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Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain
BBRC Biochemical and Biophysical Research Communications 310 (2003) 334–340 www.elsevier.com/locate/ybbrc
Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domainq Ya-Qiu Long,a,* Ribo Guo,b Juliet H. Luo,b Dajun Yang,b and Peter P. Rollerc,* a
c
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China b School of Medicine, University of Michigan, Ann Arbor, MI 48109, USA Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, FCRDC, Frederick, MD 21702, USA Received 4 July 2003
Abstract Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu2 sidechain for the absence of the phosphate functionality on Tyr0 in the peptide ligand, cyclo(CH2 CO-Glu2 -Leu-Tyr0 -Glu-Asn-Val-Gly-Met5þ -Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr + 5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with b-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC50 of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla2 ) in which the original residue of Glu2 was substituted by c-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 lM level. More significantly, both G1TE(Gla2 ) alone and the conjugate of G1TE(Gla2 ) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC50 of 50 and 2 lM, respectively. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Non-phosphorylated Grb2-SH2 antagonist; Thioether-bridged cyclic peptide; Hydrophobic residue; Sulfoxide linkage; Conformational constraint; Phage library; Global optimization; Chemotherapeutic agents; Intracellular signaling; ErbB2-related cancer
SH2 (src homology 2) domains are protein modules of approximately 100 amino acids found in more than 50 critical intracellular signaling proteins [1]. SH2 domains directly bind to phosphorylated growth factor receptors or other cytoplasmic proteins phosphorylated in response q
A preliminary account of this information was presented in: Y.-Q. Long, P.P. Roller, Potentiating effect of distant sites in cyclic peptide antagonists of the Grb2-SH2 domain, in: E. Benedetti, C. Pedone (Eds.), Peptide 2002 (Proceedings of the 27th European Peptide Symposium), Edizioni Ziino, Napoli, Italy, 2002, pp. 982–983. * Corresponding authors. Fax: +86-21-5080-7088 (Y.-Q. Long), Fax: 1-301-846-6033 (P.P. Roller). E-mail addresses: [email protected] (Y.-Q. Long), [email protected] (P.P. Roller). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.022
to extracellular signals, and thereby serve as important mediators of tyrosine kinase-dependent signal transduction [2]. Among these SH2 domains, the Grb2-SH2 domain directly mediates the activation of mitogenic Ras pathway. An aberrant Grb2-SH2 dependent Ras activation pathway has been shown to be essential for cellular transformation in a subset of human tumors [3]. Compounds which selectively antagonize the SH2 domain of Grb2 should interrupt these signaling pathways and thus are attractive targets for drug therapy in oncology [4]. Over the past few years, considerable progress has been made in designing ligands with high affinity for the Grb2SH2 domain [5–8]. However, the phosphotyrosine residue still serves as a critical recognition determinant for
Y.-Q. Long et al. / Biochemical and Biophysical Research Communications 310 (2003) 334–340
Fig. 1. The structures of the substitutes in position Y + 5 and combined modifications of the distant sites in G1TE.
effective binding to Grb2-SH2, which would confer poor cellular activity and selectivity [9,10]. Our approach is based on a phage library derived non-phosporylated disulfide-bridged cyclic decapeptide and its redox-stable thioether cyclized analog, cyclo(CH2 CO-Glu2 -Leu-Tyr0 -Glu-Asn-Val-Gly-Met5þ Tyr-Cys)-amide (termed G1TE) [11,12], which could inhibit the EGFR/Grb2-SH2 domain association in a phosphotyrosine independent manner. The non-phosphorylated cyclopeptide specifically binds to the Grb2SH2 domain, but does not bind to the homologous Src-SH2 domain [11]. The absence of a phosphate moiety in this phage peptide contributes to its remarkably high specificity and enhanced bioavailability. Unlike the phosphopeptide ligands of Grb2-SH2 in which phosphotyrosine is a major contributor to the binding, this novel non-phosphorylated cyclic peptide antagonist requires essentially all amino acids, except Gly4þ , as well as the constrained conformation in retaining high affinity binding with the Grb-SH2 domain [11,13–16]. This multipoint binding pattern of G1TE encouraged us to explore the potentiating effect of distant sites from the consensus sequence of –YXN– on the backbone to further improve the activity. In the current study, we report the functional importance of the fifth residue Met5þ , C-terminal to the consensus sequence of –Y0 -XN2þ – in G1TE, and the simultaneous optimization of the distant sites such as the Y ) 2 position, the Y + 5 position, and the cyclization linkage in G1TE (see Fig. 1) leading to potent non-phosphorylated Grb2-SH2 antagonist not relying on phosphotyrosine mimics.
Materials and methods Inhibitors The thioether-bridged cyclic peptides were synthesized in a similar procedure described previously [17]. Briefly, the linear peptide was
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synthesized on solid phase on a PAL amide resin with an ABI 433A peptide synthesizer utilizing FastMoc chemistry. After removing the Na -Fmoc group with 20% piperidine/DMF, the resin-bound protected peptide was N-terminally chloroacetylated by 3 equivalents of chloroacetic anhydride in DMF at RT overnight. Then the peptide was cleaved from the resin by using TFA containing 2.5% each (v/v) of triethylsilane and deionized water (2 h). For isolation of the product, two-thirds of the cleavage reagent mixture was evaporated under N2 stream and the residue was triturated and washed with ice-cold ether. The precipitated crude peptide was dissolved in 50 mL of water and added dropwise into 100 mL of aqueous solution, which was adjusted to pH 8–9 with 0.2 M aqueous solution of NH4 OH and NH4 OAc (1:1, mol/mol), repeatedly. After 6 h stirring at RT, the cyclization reaction was quenched by addition of 30% acetic acid until pH 4–5 to give the thioether-bridged analogs of G1TE. The oxidation of the thioether linkage into sulfoxide was accomplished using 5% H2 O2 aqueous solution. The resulting sulfoxide diastereoisomers were easily separated by RP-HPLC. The absolute configuration of the sulfoxide analogs was determined by CD spectra using documented method [18]. All final products were purified by RP-HPLC and the identity was assessed by amino acid and mass spectral analyses. HPLC conditions: Vydac C4 column (20 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated below; flow rate, 10 mL/min; and UV detector, 225 nm. FAB-MS (unit resolution, glycerol matrix) was performed on a VG Analytical 7070E-HF mass spectrometer. Amino acid analysis (6 N HCl, 110 °C, 24 h) was carried out at the Protein and Carbohydrate Structure Facility (University of Michigan, Ann Arbor, MI, USA). The PAL amide resin and Fmoc derivatives of standard amino acids were obtained from Perkin–Elmer/Applied Biosystems Division. L -Citrulline (Cit), L -a-aminoadipic acid (Adi), c-carboxy-L -glutamic acid (Gla), L -norleucine (Nle), L -ornithine (Orn), L -homophenylalanine (hPhe), L -di-n-butyl glycine (Dbg), and L -tert-butyl alanine (tBuAla) were purchased from BACHEM (Torrance, CA, USA) in Fmoc protection form. Physicochemical data for selected cyclic peptides: Peptide 11. RP-HPLC gradient 25–40% B over 35 min; Rt ¼ 18:5 min; FAB-MS (M + H)þ 1271.3 (calc. 1271.55). Peptide 12. RP-HPLC gradient 25–40% B over 35 min; Rt ¼ 21:3 min; FAB-MS (M + H)þ 1271.5 (calc. 1271.55). Peptide 13. RP-HPLC gradient 20–70% B over 27 min; Rt ¼ 16:3 min; FAB-MS (M + H)þ 1299.6 (calc. 1299.55). AAA: Asp 0.92 (1), Glu 1.21 (1), Gly 1.01 (1), Tyr 1.94 (2), Val 0.92 (1), Leu 0.99 (1). Peptide 14. RP-HPLC gradient 20–40% B over 35 min; Rt ¼ 17:7 min; FAB-MS (M + H)þ 1315.6 (calc. 1315.54). Peptide 15. RP-HPLC gradient 20–40% B over 35 min; Rt ¼ 20:0 min; FAB-MS (M + H)þ 1315.5 (calc. 1315.54). Peptide 16. RP-HPLC gradient 40–90% B over 27 min; Rt ¼ 20:6 min; FAB-MS (M + Na)þ 2823.4 (calc. 2824.3). Peptide 17. RP-HPLC gradient 50–90% B over 24 min; Rt ¼ 18:7 min; FAB-MS (M + Na)þ 2780.0 (calc. 2781.3). Binding affinity measurement of peptides to Grb2-SH2 domain using surface plasmon resonance The competitive binding affinity of ligands for the Grb2-SH2 protein was assessed using Biacore surface plasmon resonance (SPR) methods on a BIAcore 2000 instrument (Pharmacia Biosensor, Uppsala, Sweden). IC50 values were determined by mixing various concentrations of inhibitors with the recombinant GST-Grb2-SH2 domain protein and measuring the amount of binding at equilibrium to the immobilized SHC phosphopeptide(pTyr317 ), i.e., biotinyl-DDPS-pYVNVQ, in a manner described previously [11]. Briefly, the biotinylated phosphopeptide was attached to a streptavidin coated SA5 Biosensor chip and the binding assays were conducted in, pH 7.4, PBS buffer containing 0.01% P-20 surfactant (Pharmacia Biosensor).
