA or by
catalytic hydrogenation using 10% Pd/C to afford tar-
get compounds 7 and 20–39. Mixtures of diastereo-
isomers were separated by RP-HPLC.
Scheme 1. Reagents and conditions: (a) IBCF, NMM, THF, ꢀ20 ꢁC;
(b) 20% TFA/CH2Cl2; (c) IBCF, NMM, RNH2; (d) 95% aq TFA
(examples 7, 20–33, 38, 39) or 10% Pd/C, AcOH (examples 34–37).
Amines were commercially available except for the
benzyl ester of 3-aminoindan-1-carboxylate 16 and the
tert-butyl ester of 3-aminoindan-1-acetic acid 19. The
synthesis of compound 16 is described in Scheme 2.
Phenylsuccinic anhydride 11 was treated with AlCl3 to
give 3-oxoindan-1-acetic acid which was protected as a
benzyl ester to give compound 12. Treatment with
hydroxylamine hydrochloride and base afforded the
oxime 13. Treatment of 13 with diphenylchloro phos-
phine gave the iminophosphine oxide 14 which was
reduced to the aminophosphine oxide 15 with NaBH4.
Treatment of 15 with HCl in dioxan gave the benzyl
ester of 3-aminoindan-1-carboxylic acid 16 as a mixture
of all four diastereomers. Compound 19 was prepared
as summarised in Scheme 3. 3-Oxoindan-1-acetic acid
17 was treated with N,N,-diisopropyl-O-t-butylisourea
followed by hydroxylamine hydrochloride and base to
afford the oxime 18. Catalytichydrogenation of 18 gave
the tert-butylester of 3-aminoindan-1-acetic acid 19 as a
mixture of the two cis diastereoisomers.
Scheme 2. Reagents and conditions: (a) AlCl3, ClCH2CH2Cl;
.
(b) BnOH, H2SO4; (c ) NHOH HCl, NaOH, EtOH; (d) ClPPh2, NEt3,
2
CH2Cl2, ether, ꢀ78 ꢁC; (e) NaBH4, THF; (f) 4 M HCl, dioxan.
Inspection of the crystal structure of 7 showed that the
C-terminal methylamide did not make any hydrogen
bond contacts with the SH2 domain. In support of this,
incorporation of (S)-2-methylbutylamine into the struc-
ture afforded 27 which was actually slightly more active
than the parent amino acid derivative. Hence, the first
series of replacements targeted were commercially
available amines which might occupy the pocket more
fully. However, neither benzylamine (20, 1.5 mM) nor
cyclohexylmethylamine (29, 1.6 mM) showed any
improvement in activity compared to the isobutyl
group. A wide range of substituted benzylamines were
incorporated (a selection of these replacements are
shown in Table 2) which had very similar activity to the
benzylamide. One exception was the p-chloro-
benzylamide (21, 0.4 mM) which was 4-fold more active
than the benzylamide. In fact most of the replacements
(Table 2) showed an activity of between 1 and 10 mM.
Even the adamantylmethyl group (30, 1.1 mM) was of
roughly equal potency to the methylbutylamide. A
crystal structure was obtained for 30 which showed the
adamantyl group bound to the mouth of the pocket,
and the 9-aminopyridazinodiazepine displaced even
further from the peptide backbone of Eck’s structure.10
Inspection of the crystal structures shows there are a
number of water mediated interactions between the
pyridazinodiazepine bicycle and the SH2 domain and
this may allow sufficient movement of the bicycle to
accommodate a wide range of substituents in the pY+3
Scheme 3. Reagents and conditions: (a) N,N-diisopropyl-O-t-butyl-
.
isourea, CH2Cl2; (b) NH2OH HCl, NaOH, EtOH; (c) H2, Pd/C,
MeOH.
phosphorylation protocol described by Kitas et al.9
Peptides (2–5) were prepared by sequentially coupling
methylamine, the appropriate N-BOC amino acid and
either N-BOC–Tyr–(PO3Bn)–OH (for the tetra and
pentapeptides) or Ac–Tyr–(PO3Bn)–OH (for the di and
tripeptides) from C- to N-terminus using isobutyl-
chloroformate as the coupling agent. Removal of the N-
BOC protective group was effected with 4 N HCl in
ethyl acetate. Removal of the N-BOC protective group
from phosphotyrosine containing peptides was effected
with HCl in acetic acid or formic acid. Acetylation of
the N-terminus was effected with acetylchloride and N-
methylmorpholine. Finally, hydrogenation of the pro-
tecting groups with 10% Pd/C in either methanol or
formicacid afforded the desired peptides.

