Article pubs.acs.org/jmc
Development and Evaluation of Novel Phosphotyrosine Mimetic Inhibitors Targeting the Src Homology 2 Domain of Signaling Lymphocytic Activation Molecule (SLAM) Associated Protein Chi-Yuan Chu,†,∥ Chun-Ping Chang,‡,§,∥ Yun-Ting Chou,† Handoko,† Yi-Ling Hu,† Lee-Chiang Lo,*,† and Jing-Jer Lin*,‡,§ †
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan § Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan ‡
S Supporting Information *
ABSTRACT: Specific interactions between Src homology 2 (SH2) domain-containing proteins and the phosphotyrosine-containing counterparts play significant role in cellular protein tyrosine kinase (PTK) signaling pathways. The SH2 domain inhibitors could potentially serve as drug candidates in treating human diseases. Here we have incorporated a novel phosphotyrosine mimetic, which is an unusual amino acid carrying a cyclosaligenyl (cycloSal) phosphodiester moiety, into dipeptides to investigate the inhibitory effect on SH2 domain-containing proteins. A plate-based assay was also established to screen for inhibitors that disrupt the interaction between a phosphopeptide of SLAM (signaling lymphocytic activation molecule) and its interacting protein SAP (SLAM-associated protein). We identified a number of inhibitors with IC50 values in the range of 17−35 μM, implying that the cycloSal phosphodiester-carrying amino acid could mimic the phosphotyrosyl residue. Our results also raise the possibility of integrating the newly developed phosphotyrosine mimetic moiety into inhibitors designed for other SH2 domain-containing proteins.
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INTRODUCTION Selective protein−protein interactions are important in organizing the regulatory processes of eukaryotic cells. The Src homology 2 (SH2) domain is a highly conserved protein− protein interaction domain that specifically recognizes short tyrosine-phosphorylated peptide motifs.1 SH2 domains are embedded in proteins that couple to intracellular protein tyrosine kinase (PTK) signaling pathways in order to coordinate functionally diverse proteins including enzymes, adaptors, regulators, and transcription factors.2 Modulation of protein−protein interactions through specific association of SH2 domains and tyrosine-phosphorylated peptides thus leads to efficient propagation of PTK signals. SH2 domain-containing proteins therefore play important regulatory roles in PKT signaling pathways and are related to many diseases.3,4 For example, the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), which consists of a single SH2 domain and a short C-terminal tail, has been shown to play crucial roles in the development, differentiation and function of T cells, natural killer (NK) cells, NKT cells, eosinophils, platelets, and some B-cell populations.3,4 In addition, SAP is required for mediating signals from the SLAM family members in both cellular and humoral immune responses. © 2013 American Chemical Society
Furthermore, it has been shown that blocking the interaction between SH2 domain and its counter-protein inhibits the overactive signal pathways in several cancer cells.5 For example, inhibition of Stat3−Stat3 complex formation by a small molecule, S31-201, could suppress Stat3-dependent transcription activity and inhibit the growth of breast tumor in vivo as well.6 Therefore, the development of SH2 domain inhibitors designed to antagonize or modulate PTK signaling has received a significant amount of attention in drug development for treating human disease in recent years.
