Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.

Abstract Myeloid cell leukemia (Mcl-1) is a multidomain, anti-apoptotic protein, member of the Bcl-2 family proteins, an important survival factor for many cancers. Mcl-1 has a critical and distinct role(s) in maintaining cell survival and emerging as an independent and promising therapeutic target. Through high throughout screening (HTS) of 53,300 structurally diverse synthetic organic compounds, we have discovered a number of novel inhibitors of Mcl-1. The hits identified by HTS were evaluated in a series of complementary biochemical, biophysical, functional and cellular assays to eliminate false positives and to determine the specificity and mechanism of action of these new compounds and 23 compounds were found to be inhibitors of Mcl-1. The most promising compounds, 59 and 62, have diverse chemical scaffolds. We have shown that these small molecule inhibitors bind to the BH3 binding site in Mcl-1 with IC50 values of 1,200 nM and 200 nM, respectively, and compete with BH3 peptides derived from Bid, Bim, or Noxa proteins. They bind to the Mcl-1 protein showing selectivity over two other Bcl-2 family members, Bcl-2 and Bcl-xL. NMR spectroscopy shows that 59 and 62 bind to the same BH3 domain of Mcl-1 as the Bim BH3 peptide. These initial lead compounds antagonize Mcl-1 on the functional level and they induce release of cytochrome c, inhibit cell growth and induce apoptosis in pancreatic and melanoma cancer cells with high levels of Mcl-1. We have tested several analogues of 59 and 62, both synthetic and commercially available, and established initial structure-activity relationships. One of the compounds tested (77), an analogue of 59, has improved binding affinity to Mcl-1 protein, as well as potent activity in inhibition of cell growth in cancer cells. Furthermore, by using murine embryonic fibroblasts (MEFs), wild type and deficient in both Bax and Bak (double knock out), it was demonstrated that the cytotoxic activity and induction of apoptosis by several analogues of the lead compound 59, depend on Bax and/or Bak, suggesting that they function as BH3 mimetics. Collectively, these findings provide good promise for chemical modifications of the new identified lead compounds, 59 and 62, and their further optimization toward developing a new class of anticancer drugs, Mcl-1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4521.

The epithelial-derived, type II transmembrane serine protease matriptase, the mouse homologue of which is epithin, has been shown to be involved in epidermal differentiation, hair formation, and thymus function. We show in this study that epithin/matriptase (Epi/MTP) plays a significant role in mammary epithelial cell growth and morphogenesis. Epi/MTP is expressed at low level in the mouse mammary epithelium of young animals and it accumulates at the terminal end-bud of the growing ducts. The level of Epi/MTP is elevated in the mammary glands at stages when epithelial proliferation and modeling occur. It is primarily present in the luminal epithelial cells of mouse mammary ducts and lobules. Using an ex vivo three-dimensional culture system for mammary epithelial functional assays, we show that mammary epithelial growth and morphogenesis in the presence of the latent form hepatocyte growth factor (pro-HGF) are blocked either by an inhibitor of the Epi/MTP protease activity or by siRNA knockdown of the Epi/MTP expression. These studies demonstrate that Epi/MTP participates in mammary epithelial growth and modeling through activation of pro-HGF. Our findings reveal an important pathway in normal mammary epithelial morphogenesis which may participate in breast cancer progression.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. 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 micromolar range binding affinity, such as cyclic peptide 15 with an K(d)=0.359microM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.

4148 Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are key regulators of the apoptotic pathway and their increased expression in human malignancies is associated with disease progression, tumor resistance to chemotherapy, radiation and poor clinical outcome. Recently,a novel Bcl-2/Bcl-xL/Bcl-w inhibitor (ABT-737) has been developed, which is highly potent in killing tumor cells displayinghigh levels of Bcl-2 and dependent upon Bcl-2 forsurvival. However, ABT-737 exhibits a relatively low affinityfor Mcl-1 and is considerably less efficient in killing tumor cellsexpressing high Mcl-1 levels. Several groups provide strong evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and one approach for overcoming this resistance could be the combination of ABT-737 with agentscapable of down-regulating/inhibiting Mcl-1. Here, we describe a high throughput screening approach that led to the identification of several chemical classes, small molecule Mcl-1 inhibitors. The compounds were characterized in a number of assays including in vitro binding using fluorescence polarization based assay, SPR solution competitive assay, heteronuclear single quantum correlation (HSQC) NMR spectroscopy, pull-down assay, functional assay for cytochrome c and Smac release and a cell-based viability assay with human cancer cells. The most potent of the inhibitors identified was compound 65, which binds to Mcl-1 protein with IC50 of 0.3 μM, showing >100-fold selectivity over Bcl-xL and 60-fold selectivity over Bcl-2 protein. 15N HSQC spectra conclusively showed that compound 65 interacts with the BH3 domain in Mcl-1 protein and many residues in the BH3 binding groove of Mcl-1 protein were affected by 65. Furthermore, pull-down assay demonstrates that the compound 65 is able in dose dependent manner to disrupt interactions between endogenous Mcl-1 protein and biotin labeled Noxa and Bid BH3 peptides, but not between Bid BH3 peptide and other members of Bcl-2 family (Bcl-xL and Bcl-2). Functional studies showed that compound 65 effectively antagonizes recombinant Mcl-1 protein and promotes release of cytochrome c and Smac from isolated mitochondria. Compound 65 displayed cytotoxicity with IC50 from 3 μM to 8 μM against a number of human cancer cell lines with high level of Mcl-1 protein. Collectively, these data indicate that 65 represents a novel and bone fide small-molecule inhibitor of Mcl-1 and a promising lead compound for further optimization toward developing a new class of anticancer drugs by targeting the Mcl-1 protein.