1368
C. J. Hobbs et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1365–1369
Table 2. Lck SH2 domain inhibitory activity of peptidomimetics
containing alkylamides and conformationally restricted benzylamides
in the pY+3 pocket
Inspection of the crystal structure of 7 indicated that a
carboxylic acid attached to one of the conformationally
restricted benzylamines might be able to form a hydro-
gen bond to arginine 67 on the rim of the pY+3 pocket.
The 3-aminoindan-1-carboxylic acid was prepared as a
mixture of all four possible diastereomers which was
separated into the four constituent diastereomers by
preparative RP-HPLC. The 3(R)aminoindan-1(S)-car-
boxylicacid ( 37, 0.1 mM, Table 3) was the most potent
diastereoisomer binding 16-fold more tightly than the
benzylamide. Modelling suggested that substitution of
the indane with an acetic acid should also allow inter-
action with arginine 67. The aminoindaneacetic acid
was prepared as a mixture of cis diastereomers and
separated by RP-HPLC. The 3(R)-aminoindan-
1(R)-acetic acid (39, 0.033 mM) was the most potent
isomer and indeed the most potent pY+3 replacement
prepared in this series. We were unable to obtain a crystal
structure of the aminoindane acetic acid to confirm the
interaction of the carboxylic acid with arginine 67 or
indeed the binding of the phenyl ring in the pY+3 pocket.
Compd
R
IC50 (mM)a
20
21
22
23
24
25
26
27
28
29
30
31
32
33
PhCH2NH
pClPhCH2NH
pHOPhCH2NH
EtNH
PrNH
1.5
0.4
10
8.5
6.0
1.9
1.8
1.2
1.8
1.6
1.1
1.9
1.1
1.2
BuNH
PnNH
(S)-2-Methylbutylamine
NeopentylNH
Cyclohexylmethylamine
Adamantylmethylamine
1-Aminomethylnaphthalene
1-Aminonaphthalene
1-Aminoindane
Starting from the crystal structure of p56lck SH2 domain
reported by Eck et al.2 we have determined the minimum
peptide sequence needed for optimal binding to be the
tetrapeptide Ac-pY-E-E-I-NHMe. We have designed an
uncharged replacement for the Glu-Glu linker and
explored potential replacements for the isoleucine moi-
ety in the pY+3 pocket. The majority of replacements
were of similar activity to the isoleucinemethylamide.
However, we did identify a potent replacement con-
taining a carboxylic acid. Having addressed the issues of
peptide hydrolysis and potency, we will describe the
identification of phosphotyrosine replacements and
antagonists of reduced charge in a subsequent paper.
aCompounds were tested using a scintillation proximity assay.5
Table 3. Lck SH2 domain inhibitory activity of peptidomimetics
containing aminoindanes in the pY+3 pocket
Acknowledgements
Compd
a*
b*
X
IC50 (mM)a
The authors wish to thank all of their colleagues in the
Physical Methods department at RDW whose hard
work and dedication have contributed to this work.
34
35
36
37
38
39
Trans diast. A
Trans diast. B
R
S
R
S
CO2H
CO2H
CO2H
CO2H
CH2CO2H
CH2CO2H
7.7
7.3
0.1
2.0
0.03
2.8
S
R
R
S
References and Notes
aCompounds were tested using a scintillation proximity assay.5
1. Songyang, Z.; Shoelson, S. E.; Chaudhuri, M.; Gish, G.;
Pawson, T.; Hase, W. G.; King, F.; Roberts, T.; Ratnofsky,
S.; Lechleider, R. J.; Neel, B. G.; Birge, R. B.; Fajardo, J. E.;
Chou, M. M.; Hanafusa, H.; Schaffhausen, B.; Cantley, L. C.
Cell 1993, 72, 767.
pocket. A limited correlation between structure and
activity was observed in certain series of compounds.
For instance, in the alkylamides there was a gradual
increase in activity on going from the ethylamide (23,
8.5 mM) to the butylamide (25, 1.9 mM) but no further
increase in activity for the pentylamide (26, 1.8 mM).
2. Eck, M. J.; Schoelson, S. E.; Harrison, S. C. Nature 1993,
362, 87.