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Biological cells and cell culture Cell lines were obtained from the American Type Culture Collection (Rockville, MD) and Lombardi Cancer Center, Georgetown University Medical Center. Cells were routinely maintained in improved minimal essential medium (IMEM, Biofluids, Rockville, MD) with 10% fetal bovine serum. Cultures were maintained in a humidified incubator at 37 °C and 5% CO2 . (A) Inhibition of Grb2-SH2 domain binding in cell homogenates. Cell lysates were prepared from serum-treated erbB2 overexpressing breast cancer cells (MDA-MB-453), as described previously [11]. Cell lysates were incubated with G1TE(Gla2 ), G1TE(Gla2 )-carrier, or control peptide G1TE, G1TE-carrier at concentrations of 0.1, 0.4, 2, 10, and 50 lM for 30 min, and then 500 lg of protein was immunoprecipitated with anti-Grb2 antibody (Santa Cruz Biotech., Santa Cruz, CA, USA) and collected with protein A–Sepharose. Immunoprecipitated proteins were separated by SDS–PAGE on 8–16% gradient gels (Novex, San Diego, CA, USA). pTyr-containing proteins were detected by Western blotting using anti-phosphotyrosine antibodies (Upstate Biotechnology, Lake Placid, NY, USA) and visualized with ECL (Amersham, Arlington Heights, IL). Previous experiments have shown that a major tyrosine phosphorylated protein in these cells is the p185erbB2 , which is overexpressed as a consequence of gene amplification [19]. Western blotting with Grb2 MAb was done as a control. (B) Inhibition of Grb2-SH2 domain binding in whole cells [8]. ErbB2 overexpressing breast cancer cells, MDA-MB-453, were treated with desired concentrations of inhibitors for 3 h in serum-free IMEM (Gibco). Cells were washed twice with PBS to remove inhibitor and then cell lysates were prepared using 1% Triton X-100 in PBS containing 0.2 mM NaVO4 . Grb2 and associated Grb2-binding proteins were immunoprecipitated from each lysate (500 lg) with anti-Grb2 antibodies and collected using protein A–Sepharose. The following work-up is the same as that in the cell homogenate assay.
Results and discussion The phage library derived nonphosphorylated cyclic peptides, typified by G1TE, 1, comprise a novel family of agents binding to the Grb2-SH2 domain in a phosphotyrosine independent manner [11–17]. Earlier Ala mutation studies demonstrated that essentially all amino acids as well as special conformation are required for sustenance of the biological activity. The multipoint-
binding pattern of G1TE provides us with a unique opportunity and potential to further improve their activity by systematic structural optimization. Our previous structure/activity studies amply demonstrated that the carboxyl side chain of Glu in position Y ) 2 of G1TE partially compensates for the absence of the phosphate group on Tyr0 [14,15]. Since positions Y ) 2 and Y0 share an overlapping function in the Grb2-SH2 binding, the optimal combination of amino acids in the two positions produces potent peptide antagonists of Grb2-SH2, which are exemplified by G1TE(Gla2 ) (IC50 ¼ 0.64 lM) [14], G1TE(Ala2 , pTyr0 ) (IC50 ¼ 0.023 lM) [14], and G1TE(Adi2 , cmPhe0 ) (IC50 ¼ 0.70 lM) [15]. Generally speaking, when Tyr is in position Y0 which provides phenyl group but lacks the negative charge, the acidic side chain in the compensatory position of Y ) 2 can provide a significant enhancement in Grb2SH2 domain binding potency. When pTyr is present in position Y0 which carries bulky and highly charged phosphate, the acidic side chain in position Y ) 2 is unfavored and Ala substitution showed improved activity. When the pTyr mimic cmPhe is built into the position Y0 which carries less bulky but still doubly charged carboxyl groups, the acidic side chain in position Y ) 2 exhibited a slight potentiating effect on its binding. Our recent investigation on the conformational requirement of G1TE upon binding to Grb2-SH2 domain has indicated that the polarity and configuration of the cyclization linkage were important in assuming the active conformation [16]. When the flexible thioether linkage in G1TE was oxidized into the relatively rigid sulfoxide, the R-configured sulfoxide peptide isomers provide superior Grb2-SH2 domain antagonists. The stereochemistry of the sulfoxide diastereoisomers was identified by CD spectra using documented method [18]. In our continuing efforts to explore the functional importance of the distant sites on G1TE thus deriving potent non-phosphorylated Grb2-SH2 antagonists, we conducted a systematic modification in the fifth residue
Table 1 Grb2-SH2 domain inhibitory activity of the Peptides 1–9a Compound
Peptide analogs
X5þ
IC50 (lM)b
1 2 3 4 5 6 7 8 9
G1TE, control [11] G1TE(Met5þ deleted) G1TE(Met(O)5þ ) G1TE(Orn5þ ) G1TE(Adi5þ ) G1TE(Nle5þ ) [16] G1TE(hPhe5þ ) G1TE(Dbg5þ ) G1TE(tBuAla5þ )
Met Deleted Met(O) Orn a-Aminoadipic acid Nle HomoPhe Di-n-butylGly b-t-ButylAla
25 5c 315 185 165 114 236 164 120 20 26.5 3.5 16 6 107 43 14.3 0.5
a Competitive binding assays with SHC phosphopeptide, DDPSpYVNVQ, using surface plasmon resonance on a BIAcore 2000 instrument as reported earlier [11]. b Values are means of at least two independent experiments and are expressed as the concentration at which half-maximal competition was observed (IC50 ). IC50 of reference SHC(pY317) peptide: 1.1 0.3 lM. Standard deviation is given in parentheses. c This value has previously been reported [16] and is used here as a reference.
FAB-MSd aP f values were calculated from Rekker fragmental constants listed in [20]. b HPLC conditions: Vydac C18 column (10 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated; flow rate, 2.5 mL/min; and UV detector, 225 nm. c HPLC conditions: Vydac C4 column (20 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated; flow rate, 10.0 mL/min; and UV detector, 225 nm. d FAB-MS (unit resolution, glycerol matrix) was performed on a VG Analytical 7070E-HF mass spectrometer.
Rt ¼ 16:5 min (gradient 20–70% B over 27 min)c (M + H)þ 1255.3 (calc. 1255.56) Rt ¼ 18:6 min (gradient 20–70% B over 27 min)c (M + H)þ 1297.5 (calc. 1297.6) Rt ¼ 16:6 min (gradient 20–70% B over 27 min)c (M + H)þ 1290.0 (calc. 1289.5) Rt ¼ 12:3 min (gradient 20–65% B over 25 min)b (M + H)þ 1242.1 (calc. 1242.5) Rt ¼ 12:8 min (gradient 20–80% B over 30 min)b (M + H)þ 1258.7 (calc. 1259.5)
2.878 2.258 1.229
7 6 [16]
Based on the preliminary results, the following effort is focused on the hydrophobicity modification on the side chain of residue X5þ in G1TE. First of all, the sulfur atom of Met5þ was replaced with a less polar CH2 group. Encouragingly, the resulting peptide 6 turned out to have comparable activity (6, IC50 ¼ 26.5 lM). Thus further modifications included increasing the hydrophobicity of the side chain by incorporating aromatic homophenylalanine (Peptide 7) or bulky di-n-butylglycine (Peptide 8) or branched b-t-butylanaline (Peptide 9) into position Y + 5 of G1TE. Results indicate that aromatic moiety containing homophenylalanine shows an improved binding affinity (7, IC50 ¼ 16 lM) with improved hydrophobicity. But when Met5þ was replaced
1 [11]
The hydrophobicity of the side chain in position Y + 5 strengthens the intramolecular packing for a favored conformation of the cyclic peptide binding to Grb2-SH2
Peptide P a f
Judiciously altering the original residue of Met5þ with methioninesulfoxide whose side chain is more polar (Peptide 3), or ornithine whose side chain carries a basic end group (Peptide 4), or aminoadipic acid whose side chain bears an acidic end group (Peptide 5), constitutes our effort to examine the effect of polarity on the interaction of X5þ side chain in G1TE with Grb2-SH2 binding pocket. As shown in Table 1, with the retention of the chain length, the increase in polarity of the side chain results in the loss of binding affinity. Either neutral (3, IC50 ¼ 165 lM) or ionic polarity (4, IC50 ¼ 236 lM; 5, IC50 ¼ 120 lM) in position Y + 5 disfavored the interactions of G1TE analogs with the Grb2-SH2 protein. The basic or acidic head of the side chain particularly damaged the interactions.
