Feng-Di T. Lung

Nonphosphorylated Peptide Ligands for the Grb2 Src Homology 2 Domain

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10–25 μm) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.

Oligomerization of Human Gadd45a Protein

Gadd45a is an 18-kDa acidic protein that is induced by genotoxic and certain other cellular stresses. The exact function of this protein is not known. However, there is evidence for its involvement in growth control, maintenance of genomic stability, DNA repair, cell cycle control, and apoptosis. Consistently, Gadd45a has previously been shown to interact in vitro and/orin vivo with a number of proteins playing central roles in these cellular processes: proliferating cell nuclear antigen, p21Cip1/Waf1, Cdc2-CyclinB complex, MTK1, and histones. Adding to this complexity, we have found that Gadd45a self-associates in solution, both in vitro and when expressed in the cell. Moreover, Gadd45a can complex with the two other members of the Gadd45 family of stress-induced proteins, human Gadd45b (MyD118) and Gadd45g (CR6). Gel-exclusion chromatography, native gel electrophoretic analysis, enzyme-linked immunosorbent assay, and chemical cross-linking showed that recombinant Gadd45a forms dimeric, trimeric, and tetrameric species in vitro, the dimers being the predominant form. Deletion mutant and peptide scanning analyses suggest that Gadd45a has two self-association sites: within N-terminal amino acids 33–61 and within 40 C-terminal amino acids. Despite the low abundance of Gadd45a in the cell, oligomer-forming concentrations can probably be achieved in the foci-like nuclear structures formed by the protein upon overexpression. Evidence for a potential role of Gadd45a self-association in altering DNA accessibility on damaged nucleosomes is presented.

Significant compensatory role of position Y-2 conferring high affinity to non-phosphorylated inhibitors of GRB2-SH2 domain

Systematic modification of amino acid at position Y-2 of a library-derived non-phosporylated thioether-cyclized peptide, cyclo(CH2CO-Glu2-Leu-Tyr0-Glu-Asn-Val-Gly-Met-Tyr-Cys) -amide, aided by molecular modeling, demonstrates that the Glu(-2) sidechain compensates for the absence of Tyr0 phosphorylation in retaining effective binding to Grb2-SH2 domain. Replacement of Glu(-2) with gamma-carboxyglutamic acid produced a high affinity inhibitor, the first example with submicromolar affinity (IC50 = 640 nM).

Development of non-phosphorylated cyclic thioether peptide binding to the Grb2-SH2 domain

One of the critical intracellular signaling pathways involves specific interactions between growth factor receptors and the adaptor protein Grb2. These interactions normally involve specific tyrosine phosphorylated regions in receptors and other cognate proteins. Following the lead of our recent findings that a phage library based non-phosphorylated disulfide linked 11-mer peptide inhibited such interactions, we report here the synthesis of novel redox-stable cyclic peptide analogs. These include thioether cyclized and backbone cyclized structures. The thioether analog was prepared under mild conditions from an N-terminally chloroacetylated and C-terminally cysteine extended peptide precursor. The thioether peptide showed equipotent binding affinity for the Grb2-SH2 domain (IC50=10–15 μM) when compared to the disulfide cyclized lead-peptide. The bioactive thioether linked peptide was demonstrated to offer advantages to the disulfide cyclized peptides under physiological conditions.

Global Optimization of Conformational Constraint on Non-phosphorylated Cyclic Peptide Antagonists of the Grb2-SH2 Domain

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu(2) and Gly(7) in this lead peptide, cyclo(CH(2)CO-Glu(1)-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC(50)=6.5 microM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of omega-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu(1) and Adi for Glu(4) in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency (19, IC(50)=23.3 microM) relative to G1TE. Moreover, the lipophilic chain in omega-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.

NMR based solution structure and dynamics of a nonphosphorylated cyclic peptide inhibitor for the Grb2 SH2 domain

Grb2 is an adaptor protein with a domain structure of SH3-SH2-SH3. Grb2 SH2 binds pTyr-peptides with the consensus sequence pYXNX within several proteins. Binding of Grb2 SH2 to receptors triggers the kinase cascade which is essential for cell growth and differentiation. The design of Grb2 SH2 inhibitors holds the promise of targeted inhibition of this pathway. Recently, we discovered a nonphosphorylated cyclic peptide ligand, G1TE, for the Grb2 SH2 domain [1,2]. Nonphosphorylated G1TE defines a new type of SH2 domain binding motif that may advance the design of Grb2 inhibitors. In order to gain further insight into these specific protein-protein interactions, we have determined the solution structure and dynamics of G1TE using two dimensional NMR and isotope labeling techniques. Results of conformational studies provide a molecular basis for the structurebased design of Grb2 SH2 inhibitors.

Structure-Based Design, Synthesis, and Surface Plasmon Resonance (SPR) Detection of Antagonists of the Grb2 SH2 Domain

One of the critical intracellular signal transduction pathway involves the binding of Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factors such as EGFR and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for development of potential anti-cancer agents. Previously, we reported a series of potent peptides and the detection of their inhibitory effects on the Grb2-SH2 domain. We report here our recent experimental design for detecting the interaction between Grb2-SH2 domain and its ligands using surface plasmon resonance (SPR) technology. Binding interactions between peptides and the Grb2-SH2 domain were measured and analyzed using BIACORE X instrument, which provide detailed information of the real-time detection of the binding interaction. We also established an SPR-based assay to determine the inhibitory effects of a series of designed peptide analogs, and discovered some potent antagonists of the Grb2 SH2 domain. Results of this study should provide important information for further development of peptide or peptidomimetics with high affinity for the Grb2 SH2 domain.