3. A number of structures of p56lck SH2 domain have
subsequently appeared in the literature. (a) Eck, M. J.;
Atwell, S. K.; Shoelson, S. E.; Harrison, S. C. Nature 1994,
368, 764. (b) Mikol, V.; Baumann, G.; Keller, T. H.;
Manning, U.; Zurini, M. G. M. J. Mol. Biol. 1995, 246,
344, as well as a number of structures of the closely related
p60src SH2 domain. (c) Shakespeare, W.; Yang, M.; Boha-
cek, R.; Carasoli, F.; Stebbins, K.; Sundaramoorthi, R.;
Azimioara, M.; Yu, C.; Pradeepan, S.; Metcalf III, C.;
Haraldson, C.; Merry, T.; Dalgarno, D.; Narula, S.;
Hatada, M.; Lu, X.; van Schravendijk, M.-R.; Adams, S.;
A series of benzylamides was prepared in an attempt to
improve the potency of the series, including the con-
formationally restricted 1-aminoindanylamide (33, 1.2
mM) and 1-aminonaphthylamide (32, 1.1 mM) and the
benzfused derivative 1-aminomethylnaphthylamide (31,
1.9 mM). All were essentially equipotent with the
benzylamide.

C. J. Hobbs et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1365–1369
1369
Violette, S.; Smith, J.; Guan, W.; Bartlett, C.; Herson, J.;
Iuliucci, J.; Weigele, M.; Sawyer, T. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 9373. (d) Buchanan, J. L.; Bohacek, R. S.;
Luke, G. P.; Hatada, M.; Lu, X.; Dalgarno, D. C.; Narula,
S. S.; Yuan, R.; Holt, D. A. Bioorg. Med. Chem. Lett.
1999, 9, 2353. Plummer, M. S.; Holland, D. R.; Shahri-
pour, A.; Lunney, E. A.; Fergus, J. H.; Marks, J. S.;
McConnell, P.; Mueller, W. T.; Sawyer, T. K. J. Med.
Chem. 1997, 40, 3719.
concn of 7 nM and 100 mg of streptavidin coated SPA beads
(Amersham Pharmacia Biotech) per well. The buffer used to
dilute beads and protein reagents was 100 mM Tris–HCl pH
8.0, 100 mM NaCl, 4 mM DTT, 100 mg/mL BSA. Titration
curves of inhibitors or control compounds were diluted in
DMSO and added to the assay so that the final concn of
diluant was 5%. Finally the assay was initiated by adding 50
ng of GST-lck SH2 fusion protein, 150 ng of anti-GST IgG
(Molecular Probes, Inc.) and 50 nCi of 125I anti rabbit IgG,
antibody. Binding was allowed to reach equilibrium for a
minimum of 4 h prior to counting on a Wallac Microbeta
counter.
6. A number of groups have addressed these issues in recent
years. For a recent review on the identification of SH2 domain
antagonists including p56lck, see; Garcia-Echeverria, C. Curr.
Med. Chem. 2001, 8, 1589.
7. Attwood, M. R.; Hassall, C. H.; Krohn, A.; Lawton, G.;
Redshaw, S. J. Chem. Soc., Perkin Trans. 1 1986, 6, 1011.
8. The atomic coordinates of compound 7 bound to p56lck
SH2 domain have been deposited to the Protein Data Bank
and the EBI deposition code is EBI-8436.
4. Tong, L.; Warren, T. C.; King, J.; Betageri, R.; Rose, J.;
Jakes, S. J. Mol. Biol. 1996, 256, 601.
5. The SH2 domain from amino acid residues 127 to 229 of
p56lck was expressed as a GST fusion protein by subcloning
into the vector pGEX-3X (Amersham Pharmacia Biotech.).
After induction with isopropylthiogalactoside, soluble GST-
SH2 protein was initially purified on a Glutathione Sepharose
column (Amersham Pharmacia Biotech) followed by phospho-
Tyrosine Agarose affinity chromatography (Sigma Ltd.). A
final buffer exchange was carried out on a PD10 column
(Amersham Pharmacia Biotech).
The assay employed a scintillation proximity readout and
monitored the binding of the SH2 fusion protein to the pY
containing peptide LEDFFTATEGQpYQPQP. This peptide
corresponds to the region around the autoregulatory site at Y
505 on p56lck. The assay was run on a microtitre format and
used a biotinylated version of the above peptide at a final
9. Kitas, E. A.; Knorr, R.; Trzeciak, A.; Bannwarth, W. Helv.
Chim. Acta 1991, 74, 1314.
10. The atomic coordinates of compound 30 bound to p56lck
SH2 domain have been deposited to the Protein Data Bank
and the EBI deposition code is EBI-8445.