Table 2 P f valuesa of the side chain in position Y + 5 in peptides 1, 6–9 and their physicochemical data
Polar amino acids are not favored for the side chain interactions in position Y + 5
Physico-chemical data RP-HPLC
4.516
8
9
X5þ . In the present paper, a series of G1TE analogs with various substitutions in position Y + 5 were synthesized and the Grb2-SH2 domain inhibitory activity was assessed using Biacore SPR methodology Table 1. Our earlier Ala scan studies indicated that replacement of Met5þ with Ala remarkably reduced the inhibitory activity by 60-fold [13]. Accordingly, the Met5þ deleted peptide 2 showed a dramatic decrease of the binding affinity (2, IC50 ¼ 315 lM). The initial results implicated that the amino acid in position Y + 5 plays an important role for the effective binding of G1TE to Grb2 protein. It might function by directly contacting with the Grb2 binding pocket or by stabilizing the favored conformation via the ligand side-chain interactions. Therefore, variations in the side chain of residue X5þ with respect to the polarity and the hydrophobicity were examined to determine the favorable interaction for improved binding.
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by the more hydrophobic di-n-butylglycine, a reduced binding affinity was observed (8, IC50 ¼ 107 lM), suggesting that the bulky and flexible groups interfere with the optimal orientation of the side chain in position Y + 5 for effective binding. Not surprisingly, the relatively rigid and still very hydrophobic b-t-butylalanine gained a remarkable increase in the binding affinity (9, IC50 ¼ 14.3 lM), improved by a factor of 8.4-fold relative to its polar counterpart (5, IC50 ¼ 120 lM). Even compared to its parent compound 1, Peptide 9 showed a 2.0-fold enhancement in the binding potency just by slight alteration of the side chain in position Y + 5. Comparison of the hydrophobicity of the side chains in position Y + 5 on selected peptides (Table 2), which can be roughly evaluated by Rekker’s f value [20], indicated that shape as well as hydrophobicity is responsible for the potentiating effect. Our molecular modeling has confirmed that the Met5þ residue of G1TE formed a hydrophobic packing with Leu1 , Cys7þ , and the N-acetyl group within G1TE [18]. This intramolecular packing stabilizes the favorable backbone conformation of the cyclic peptide for other residues effectively binding to the Grb2-SH2 domain. Thus a more hydrophobic and less flexible amino acid in position Y + 5 results in favorable and well-defined van der Waals interactions with Leu1 and Cys7þ , correspondingly enhancing the effective interaction of G1TE analogs with the Grb2SH2 protein. Combined potentiating effect of the distant sites confers high affinity to non-phosphorylated and non-pTyr-mimetic containing Grb2-SH2 antagonists Previous study has disclosed that the overlapping function of amino acids Tyr0 and Glu2 in G1TE secured effective binding to Grb2-SH2 domain, even without the presence of a phosphotyrosyl group [14,15]. The systematic modification in the fifth residue X5þ demonstrated that a hydrophobic side chain is favorable
for the intramolecular packing to stabilize the favored backbone conformation. Besides, the polarity of the cyclization linkage was implicated as crucial in maintaining such an active conformation for effective binding to Grb2-SH2 [16]. We are intrigued to examine the synergistic effect of simultaneous optimization of the distant sites on G1TE. A series of relevant thioether cyclized peptides were synthesized for this purpose (Table 3). Selected analogs were also oxidized with aqueous H2 O2 to provide pairs of sulfoxide-cyclized analogs that could be separated by HPLC. The stereochemistry of the sulfoxide diastereoisomers was identified by CD spectra [18]. The faster eluting sulfoxide diastereoisomer was found to be R-configured, and the slower eluting sulfoxide was the S-configured analog. Consistently, the R-configured sulfoxide analog was remarkably more potent than its cognate pair (e.g., 11 versus 12, 14 versus 15), whereas the S-configured sulfoxide is even less active than the thioether analog (e.g., 12 versus 9, 15 versus 13). As previously indicated [13], substitution of Glu2 with Gla2 in G1TE significantly favored the interaction with Grb2-SH2 (10, IC50 ¼ 0.77 lM). Current results show that modification of distant sites in the non-phosphorylated cyclic antagonist, i.e., residue tBuAla5þ and the sulfoxide linkage, displayed a slight enhancing effect (9, IC50 ¼ 14.3 lM; 11, IC50 ¼ 11.7 lM). More significantly, simultaneous structural modification of the three sites within G1TE provided peptide 14, with a 113-fold enhancement in Grb2-SH2 domain antagonist potency (14, IC50 ¼ 0.22 lM) relative to the parent compound, G1TE (1, IC50 ¼ 25.0 lM). Thus, by judiciously combining the optimal modulations of the functional groups on the distant sites, we have obtained a highly potent non-phosphorylated Grb2-SH2 inhibitor that is also devoid of pTyr mimics. This new inhibitor constitutes a novel experimental agent for controlling Grb2-SH2 domain mediated cellular signaling processes.
Table 3 Grb2-SH2 domain inhibitory activity of G1TE analogs with global optimization on distant sitesa Compd.
Peptide analog
1 9 10 11 12 13 14
G1TE [11] G1TE(tBuAla5þ ) G1TE(Gla2 ) [14] G1TE(tBuAla5þ )-sulfoxide linker, R-configured sulfoxide G1TE(tBuAla5þ )-sulfoxide linker, S-configured sulfoxide G1TE(Gla2 , tBuAla5þ ) G1TE(Gla2 , tBuAla5þ )-sulfoxide linker, R-configured sulfoxide
15
G1TE(Gla2 , tBuAla5þ )-sulfoxide linker, S-configured sulfoxide
a
Variations on the distant sites tButyl-Ala5þ Gla2 tButyl-Ala5þ and sulfoxide linkage Gla2 and tButyl-Ala5þ Gla2 and tButyl-Ala5þ and sulfoxide linkage
IC50 (lM)b 25 5 14.3 5.3 0.77 0.23c 11.7 1.3 96.0 24.0 1.06 0.84 0.22 0.10 9.10 2.90
Competitive binding assays with SHC phosphopeptide, DDPSpYVNVQ, using surface plasmon resonance methodology on a BIAcore 2000 instrument as reported previously [11]. b Binding affinities of the relevant peptides are average values from at least three independent experiments. c This compound has previously been reported with an IC50 value of 0.64 lM [14] and in current study it was reevaluated with an IC50 value of 0.77 lM which was a mean value from seven experiments.