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.

A large number of cyclic peptides, which exhibit a range of biological activities, have been found in nature. The reason of cyclic peptides that attracted world wide attention and made them extensively studied, is their enhanced metabolic stabilities, biological specificities, bioavailability, and conformational constrained structural feature, which make them important leads for drug discovery, and as actual drugs. Many cyclic peptides show very promising biological activities, including anticancer, antibacterial, antiviral, antifungal, anti-inflammatory, and anti-clotting or anti-atherogenic properties. Peptide cyclization becomes an effective and commonly employed strategy for peptide modifications. During the past several decades, great efforts have been made to develop more efficient methods for the synthesis of cyclic peptides and peptidomimetics. This review will be focused on the major progress of the novel recently developed peptide cyclization approaches and the profitable applications since 2002.

The equivalent strain energy density (ESED) method was originally suggested and is still widely used to calculate the stresses and strains at notch root. It is well recognized, however, that the ESED method tends to underestimate the notch-tip stresses and strains. In order to obtain better predictions, in the present paper, a factor ((1 + νe)/(1 + νeff)) was introduced to modify the dissipated heat energy contained in the ESED method. A computational modeling technique coupling with Jiang-Sehitoglu plasticity model was also developed to calculate the multiaxial elastic-plastic notch-tip stress-strain responses of notched components. Extensive validations of the proposed method show that the calculated stresses and strains are in accord with experimental data and show reasonable accuracy.

We have designed and synthesized a cyclic, bivalent Smac mimetic (compound 3) and characterized its interaction with the X-linked inhibitor of apoptosis protein (XIAP). Compound 3 binds to XIAP containing both BIR2 and BIR3 domains with a biphasic dose-response curve representing two binding sites with IC 50 values of 0.5 and 406 nM, respectively. Compound 3 binds to XIAPs containing the BIR3-only and BIR2-only domain with K i values of 4 nM and 4.4 microM, respectively. Gel filtration experiments using wild-type and mutated XIAPs showed that 3 forms a 1:2 stoichiometric complex with XIAP containing the BIR3-only domain. However, it forms a 1:1 stoichiometric complex with XIAP containing both BIR2 and BIR3 domains, and both BIR domains are involved in the binding. Compound 3 efficiently antagonizes inhibition of XIAP in a cell-free functional assay and is >200 times more potent than its corresponding monovalent compound 2. Determination of the crystal structure of 3 in complex with the XIAP BIR3 domain confirms that 3 induces homodimerization of the XIAP BIR3 domain and provides a structural basis for the cooperative binding of one molecule of compound 3 to two XIAP BIR3 molecules. On the basis of this crystal structure, a binding model of XIAP containing both BIR2 and BIR3 domains and 3 was constructed, which sheds light on the ability of 3 to relieve the inhibition of XIAP with not only caspase-9 but also caspase-3/-7. Compound 3 is cell-permeable, effectively activates caspases in whole cells, and potently inhibits cancer cell growth. Compound 3 is a useful biochemical and pharmacological tool for further elucidating the role of XIAP in regulation of apoptosis and represents a promising lead compound for the design of potent, cell-permeable Smac mimetics for cancer treatment.

Structure-based strategy was employed to design flavonoid compounds to mimic the Bim BH3 peptide as a new class of inhibitors of the anti-apoptotic Bcl-2 proteins. The most potent compound, 4 (BI-33), binds to Bcl-2 and Mcl-1 with Ki values of 17 and 18 nM, respectively. Compound 4 inhibits cell growth in the MDA-MB-231 breast cancer cell line with an IC50 value of 110 nM and effectively induces apoptosis.

XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently, targeting both its BIR2 and BIR3 domains. We report the design, synthesis, and characterization of a nonpeptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000 times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultrapotent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in the HL-60 leukemia cell line. The potency of bivalent SM-164 in binding, functional, and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics.

