Peng Li

Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database.

The X-linked inhibitor of apoptosis (XIAP) is a promising new molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells to chemotherapeutic agents and radiation therapy. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Through computational structure-based screening of an in-house traditional herbal medicine three-dimensional structure database of 8221 individual natural products, followed by biochemical testing of selected candidate compounds, we discovered embelin from the Japanese Ardisia herb as a small-molecular weight inhibitor that binds to the XIAP BIR3 domain. We showed that embelin binds to the XIAP BIR3 protein with an affinity similar to that of the natural Smac peptide using a fluorescence polarization-based binding assay. Our NMR analysis further conclusively confirmed that embelin interacts with several crucial residues in the XIAP BIR3 domain with which Smac and caspsase-9 bind. Embelin inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP. In stably XIAP-transfected Jurkat cells, embelin effectively overcomes the protective effect of XIAP to apoptosis and enhances the etoposide-induced apoptosis and has a minimal effect in Jurkat cells transfected with vector control. Taken together, our results showed that embelin is a fairly potent, nonpeptidic, cell-permeable, small-molecule inhibitor of XIAP and represents a promising lead compound for designing an entirely new class of anticancer agents that target the BIR3 domain of XIAP.

Cyclization strategies in peptide derived drug design.

The choice of peptides as prototype modulators of biological function, is justified on the grounds that peptides are natural constituents of living systems. They exist as hormones, biochemical inhibitors, antigens, growth factors, transmembrane carriers and, indeed they are comprised of the building blocks of all proteins. As such, the natural and mutated analogs of these functional entities provide a rich variety of pharmacophore models for further development. Peptidomimetic modification of active peptides can provide biostable analogs. Moreover, cyclization of linear peptides is frequently used as an attractive venue to provide both conformationally more restricted as well as more biostable analogs. The objective of this review is to report an updated summary of the more recently developed methodologies for the design and synthesis of cyclized peptides, citing selected examples of the effect of cyclization on both proteolytic stability and biological activity.

Synthesis and evaluation of the sunflower derived trypsin inhibitor as a potent inhibitor of the type II transmembrane serine protease, matriptase

We report here the synthesis of a 14-amino acid long bicyclic peptide, previously isolated from sunflower seeds. This peptide, termed sunflower trypsin inhibitor (SFTI-1), is one of the most potent naturally occurring small-molecule trypsin inhibitors. In addition to inhibiting trypsin, the synthetic SFTI-1 is also a very potent inhibitor, with a K(i) of 0.92nM, of the recently identified epithelial serine protease, termed matriptase.

Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7.

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (YXN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY+2) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.

Structure-based design of thioether-bridged cyclic phosphopeptides binding to Grb2-SH2 domain.

A series of phosphotyrosine containing cyclic peptides was designed and synthesized based upon the phage library derived cyclopeptide, G1TE. Considering the type-I beta-turn feature of peptidic ligand binding to Grb2 SH2 domain, we introduce alpha,alpha-disubstituted cyclic amino acid, Ach, into the 4th position of the cyclic peptide to induce a local right handed 3(10) helical conformation. In order to stabilize the favorable binding conformation, the bulky and hydrophobic amino acids, neopentylglycine (NPG) and phenylalanine, were introduced into the 8th and 2nd positions of the peptide ligand, respectively. To facilitate the sidechain of pTyr3 reaching into the phosphotyrosine binding pocket, a less bulky alanine was preferred in position 1. Based upon these global modifications, a highly potent peptide ligand 12 was discovered with an IC(50)=1.68 nM, evaluated by ELISA binding essay. Ligand 12 is at least 10(5) more potent than the lead peptide, termed G1TE.

Potent Grb2–SH2 domain antagonists not relying on phosphotyrosine mimics

Development of Grb2-SH2 domain antagonists is an effective approach to inhibit the growth of malignant cells by modulating Grb2-related Ras signaling. We report here potent Grb2-SH2 domain antagonists that do not rely on phosphotyrosine or its mimics. These non-phosphorylated antagonists were developed and further modified by constraining the backbone conformation and optimizing amino acid side chains of a phage library-derived peptide, G1TE. After extensive SAR studies and structural optimization, non-phosphorylated peptide 12 was discovered with an IC(50) of 75 nM. This potent peptidomimetic provides a novel template for the development of non-pTyr containing Grb2-SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.

Structural basis for a non-phosphorus-containing cyclic peptide binding to Grb2-SH2 domain with high affinity

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor bound protein 2 (Grb2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferative agents due to its potential to shut down the Ras activation pathway. Generally, the pTyr-X-Asn minimal binding motif is required for a high-affinity ligand binding to the Grb2-SH2 domain. Using phage-display techniques, we discovered a non-pTyr-containing cyclic peptide G1 with moderate binding affinity from 10(7) different sequences. To understand the structural basis for the high-affinity binding of these novel non-phosphorus-containing inhibitors to the Grb2-SH2 domain, we extensively studied herein the unique functional requirements of the acidic side chain in Tyr-2 position due to the absence of the phosphate group in these non-phosphorylated peptides. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low nanomolar range binding affinity, such as cyclic peptide 20 with an IC(50)=0.026 microM, which is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. Whole cell assays indicate that peptide 20 can penetrate the cell membranes and inhibit the association of Grb2 with p185(erbB2) in erbB2-overexpressing MDA-MA-453 cancer cells at low micromolar concentrations.

Small nonphosphorylated Grb2–SH2 domain antagonists evaluated by surface plasmon resonance technology

The growth factor receptor‐binding protein–Src homology 2 (Grb2–SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, which involves cell proliferation and differentiation. Therefore, the Grb2–SH2 domain has been chosen as our target for development of potential antiproliferative agents. Herein, we report the study of the inhibitory effects of small nonphosphorylated peptide analogs interacting with the Grb2–SH2 domain protein by surface plasmon resonance (SPR) technology. A set of 8 related peptide analogs were synthesized, purified, and characterized. Their inhibitory effects on Grb2–SH2 were evaluated by the SPR technology developed with the BIACORE X instrument. The lead peptide, Fmoc–Glu–Tyr–Aib–Asn–NH2 (Fmoc–E–Y–Aib–N; Fmoc: 9‐fluorenylmethyoxycarbonyl; Aib = α‐amino isobutyric acid) inhibited Grb2–SH2 domain function with an IC50 value of 8.7 μM. A molecular modeling study of the lead peptide indicated that the glutamate in the Fmoc peptide is ideally positioned to form a strong salt bridge to Arg 67 in the Grb2–SH2 domain, using both its backbone carbonyl and its acidic group. Residue Glu 89 in Grb2–SH2 flips inward to fill the binding site and partially replace the phosphate group as a hydrogen‐bond acceptor. Results of these studies provide important information for further development of potent nonphosphorylated peptide inhibitors of the Grb2–SH2 domain.

Bicyclic Peptide Inhibitors of an Epithelial Cell-Derived Transmembrane Protease, Matriptase

Matriptase is a recently identified type II transmembrane protease that is found on the surface of epithelial cells [1,2]. It is overexpressed in most cancer cells, including human breast cancer cells. It is a multidomain protein with a C-terminal extracellular region containing the protease domain. It activates urokinase-type of plasminogen activator (uPA), and the protease activated receptor (Par-2). It has been demonstrated that matriptase can proteolytically activate hepatocyte growth factor (HGF/Scatter Factor) to its active form, and thus it may function in epithelial cell migration, cancer invasion and metastasis [2]. Developing inhibitors of matriptase provides for a therapeutic approach to metastatic diseases, and in particular, cancer.