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G1TE(Gla2 )- and G1TE(Gla2 )-carrier conjugates effectively inhibit the association of oncogenic ErbB2 and Grb2 in whole cell assay as well as in cell homogenate assay Biacore data present in Tables 1 and 3 reflect the binding of isolated Grb2-SH2 domain fusion protein to a reference phosphopeptide SHC(pY317), i.e., DDPSpYVNVQ. This type of data is a useful indicator of the protein–inhibitor interaction. However, in physiological contexts, binding interactions occur between the full Grb2 protein (which consists of an SH2 domain and two SH3 domains) and phosphorylated proteins, which include SHC and erbB2 growth factor receptor cytoplasmic domains. Therefore, in order to evaluate the effectiveness of synthetic peptides to inhibit the interaction of native Grb2 protein with activated cellular growth factor receptor, cell homogenate assays were conducted. MDA-MB-453 cells were used, which are derived from a human breast cancer where there is amplification of the erbB2 gene [21]. Peptide 10 was selected for this purpose, which showed submicromolar binding affinity to the Grb2 target protein in our in vitro SPR-based competitive binding assays (Table 2). We incubated the active peptide 10 with cell lysates of the MDA-MB-453 cell lines which overexpress the oncogenic p185(erbB2) receptor protein. As shown in Fig. 2, Peptide 10 was effective in inhibiting protein/protein association in a dose dependent manner, at the half maximal concentration of 2.0 0.8 lM, whereas the prototype G1TE 1 served as a reference. Furthermore, to facilitate the cellular internalization of the synthetic inhibitor, a peptide carrier with the sequence of AAVALLPAVLLALLAP was conjugated to the C-terminal of peptide 10, affording the conjugated peptide G1TE(Gla2 )-carrier 16. In previous studies the location of the carrier sequence in the N-termini or C-termini has been shown not to influence the peptide import function [22]. Encouragingly, even in the presence of the hydrophobic 16-mer carrier, the inhibitory potency of peptide
Fig. 3. Effect of inhibitors on interaction of Grb2 with tyrosine phosphorylated P185erbB2 in human breast cancer cells, MDA-MB-453 as described in Materials and methods. MDA-MB-453 cells were plated in 100 mm dishes cultured in IME with 10% FBS medium overnight. Cells were treated with desired concentrations of inhibitors for 3 h, washed, and lysed and 500 mg of protein immunoprecipitate was treated with Grb2 antibody. Co-immunoprecipitated pTyr-containing P185erbB2 was detected using pTyr antibody (Py99) and immunoblotting. Western blotting with Grb2 Mab was done as a control.
16 shows promising results in the cell homogenate assay (16, IC50 ¼ 2.0 lM). On the basis of cell lysate assay, we further examined the inhibitory potency of active peptide 10 and its conjugate 16 in whole cells in which inhibition of binding of full length Grb2 to native erbB2 was measured following treatment of cells with synthetic inhibitors. In cellular systems ligands must cross cell membranes prior to interacting with targets. Shown in Fig. 3 are results from these assays. As expected, the hydrophobic carrier enhances cellular membrane penetration, thus the conjugate peptide 16 exhibited significant intracellular inhibition of Grb2-SH2 binding to erbB2 receptor in low micromolar concentration. Surprisingly, the inhibition of cognate Grb2-SH2 domain binding was also observed on peptide 10 with an approx. IC50 of 50 lM. As a comparison, the parent peptide G1TE 1 and its conjugate 17 with the same hydrophobic carrier did not display observable inhibitory activity in the whole cell assay.
Conclusions
Fig. 2. Inhibition of complexes formed between the Grb2 protein and GF-receptor protein P185erbB2 in MDA-MB-453 cell homogenates, on treatment with peptide 10, G1TE(Gla2 ) at a concentration range of 0.1–50 lM, and the conjugate peptide 16 at the same concentration range. The parent peptide G1TE and its conjugate peptide 17 were employed as a reference at a concentration range of 0.4–250 lM (see Materials and methods for details).
As a novel non-phosphorylated cyclic peptide ligand of Grb2-SH2, cyclo(CH2 CO-Glu2 -Leu- Tyr0 -Glu-AsnVal-Gly-Met-Tyr-Cys)-amide, G1TE attributed its effective binding to the highly favored interactions of its structural elements interacting with the binding pocket of the target protein with the aid of its constrained conformation. In this study, we demonstrated that the fifth residue C-terminal to the consensus sequence –YXN– of G1TE is also involved in important binding interactions with the Grb2-SH2 protein. A systematic modification on the side chain of amino acids at position Y + 5 served to fine tune its binding affinity in accordance with the change of the polarity or hydrophobicity. When increasing the polarity of the side chain by
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replacing methionine with S-methioninesulfoxide or ornithine or a-aminoadipic acid, the resulting modified polar side chain(s) exhibited reduced potency. However, the binding affinity increased accordingly with the hydrophobicity of the side chain of X5þ , whereas the bulky group at that position distorted the optimal orientation, thus leading to a loss of the intramolecular packing. When hydrophobic and rigid b-t-butylalanine was incorporated into position Y + 5, the binding potency was enhanced 2.0-fold relative to the parent peptide 1. By simultaneous optimization of the distant sites of G1TE, we obtained ultimately a potent non-phosphorylated Grb2-SH2 inhibitor, G1TE(Gla2 , tBuAla5þ )-sulfoxide (R-configured) with an IC50 of 220 nM. More significantly, G1TE(Gla2 ) and the conjugate of G1TE(Gla2 ) with a peptide carrier can effectively inhibit the intracellular association of erbB2 and the Grb2 protein in MDA-MB-453 breast cancer cell lines with an IC50 of 50 and 2 lM, respectively. These studies provide an improved understanding of the molecular binding mechanism of this novel agent and suggest a novel template for the development of chemotherapeutic agents targeting Grb2 protein for the treatment of cancer caused through erbB2 overexpression.
Acknowledgments We express our thanks for the financial support from Chinese Academy of Sciences (KSCX1-SW-11) and the Department of Personnel of China (Y.-Q. Long).
References [1] B. Schaffhausen, SH2 domain-structure and function, BBA-Rev. Cancer 1242 (1995) 61–75. [2] (a) C.A. Koch, D. Anderson, M.F. Moran, C. Ellis, T. Pawson, SH2 and SH3 domains—elements that control interactions of cytoplasmic signaling proteins, Science 252 (1991) 668–674; (b) T. Pawson, J. Schlessinger, SH2 and SH3 domains, Curr. Biol. 3 (1993) 434–442; (c) D. Cowburn, SRC homology adapter proteins—more than the sum of the parts, Structure 3 (1995) 429–430; (d) P. Chardin, D. Cussac, S. Maignan, A. Ducruix, The Grb2 adapter, FEBS Lett. 369 (1995) 47–71. [3] A.M. Tari, M.C. Hung, K. Li, G. Lopez-Berestein, Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells, Oncogene 18 (1999) 1325–1332. [4] H. Fretz, P. Furet, C. Garcia-Echeverria, J. Rahuel, J. Schoepfer, Structure-based design of compounds inhibiting Grb2-SH2 mediated protein–protein interactions in signal transduction pathways, Curr. Pharm. Design 6 (2000) 1777–1796. [5] J. Schoepfer, H. Fretz, B. Gay, P. Furet, C. Garcia-Echeverria, N. End, G. Caravatti, Highly potent inhibitors of the Grb2-SH2 domain, Bioorg. Med. Chem. Lett. 9 (1999) 221–226. [6] P. Furet, B. Gay, G. Caravatti, C. Garcia-Echeverria, J. Rahuel, J. Schoepfer, H. Fretz, Structure-based design and synthesis of high affinity tripeptide ligands of the Grb2-SH2 domain, J. Med. Chem. 41 (1998) 3442–3449.