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RESULTS AND DISCUSSION Chemistry. To date, a number of inhibitors targeting various SH2 domain proteins have been designed and tested. For example, peptides or cyclic peptides carrying phosphotyrosyl mimetics were shown to be effectively bound to the SH2 domain of Grb2.7−11 It is apparent that an appropriate phosphotyrosyl mimetic is crucial for high-affinity binding of these inhibitors, which makes exploring novel molecular skeletons mimicking phosphotyrosine residue an indispensible assignment. Herein we report the development of an unusual Received: November 1, 2012 Published: March 7, 2013 2841
dx.doi.org/10.1021/jm301610q | J. Med. Chem. 2013, 56, 2841−2849
Journal of Medicinal Chemistry
Article
Boc protecting group of 7 was removed with TFA, followed by reaction with Fmoc-OSu to afford building block 8 (60% yield in two steps). The synthetic strategy for the construction of series A dipeptides (compounds 9−39) is shown in Scheme 2. Since a 9-fluorenylmethyloxycarbonyl (Fmoc) group will be retained at the N-terminus of the final dipeptide products (Scheme 1), we started with coupling reactions of building block 6 with the corresponding Fmoc-amino acid derivatives (Fmoc-AA-OH) that were commonly used in the solid-phase peptide synthesis (SPPS) under standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/1-hydroxybenzotriazole (EDC/HOBt) conditions. Nineteen Fmoc-amino acid derivatives were used for the coupling. The side-chain protecting groups for the appropriate amino acids included tert-butyloxycarbonyl (Boc) (for Lys and Trp), tert-butyl (tBu) (for Asp, Glu, Ser, Thr, and Tyr), triphenylmethyl (trityl, Trt) (for Asn, Gln, and His), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) (for Arg). In addition to the 19 naturally occurring amino acids, we also performed direct coupling between 6 and 8 to give 39 (Table 1). It should be emphasized that most of the coupling products could be purified by crystallization from CHCl3/ether, which largely simplified the purification process. For those amino acids with side-chain protection, we could later convert them to the corresponding deprotected forms with TFA/ triisopropylsilane (TIS)/water (95/2.5/2.5). For the synthesis of series B dipeptides (compounds 40− 67), we first converted the 19 commercially available Fmoc-AAOH into the corresponding Fmoc-amino acid amides (FmocAA-NH2) by treatment with Boc2O/ammonium bicarbonate/ pyridine in either 1,4-dioxane or N,N-dimethylformamide (DMF) (Scheme 3).13 The Fmoc groups were then removed with 20% diethylamine in DMF to prepare the free amines, which were used directly for the coupling with building block 8 under EDC/HOBt or N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) conditions to prepare the dipeptides. Fortunately, the same simple crystallization purification process that was used for series A could also be applied to the coupling products of this series. For those amino acids with side-chain protection, three different acidic conditions were applied to convert them to the corresponding deprotected forms [neat TFA for compounds 49 and 53, TFA/H2O (95/5) for 55, and TFA/TIS/H2O (95/ 2.5/2.5) for all others]. It should be noted that His residue failed to produce coupling product. We therefore prepared only 28 series B dipeptides. All final products were characterized by NMR, IR, and mass spectrometry (Supporting Information). Biological Analysis. Establishing an Assay System to Monitor SAP−Substrate Interaction. It is well documented that the SLAM receptor and SAP family members play crucial roles in immune cell interactions. Upon contact with other immune cells, the SLAM family receptors utilize their cytoplasmic domain to physically associate with members of the SAP family adaptors. The SLAM receptor and SAP family members are required for the regulation of T-cell-dependent humoral immune responses and have been implicated as targets in the pathogenesis of autoimmune diseases.3,4 We have therefore used SAP as a model in this study. To identify small-molecular-weight compounds that inhibit SAP−SLAM interactions, an assay system was first established by use of a recombinant SAP protein (Figure 2A). The SAP gene encodes a 128-amino-acid protein that consists of a single SH2 domain and a short C-terminal tail.14−16 We have
amino acid carrying a cyclosaligenyl (cycloSal) phosphodiester moiety (cpY) that serves as a mimetic for the phosphotyrosyl residue (pY) (Figure 1). Combinatorial syntheses were
Figure 1. Structures of phosphotyrosyl moiety 1 (pY) and a phosphotyrosyl mimetic 2 (cpY), as well as two series of Fmoccapped, 2-carrying dipeptides (series A and B) in which R stands for the side chain (protected and unprotected forms) of an arbitrary amino acid (except Cys).
performed to prepare two series of Fmoc-protected, cpYcontaining dipeptide libraries. The dipeptide libraries were then tested for their inhibitory effects toward SAP−SLAM interaction via a newly established plate-based assay system. Several candidates in the dipeptide libraries showed potent inhibitory activity at levels