PR-2 XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac/DIABLO (second mitochondria-derived activator of caspase or direct IAP binding protein with low pI), a protein released from mitochondria in response to apoptotic stimuli, in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We designed, synthesized and evaluated a series of non-peptide, cell-permeable, bivalent small-molecules which mimics Smac protein for targeting XIAP. SM-164 is one of the most potent Smac mimetics we have designed. Our binding and gel filtration studies shows SM-164 concurrently targets both the BIR2 and BIR3 domains in XIAP and binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000-times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. In our cell-free caspases functional assays, SM-164 completely restores the activity of caspase-9 and caspases-3 and -7 inhibited by XIAP at the same molar concentraction as XIAP. In cell-based assays, SM-164 effectively induces apoptosis at concentrations as low as 1 nM in several cancer cell lines and has a minimal toxicity to normal cells at 10,000 nM. The potency of SM-164 in binding, functional and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics.

STAT3 is a promising molecular target for the design of new anticancer drugs. In this paper, we report the design and synthesis of a conformationally constrained macrocyclic peptidomimetic 2 via click chemistry. Compound 2 was determined to bind to STAT3 with a Ki value of 7.3μM in a competitive fluorescence-polarization-based binding assay, representing a promising initial lead compound for further optimization.

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2. PTX is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer, yet patients have to suffer some side effects caused by its systemic toxicity. The aim of our conjugate is to specifically deliver antitumor agent PTX to breast cancer cells that overexpress oncogenic ErbB2 with the purpose to reduce toxicity and enhance selective killing of cancer cells. In this study, a concise and efficient synthetic route for the preparation of the PTX-EC-1 conjugate has been developed in 6% overall yield. This synthetic approach provides a general method for conjugating a highly functionalized and disulfide-bridge containing cyclopeptide to Taxol or other antitumor agents.

We report herein a new class of small-molecule inhibitors of antiapoptotic Bcl-2 proteins. The most potent compound, 7, binds to Bcl-2, Bcl-xL, and Mcl-1 proteins with Ki of 110, 638, and 150 nM, respectively. Compound 7 is highly effective in induction of cell death in breast cancer cells with high levels of Bcl-2, Bcl-xL, and Mcl-1 proteins and represents a promising lead compound for the design of new anticancer drugs.

Matriptase, initially isolated from human breast cancer cells in culture, is a member of the emerging class of type II transmembrane serine proteases. Matriptase blockade could potentially modulate tumorigenesis and metastasis in vivo. Sunflower trypsin inhibitor-1 (1, SFTI-1), isolated from sunflower seeds, exhibits very potent matriptase inhibitory activity. On the basis of these findings, we designed and synthesized 13 analogues of the naturally occurring peptide 1 with the intention to explore the structure-activity relationships of this type of bicyclic peptides and to improve inhibitory selectivity and metabolic stability of the disulfide-bridge-containing peptide 1. We discovered that the methylenedithioether-bridged compound 14 demonstrates very potent binding affinity to matriptase. Compound 8 exhibits much better selectivity for inhibition of matriptase versus thrombin, whereas compound 2 becomes a more potent thrombin inhibitor, which can be potentially used as an anticoagulant for prophylaxis and therapy of thromboembolism.

A214 Mitochondrial permeabilization is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins anchoring to or associated with the outer membrane of mitochondria delicately regulate this step through protein-protein interactions, which makes the mitochondria an ideal model for studying molecules that target the Bcl-2 pro-survival proteins. Purpose: to establish a reliable and specific functional assay using mitochondria isolated from cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins in the Bcl-2 family as well as the small molecular Bcl-2 inhibitors. Materials and Methods: mitochondria isolated from human breast cancer cell lines with distinct expression of Bcl-2 pro-survival proteins were dually protected by adding exogenous recombinant proteins before incubating with different BH3 peptides alone or in combination at 37°C for 1 hour. Smac and cytochrome c were tested on both supernatants and mitochondrial pellets using immunoblotting. Cell-permeable BH3 peptides were used in a cell-based apoptotic assay to further confirm their selective killing in cells with different Bcl-2 pro-survival proteins status. Non-peptide small-molecule inhibitors were also tested in the same conditions. Results and Conclusions: it is the first time using isolated human cancer cell mitochondria to comprehensively evaluate peptides and non-peptide small molecule inhibitors against three different Bcl-2 members (Bcl-2, Bcl-xL and Mcl-1). Noxa and Bad BH3 peptides exhibit their bona fide antagonistic capability against either Bcl-2/xL or Mcl-1 proteins respectively, whereas Bim BH3 peptide can antagonize all of three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergize with each other in release of cytochorme c and Smac proteins. These functional assays were then used to characterize our novel small molecule inhibitors of Bcl-2/Bcl-xL/Mcl-1 for their antagonism and specificity on a functional level.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.