[7] Z.-J. Yao, C.R. King, J. Kelley, G.W. Milne, J.H. Voigt, T.R. Burke Jr., Potent inhibition of Grb2 SH2 domain binding by nonphosphate-containing ligands, J. Med. Chem. 42 (1999) 25–35. [8] Y. Gao, J. Luo, Z.-J. Yao, R. Guo, H. Zou, J. Kelley, J.H. Voigt, D. Yang, T.R. Burke Jr., Inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. 2. 4-(2-malonyl)phenylalanine as a potent phosphotyrosyl mimetic, J. Med. Chem. 43 (2000) 911–920. [9] W.C. Shakespeare, SH2 domain inhibition: a problem solved?, Curr. Opin. Chem. Biol. 5 (2001) 409–415. [10] H.W.H.G. Kessels, A.C. Ward, T.N.M. Schumacher, Specificity and affinity motifs for Grb2 SH2-ligand interactions, Proc. Natl. Acad. Sci. USA 99 (2002) 8524–8529. [11] L. Oligino, F.-D.T. Lung, L. Sastry, J. Bigelow, T. Cao, M. Curran, T.R. Burke Jr., S. Wang, D. Krag, P.P. Roller, C.R. King, Nonphosphorylated peptide ligands for the Grb2 Src Homology 2 domain, J. Biol. Chem. 272 (1997) 29046. [12] Ligand residues are numbered relative to the position of phosphotyrosine, or putative phosphotyrosine, which is denoted 0. Positive numbers are used for amino acids C-terminal to phosphotyrosine, and negative numbers for amino acids Nterminal to phosphotyrosine. In our case, the consensus sequence for binding to Grb2-SH2 is YXN, in which Y is denoted 0. [13] F.-D.T. Lung, Y.-Q. Long, C.R. King, X.-W. Wu, S. Wang, P.P. Roller, Functional preference of the constituent amino acid residues in a phage-library based non-phosphorylated inhibitor of Grb2-SH2 domain, J. Pept. Res. 57 (2001) 447–454. [14] Y.-Q. Long, J.H. Voigt, F.-D.T. Lung, C.R. King, P.P. Roller, Significant compensatory role of position Y-2 conferring high affinity to non-phosphorylated inhibitors of Grb2-SH2 domain, Bioorg. Med. Chem. Lett. 9 (1999) 2267–2272. [15] Y.-Q. Long, Z.-J. Yao, J.H. Voigt, F.-D.T. Lung, J.H. Luo, T.R. Burke Jr., C.R. King, D.-J. Yang, P.P. Roller, Structural requirements for Tyr in the consensus sequence Y-E-N of a novel non-phosphorylated inhibitor to the Grb2-SH2 domain, Biochem. Biophys. Res. Commun. 264 (1999) 902–908. [16] Y.-Q. Long, F.-D.T. Lung, P.P. Roller, Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain, Bioorg. Med. Chem. 11 (2003) 3929–3936. [17] F.-D.T. Lung, C.R. King, P.P. Roller, Development of nonphosphorylated cyclic thioether peptide binding to the Grb2-SH2 domain, Lett. Pept. Sci. 6 (1999) 45–49. [18] P. Li, M. Zhang, Y.-Q. Long, M.L. Peach, H. Liu, D. Yang, M. Nicklaus, P.P. Roller, Potent Grb2-SH2 domain antagonists not relying on phosphotyrosine mimics, Bioorg. Med. Chem. Lett. 13 (2003) 2173–2177. [19] M.H. Kraus, N.C. Popescu, S.C. Amsbough, C.R. King, Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms, EMBO J. 6 (1987) 605–610. [20] Rekker’s f values or hydrophobic fragmental constants are calculated from octanol–water partition coefficients. The total hydrophobicity of an organic molecule can be roughly estimated from a summation of the f values of all the “fragments” of that molecule. For illustration, the parenthesized f values are given by Rekker for the following fragments: CH3 (0.701), CH2 (0.519), C (0.155), H (0.182), C6 H5 (1.840), (al)S ()0.51); (a) See: R.F. Rekker, The Hydrophobic Fragmental Constants, vol. 1, Elsevier, Amsterdam, 1997; (b) R.F. Rekker, H.M. deKort, Eur. J. Med. Chem. 14 (1979) 479–488. [21] L. Sastry, T. Cao, R. King, Multiple Grb2-protein complexes in human cancer cells, Int. J. Cancer 70 (1997) 208–213. [22] C. Du, S. Yao, M. Rojas, Y.-Z. Lin, Conformational and topological requirements of cell-permeable peptide function, J. Pept. Res. 51 (1998) 235–243.
Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domainq Ya-Qiu Long,a,* Ribo Guo,b Juliet H. Luo,b Dajun Yang,b and Peter P. Rollerc,* a
c
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China b School of Medicine, University of Michigan, Ann Arbor, MI 48109, USA Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, FCRDC, Frederick, MD 21702, USA Received 4 July 2003
Abstract Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu2 sidechain for the absence of the phosphate functionality on Tyr0 in the peptide ligand, cyclo(CH2 CO-Glu2 -Leu-Tyr0 -Glu-Asn-Val-Gly-Met5þ -Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr + 5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with b-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC50 of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla2 ) in which the original residue of Glu2 was substituted by c-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 lM level. More significantly, both G1TE(Gla2 ) alone and the conjugate of G1TE(Gla2 ) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC50 of 50 and 2 lM, respectively. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Non-phosphorylated Grb2-SH2 antagonist; Thioether-bridged cyclic peptide; Hydrophobic residue; Sulfoxide linkage; Conformational constraint; Phage library; Global optimization; Chemotherapeutic agents; Intracellular signaling; ErbB2-related cancer
SH2 (src homology 2) domains are protein modules of approximately 100 amino acids found in more than 50 critical intracellular signaling proteins [1]. SH2 domains directly bind to phosphorylated growth factor receptors or other cytoplasmic proteins phosphorylated in response q
A preliminary account of this information was presented in: Y.-Q. Long, P.P. Roller, Potentiating effect of distant sites in cyclic peptide antagonists of the Grb2-SH2 domain, in: E. Benedetti, C. Pedone (Eds.), Peptide 2002 (Proceedings of the 27th European Peptide Symposium), Edizioni Ziino, Napoli, Italy, 2002, pp. 982–983. * Corresponding authors. Fax: +86-21-5080-7088 (Y.-Q. Long), Fax: 1-301-846-6033 (P.P. Roller). E-mail addresses: [email protected] (Y.-Q. Long), [email protected] (P.P. Roller). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.022
to extracellular signals, and thereby serve as important mediators of tyrosine kinase-dependent signal transduction [2]. Among these SH2 domains, the Grb2-SH2 domain directly mediates the activation of mitogenic Ras pathway. An aberrant Grb2-SH2 dependent Ras activation pathway has been shown to be essential for cellular transformation in a subset of human tumors [3]. Compounds which selectively antagonize the SH2 domain of Grb2 should interrupt these signaling pathways and thus are attractive targets for drug therapy in oncology [4]. Over the past few years, considerable progress has been made in designing ligands with high affinity for the Grb2SH2 domain [5–8]. However, the phosphotyrosine residue still serves as a critical recognition determinant for
Y.-Q. Long et al. / Biochemical and Biophysical Research Communications 310 (2003) 334–340
Fig. 1. The structures of the substitutes in position Y + 5 and combined modifications of the distant sites in G1TE.
effective binding to Grb2-SH2, which would confer poor cellular activity and selectivity [9,10]. Our approach is based on a phage library derived non-phosporylated disulfide-bridged cyclic decapeptide and its redox-stable thioether cyclized analog, cyclo(CH2 CO-Glu2 -Leu-Tyr0 -Glu-Asn-Val-Gly-Met5þ Tyr-Cys)-amide (termed G1TE) [11,12], which could inhibit the EGFR/Grb2-SH2 domain association in a phosphotyrosine independent manner. The non-phosphorylated cyclopeptide specifically binds to the Grb2SH2 domain, but does not bind to the homologous Src-SH2 domain [11]. The absence of a phosphate moiety in this phage peptide contributes to its remarkably high specificity and enhanced bioavailability. Unlike the phosphopeptide ligands of Grb2-SH2 in which phosphotyrosine is a major contributor to the binding, this novel non-phosphorylated cyclic peptide antagonist requires essentially all amino acids, except Gly4þ , as well as the constrained conformation in retaining high affinity binding with the Grb-SH2 domain [11,13–16]. This multipoint binding pattern of G1TE encouraged us to explore the potentiating effect of distant sites from the consensus sequence of –YXN– on the backbone to further improve the activity. In the current study, we report the functional importance of the fifth residue Met5þ , C-terminal to the consensus sequence of –Y0 -XN2þ – in G1TE, and the simultaneous optimization of the distant sites such as the Y ) 2 position, the Y + 5 position, and the cyclization linkage in G1TE (see Fig. 1) leading to potent non-phosphorylated Grb2-SH2 antagonist not relying on phosphotyrosine mimics.
Materials and methods Inhibitors The thioether-bridged cyclic peptides were synthesized in a similar procedure described previously [17]. Briefly, the linear peptide was
335
synthesized on solid phase on a PAL amide resin with an ABI 433A peptide synthesizer utilizing FastMoc chemistry. After removing the Na -Fmoc group with 20% piperidine/DMF, the resin-bound protected peptide was N-terminally chloroacetylated by 3 equivalents of chloroacetic anhydride in DMF at RT overnight. Then the peptide was cleaved from the resin by using TFA containing 2.5% each (v/v) of triethylsilane and deionized water (2 h). For isolation of the product, two-thirds of the cleavage reagent mixture was evaporated under N2 stream and the residue was triturated and washed with ice-cold ether. The precipitated crude peptide was dissolved in 50 mL of water and added dropwise into 100 mL of aqueous solution, which was adjusted to pH 8–9 with 0.2 M aqueous solution of NH4 OH and NH4 OAc (1:1, mol/mol), repeatedly. After 6 h stirring at RT, the cyclization reaction was quenched by addition of 30% acetic acid until pH 4–5 to give the thioether-bridged analogs of G1TE. The oxidation of the thioether linkage into sulfoxide was accomplished using 5% H2 O2 aqueous solution. The resulting sulfoxide diastereoisomers were easily separated by RP-HPLC. The absolute configuration of the sulfoxide analogs was determined by CD spectra using documented method [18]. All final products were purified by RP-HPLC and the identity was assessed by amino acid and mass spectral analyses. HPLC conditions: Vydac C4 column (20 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated below; flow rate, 10 mL/min; and UV detector, 225 nm. FAB-MS (unit resolution, glycerol matrix) was performed on a VG Analytical 7070E-HF mass spectrometer. Amino acid analysis (6 N HCl, 110 °C, 24 h) was carried out at the Protein and Carbohydrate Structure Facility (University of Michigan, Ann Arbor, MI, USA). The PAL amide resin and Fmoc derivatives of standard amino acids were obtained from Perkin–Elmer/Applied Biosystems Division. L -Citrulline (Cit), L -a-aminoadipic acid (Adi), c-carboxy-L -glutamic acid (Gla), L -norleucine (Nle), L -ornithine (Orn), L -homophenylalanine (hPhe), L -di-n-butyl glycine (Dbg), and L -tert-butyl alanine (tBuAla) were purchased from BACHEM (Torrance, CA, USA) in Fmoc protection form. Physicochemical data for selected cyclic peptides: Peptide 11. RP-HPLC gradient 25–40% B over 35 min; Rt ¼ 18:5 min; FAB-MS (M + H)þ 1271.3 (calc. 1271.55). Peptide 12. RP-HPLC gradient 25–40% B over 35 min; Rt ¼ 21:3 min; FAB-MS (M + H)þ 1271.5 (calc. 1271.55). Peptide 13. RP-HPLC gradient 20–70% B over 27 min; Rt ¼ 16:3 min; FAB-MS (M + H)þ 1299.6 (calc. 1299.55). AAA: Asp 0.92 (1), Glu 1.21 (1), Gly 1.01 (1), Tyr 1.94 (2), Val 0.92 (1), Leu 0.99 (1). Peptide 14. RP-HPLC gradient 20–40% B over 35 min; Rt ¼ 17:7 min; FAB-MS (M + H)þ 1315.6 (calc. 1315.54). Peptide 15. RP-HPLC gradient 20–40% B over 35 min; Rt ¼ 20:0 min; FAB-MS (M + H)þ 1315.5 (calc. 1315.54). Peptide 16. RP-HPLC gradient 40–90% B over 27 min; Rt ¼ 20:6 min; FAB-MS (M + Na)þ 2823.4 (calc. 2824.3). Peptide 17. RP-HPLC gradient 50–90% B over 24 min; Rt ¼ 18:7 min; FAB-MS (M + Na)þ 2780.0 (calc. 2781.3). Binding affinity measurement of peptides to Grb2-SH2 domain using surface plasmon resonance The competitive binding affinity of ligands for the Grb2-SH2 protein was assessed using Biacore surface plasmon resonance (SPR) methods on a BIAcore 2000 instrument (Pharmacia Biosensor, Uppsala, Sweden). IC50 values were determined by mixing various concentrations of inhibitors with the recombinant GST-Grb2-SH2 domain protein and measuring the amount of binding at equilibrium to the immobilized SHC phosphopeptide(pTyr317 ), i.e., biotinyl-DDPS-pYVNVQ, in a manner described previously [11]. Briefly, the biotinylated phosphopeptide was attached to a streptavidin coated SA5 Biosensor chip and the binding assays were conducted in, pH 7.4, PBS buffer containing 0.01% P-20 surfactant (Pharmacia Biosensor).
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Biological cells and cell culture Cell lines were obtained from the American Type Culture Collection (Rockville, MD) and Lombardi Cancer Center, Georgetown University Medical Center. Cells were routinely maintained in improved minimal essential medium (IMEM, Biofluids, Rockville, MD) with 10% fetal bovine serum. Cultures were maintained in a humidified incubator at 37 °C and 5% CO2 . (A) Inhibition of Grb2-SH2 domain binding in cell homogenates. Cell lysates were prepared from serum-treated erbB2 overexpressing breast cancer cells (MDA-MB-453), as described previously [11]. Cell lysates were incubated with G1TE(Gla2 ), G1TE(Gla2 )-carrier, or control peptide G1TE, G1TE-carrier at concentrations of 0.1, 0.4, 2, 10, and 50 lM for 30 min, and then 500 lg of protein was immunoprecipitated with anti-Grb2 antibody (Santa Cruz Biotech., Santa Cruz, CA, USA) and collected with protein A–Sepharose. Immunoprecipitated proteins were separated by SDS–PAGE on 8–16% gradient gels (Novex, San Diego, CA, USA). pTyr-containing proteins were detected by Western blotting using anti-phosphotyrosine antibodies (Upstate Biotechnology, Lake Placid, NY, USA) and visualized with ECL (Amersham, Arlington Heights, IL). Previous experiments have shown that a major tyrosine phosphorylated protein in these cells is the p185erbB2 , which is overexpressed as a consequence of gene amplification [19]. Western blotting with Grb2 MAb was done as a control. (B) Inhibition of Grb2-SH2 domain binding in whole cells [8]. ErbB2 overexpressing breast cancer cells, MDA-MB-453, were treated with desired concentrations of inhibitors for 3 h in serum-free IMEM (Gibco). Cells were washed twice with PBS to remove inhibitor and then cell lysates were prepared using 1% Triton X-100 in PBS containing 0.2 mM NaVO4 . Grb2 and associated Grb2-binding proteins were immunoprecipitated from each lysate (500 lg) with anti-Grb2 antibodies and collected using protein A–Sepharose. The following work-up is the same as that in the cell homogenate assay.
Results and discussion The phage library derived nonphosphorylated cyclic peptides, typified by G1TE, 1, comprise a novel family of agents binding to the Grb2-SH2 domain in a phosphotyrosine independent manner [11–17]. Earlier Ala mutation studies demonstrated that essentially all amino acids as well as special conformation are required for sustenance of the biological activity. The multipoint-
binding pattern of G1TE provides us with a unique opportunity and potential to further improve their activity by systematic structural optimization. Our previous structure/activity studies amply demonstrated that the carboxyl side chain of Glu in position Y ) 2 of G1TE partially compensates for the absence of the phosphate group on Tyr0 [14,15]. Since positions Y ) 2 and Y0 share an overlapping function in the Grb2-SH2 binding, the optimal combination of amino acids in the two positions produces potent peptide antagonists of Grb2-SH2, which are exemplified by G1TE(Gla2 ) (IC50 ¼ 0.64 lM) [14], G1TE(Ala2 , pTyr0 ) (IC50 ¼ 0.023 lM) [14], and G1TE(Adi2 , cmPhe0 ) (IC50 ¼ 0.70 lM) [15]. Generally speaking, when Tyr is in position Y0 which provides phenyl group but lacks the negative charge, the acidic side chain in the compensatory position of Y ) 2 can provide a significant enhancement in Grb2SH2 domain binding potency. When pTyr is present in position Y0 which carries bulky and highly charged phosphate, the acidic side chain in position Y ) 2 is unfavored and Ala substitution showed improved activity. When the pTyr mimic cmPhe is built into the position Y0 which carries less bulky but still doubly charged carboxyl groups, the acidic side chain in position Y ) 2 exhibited a slight potentiating effect on its binding. Our recent investigation on the conformational requirement of G1TE upon binding to Grb2-SH2 domain has indicated that the polarity and configuration of the cyclization linkage were important in assuming the active conformation [16]. When the flexible thioether linkage in G1TE was oxidized into the relatively rigid sulfoxide, the R-configured sulfoxide peptide isomers provide superior Grb2-SH2 domain antagonists. The stereochemistry of the sulfoxide diastereoisomers was identified by CD spectra using documented method [18]. In our continuing efforts to explore the functional importance of the distant sites on G1TE thus deriving potent non-phosphorylated Grb2-SH2 antagonists, we conducted a systematic modification in the fifth residue
Table 1 Grb2-SH2 domain inhibitory activity of the Peptides 1–9a Compound
Peptide analogs
X5þ
IC50 (lM)b
1 2 3 4 5 6 7 8 9
G1TE, control [11] G1TE(Met5þ deleted) G1TE(Met(O)5þ ) G1TE(Orn5þ ) G1TE(Adi5þ ) G1TE(Nle5þ ) [16] G1TE(hPhe5þ ) G1TE(Dbg5þ ) G1TE(tBuAla5þ )
Met Deleted Met(O) Orn a-Aminoadipic acid Nle HomoPhe Di-n-butylGly b-t-ButylAla
25 5c 315 185 165 114 236 164 120 20 26.5 3.5 16 6 107 43 14.3 0.5
a Competitive binding assays with SHC phosphopeptide, DDPSpYVNVQ, using surface plasmon resonance on a BIAcore 2000 instrument as reported earlier [11]. b Values are means of at least two independent experiments and are expressed as the concentration at which half-maximal competition was observed (IC50 ). IC50 of reference SHC(pY317) peptide: 1.1 0.3 lM. Standard deviation is given in parentheses. c This value has previously been reported [16] and is used here as a reference.
FAB-MSd aP f values were calculated from Rekker fragmental constants listed in [20]. b HPLC conditions: Vydac C18 column (10 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated; flow rate, 2.5 mL/min; and UV detector, 225 nm. c HPLC conditions: Vydac C4 column (20 250 mm); solvent gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile in water with gradient indicated; flow rate, 10.0 mL/min; and UV detector, 225 nm. d FAB-MS (unit resolution, glycerol matrix) was performed on a VG Analytical 7070E-HF mass spectrometer.
Rt ¼ 16:5 min (gradient 20–70% B over 27 min)c (M + H)þ 1255.3 (calc. 1255.56) Rt ¼ 18:6 min (gradient 20–70% B over 27 min)c (M + H)þ 1297.5 (calc. 1297.6) Rt ¼ 16:6 min (gradient 20–70% B over 27 min)c (M + H)þ 1290.0 (calc. 1289.5) Rt ¼ 12:3 min (gradient 20–65% B over 25 min)b (M + H)þ 1242.1 (calc. 1242.5) Rt ¼ 12:8 min (gradient 20–80% B over 30 min)b (M + H)þ 1258.7 (calc. 1259.5)
2.878 2.258 1.229
7 6 [16]
Based on the preliminary results, the following effort is focused on the hydrophobicity modification on the side chain of residue X5þ in G1TE. First of all, the sulfur atom of Met5þ was replaced with a less polar CH2 group. Encouragingly, the resulting peptide 6 turned out to have comparable activity (6, IC50 ¼ 26.5 lM). Thus further modifications included increasing the hydrophobicity of the side chain by incorporating aromatic homophenylalanine (Peptide 7) or bulky di-n-butylglycine (Peptide 8) or branched b-t-butylanaline (Peptide 9) into position Y + 5 of G1TE. Results indicate that aromatic moiety containing homophenylalanine shows an improved binding affinity (7, IC50 ¼ 16 lM) with improved hydrophobicity. But when Met5þ was replaced
1 [11]
The hydrophobicity of the side chain in position Y + 5 strengthens the intramolecular packing for a favored conformation of the cyclic peptide binding to Grb2-SH2
Peptide P a f
Judiciously altering the original residue of Met5þ with methioninesulfoxide whose side chain is more polar (Peptide 3), or ornithine whose side chain carries a basic end group (Peptide 4), or aminoadipic acid whose side chain bears an acidic end group (Peptide 5), constitutes our effort to examine the effect of polarity on the interaction of X5þ side chain in G1TE with Grb2-SH2 binding pocket. As shown in Table 1, with the retention of the chain length, the increase in polarity of the side chain results in the loss of binding affinity. Either neutral (3, IC50 ¼ 165 lM) or ionic polarity (4, IC50 ¼ 236 lM; 5, IC50 ¼ 120 lM) in position Y + 5 disfavored the interactions of G1TE analogs with the Grb2-SH2 protein. The basic or acidic head of the side chain particularly damaged the interactions.
Table 2 P f valuesa of the side chain in position Y + 5 in peptides 1, 6–9 and their physicochemical data
Polar amino acids are not favored for the side chain interactions in position Y + 5
Physico-chemical data RP-HPLC
4.516
8
9
X5þ . In the present paper, a series of G1TE analogs with various substitutions in position Y + 5 were synthesized and the Grb2-SH2 domain inhibitory activity was assessed using Biacore SPR methodology Table 1. Our earlier Ala scan studies indicated that replacement of Met5þ with Ala remarkably reduced the inhibitory activity by 60-fold [13]. Accordingly, the Met5þ deleted peptide 2 showed a dramatic decrease of the binding affinity (2, IC50 ¼ 315 lM). The initial results implicated that the amino acid in position Y + 5 plays an important role for the effective binding of G1TE to Grb2 protein. It might function by directly contacting with the Grb2 binding pocket or by stabilizing the favored conformation via the ligand side-chain interactions. Therefore, variations in the side chain of residue X5þ with respect to the polarity and the hydrophobicity were examined to determine the favorable interaction for improved binding.
2.777
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by the more hydrophobic di-n-butylglycine, a reduced binding affinity was observed (8, IC50 ¼ 107 lM), suggesting that the bulky and flexible groups interfere with the optimal orientation of the side chain in position Y + 5 for effective binding. Not surprisingly, the relatively rigid and still very hydrophobic b-t-butylalanine gained a remarkable increase in the binding affinity (9, IC50 ¼ 14.3 lM), improved by a factor of 8.4-fold relative to its polar counterpart (5, IC50 ¼ 120 lM). Even compared to its parent compound 1, Peptide 9 showed a 2.0-fold enhancement in the binding potency just by slight alteration of the side chain in position Y + 5. Comparison of the hydrophobicity of the side chains in position Y + 5 on selected peptides (Table 2), which can be roughly evaluated by Rekker’s f value [20], indicated that shape as well as hydrophobicity is responsible for the potentiating effect. Our molecular modeling has confirmed that the Met5þ residue of G1TE formed a hydrophobic packing with Leu1 , Cys7þ , and the N-acetyl group within G1TE [18]. This intramolecular packing stabilizes the favorable backbone conformation of the cyclic peptide for other residues effectively binding to the Grb2-SH2 domain. Thus a more hydrophobic and less flexible amino acid in position Y + 5 results in favorable and well-defined van der Waals interactions with Leu1 and Cys7þ , correspondingly enhancing the effective interaction of G1TE analogs with the Grb2SH2 protein. Combined potentiating effect of the distant sites confers high affinity to non-phosphorylated and non-pTyr-mimetic containing Grb2-SH2 antagonists Previous study has disclosed that the overlapping function of amino acids Tyr0 and Glu2 in G1TE secured effective binding to Grb2-SH2 domain, even without the presence of a phosphotyrosyl group [14,15]. The systematic modification in the fifth residue X5þ demonstrated that a hydrophobic side chain is favorable
for the intramolecular packing to stabilize the favored backbone conformation. Besides, the polarity of the cyclization linkage was implicated as crucial in maintaining such an active conformation for effective binding to Grb2-SH2 [16]. We are intrigued to examine the synergistic effect of simultaneous optimization of the distant sites on G1TE. A series of relevant thioether cyclized peptides were synthesized for this purpose (Table 3). Selected analogs were also oxidized with aqueous H2 O2 to provide pairs of sulfoxide-cyclized analogs that could be separated by HPLC. The stereochemistry of the sulfoxide diastereoisomers was identified by CD spectra [18]. The faster eluting sulfoxide diastereoisomer was found to be R-configured, and the slower eluting sulfoxide was the S-configured analog. Consistently, the R-configured sulfoxide analog was remarkably more potent than its cognate pair (e.g., 11 versus 12, 14 versus 15), whereas the S-configured sulfoxide is even less active than the thioether analog (e.g., 12 versus 9, 15 versus 13). As previously indicated [13], substitution of Glu2 with Gla2 in G1TE significantly favored the interaction with Grb2-SH2 (10, IC50 ¼ 0.77 lM). Current results show that modification of distant sites in the non-phosphorylated cyclic antagonist, i.e., residue tBuAla5þ and the sulfoxide linkage, displayed a slight enhancing effect (9, IC50 ¼ 14.3 lM; 11, IC50 ¼ 11.7 lM). More significantly, simultaneous structural modification of the three sites within G1TE provided peptide 14, with a 113-fold enhancement in Grb2-SH2 domain antagonist potency (14, IC50 ¼ 0.22 lM) relative to the parent compound, G1TE (1, IC50 ¼ 25.0 lM). Thus, by judiciously combining the optimal modulations of the functional groups on the distant sites, we have obtained a highly potent non-phosphorylated Grb2-SH2 inhibitor that is also devoid of pTyr mimics. This new inhibitor constitutes a novel experimental agent for controlling Grb2-SH2 domain mediated cellular signaling processes.
Table 3 Grb2-SH2 domain inhibitory activity of G1TE analogs with global optimization on distant sitesa Compd.
Peptide analog
1 9 10 11 12 13 14
G1TE [11] G1TE(tBuAla5þ ) G1TE(Gla2 ) [14] G1TE(tBuAla5þ )-sulfoxide linker, R-configured sulfoxide G1TE(tBuAla5þ )-sulfoxide linker, S-configured sulfoxide G1TE(Gla2 , tBuAla5þ ) G1TE(Gla2 , tBuAla5þ )-sulfoxide linker, R-configured sulfoxide
15
G1TE(Gla2 , tBuAla5þ )-sulfoxide linker, S-configured sulfoxide
a
Variations on the distant sites tButyl-Ala5þ Gla2 tButyl-Ala5þ and sulfoxide linkage Gla2 and tButyl-Ala5þ Gla2 and tButyl-Ala5þ and sulfoxide linkage
IC50 (lM)b 25 5 14.3 5.3 0.77 0.23c 11.7 1.3 96.0 24.0 1.06 0.84 0.22 0.10 9.10 2.90
Competitive binding assays with SHC phosphopeptide, DDPSpYVNVQ, using surface plasmon resonance methodology on a BIAcore 2000 instrument as reported previously [11]. b Binding affinities of the relevant peptides are average values from at least three independent experiments. c This compound has previously been reported with an IC50 value of 0.64 lM [14] and in current study it was reevaluated with an IC50 value of 0.77 lM which was a mean value from seven experiments.
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G1TE(Gla2 )- and G1TE(Gla2 )-carrier conjugates effectively inhibit the association of oncogenic ErbB2 and Grb2 in whole cell assay as well as in cell homogenate assay Biacore data present in Tables 1 and 3 reflect the binding of isolated Grb2-SH2 domain fusion protein to a reference phosphopeptide SHC(pY317), i.e., DDPSpYVNVQ. This type of data is a useful indicator of the protein–inhibitor interaction. However, in physiological contexts, binding interactions occur between the full Grb2 protein (which consists of an SH2 domain and two SH3 domains) and phosphorylated proteins, which include SHC and erbB2 growth factor receptor cytoplasmic domains. Therefore, in order to evaluate the effectiveness of synthetic peptides to inhibit the interaction of native Grb2 protein with activated cellular growth factor receptor, cell homogenate assays were conducted. MDA-MB-453 cells were used, which are derived from a human breast cancer where there is amplification of the erbB2 gene [21]. Peptide 10 was selected for this purpose, which showed submicromolar binding affinity to the Grb2 target protein in our in vitro SPR-based competitive binding assays (Table 2). We incubated the active peptide 10 with cell lysates of the MDA-MB-453 cell lines which overexpress the oncogenic p185(erbB2) receptor protein. As shown in Fig. 2, Peptide 10 was effective in inhibiting protein/protein association in a dose dependent manner, at the half maximal concentration of 2.0 0.8 lM, whereas the prototype G1TE 1 served as a reference. Furthermore, to facilitate the cellular internalization of the synthetic inhibitor, a peptide carrier with the sequence of AAVALLPAVLLALLAP was conjugated to the C-terminal of peptide 10, affording the conjugated peptide G1TE(Gla2 )-carrier 16. In previous studies the location of the carrier sequence in the N-termini or C-termini has been shown not to influence the peptide import function [22]. Encouragingly, even in the presence of the hydrophobic 16-mer carrier, the inhibitory potency of peptide
Fig. 3. Effect of inhibitors on interaction of Grb2 with tyrosine phosphorylated P185erbB2 in human breast cancer cells, MDA-MB-453 as described in Materials and methods. MDA-MB-453 cells were plated in 100 mm dishes cultured in IME with 10% FBS medium overnight. Cells were treated with desired concentrations of inhibitors for 3 h, washed, and lysed and 500 mg of protein immunoprecipitate was treated with Grb2 antibody. Co-immunoprecipitated pTyr-containing P185erbB2 was detected using pTyr antibody (Py99) and immunoblotting. Western blotting with Grb2 Mab was done as a control.
16 shows promising results in the cell homogenate assay (16, IC50 ¼ 2.0 lM). On the basis of cell lysate assay, we further examined the inhibitory potency of active peptide 10 and its conjugate 16 in whole cells in which inhibition of binding of full length Grb2 to native erbB2 was measured following treatment of cells with synthetic inhibitors. In cellular systems ligands must cross cell membranes prior to interacting with targets. Shown in Fig. 3 are results from these assays. As expected, the hydrophobic carrier enhances cellular membrane penetration, thus the conjugate peptide 16 exhibited significant intracellular inhibition of Grb2-SH2 binding to erbB2 receptor in low micromolar concentration. Surprisingly, the inhibition of cognate Grb2-SH2 domain binding was also observed on peptide 10 with an approx. IC50 of 50 lM. As a comparison, the parent peptide G1TE 1 and its conjugate 17 with the same hydrophobic carrier did not display observable inhibitory activity in the whole cell assay.
Conclusions
Fig. 2. Inhibition of complexes formed between the Grb2 protein and GF-receptor protein P185erbB2 in MDA-MB-453 cell homogenates, on treatment with peptide 10, G1TE(Gla2 ) at a concentration range of 0.1–50 lM, and the conjugate peptide 16 at the same concentration range. The parent peptide G1TE and its conjugate peptide 17 were employed as a reference at a concentration range of 0.4–250 lM (see Materials and methods for details).
As a novel non-phosphorylated cyclic peptide ligand of Grb2-SH2, cyclo(CH2 CO-Glu2 -Leu- Tyr0 -Glu-AsnVal-Gly-Met-Tyr-Cys)-amide, G1TE attributed its effective binding to the highly favored interactions of its structural elements interacting with the binding pocket of the target protein with the aid of its constrained conformation. In this study, we demonstrated that the fifth residue C-terminal to the consensus sequence –YXN– of G1TE is also involved in important binding interactions with the Grb2-SH2 protein. A systematic modification on the side chain of amino acids at position Y + 5 served to fine tune its binding affinity in accordance with the change of the polarity or hydrophobicity. When increasing the polarity of the side chain by
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replacing methionine with S-methioninesulfoxide or ornithine or a-aminoadipic acid, the resulting modified polar side chain(s) exhibited reduced potency. However, the binding affinity increased accordingly with the hydrophobicity of the side chain of X5þ , whereas the bulky group at that position distorted the optimal orientation, thus leading to a loss of the intramolecular packing. When hydrophobic and rigid b-t-butylalanine was incorporated into position Y + 5, the binding potency was enhanced 2.0-fold relative to the parent peptide 1. By simultaneous optimization of the distant sites of G1TE, we obtained ultimately a potent non-phosphorylated Grb2-SH2 inhibitor, G1TE(Gla2 , tBuAla5þ )-sulfoxide (R-configured) with an IC50 of 220 nM. More significantly, G1TE(Gla2 ) and the conjugate of G1TE(Gla2 ) with a peptide carrier can effectively inhibit the intracellular association of erbB2 and the Grb2 protein in MDA-MB-453 breast cancer cell lines with an IC50 of 50 and 2 lM, respectively. These studies provide an improved understanding of the molecular binding mechanism of this novel agent and suggest a novel template for the development of chemotherapeutic agents targeting Grb2 protein for the treatment of cancer caused through erbB2 overexpression.
Acknowledgments We express our thanks for the financial support from Chinese Academy of Sciences (KSCX1-SW-11) and the Department of Personnel of China (Y.-Q. Long).
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