Near-infrared optical imaging in glioblastoma xenograft with ligand-targeting alpha 3 integrin.

PURPOSE: Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell surface-binding ligands for glioblastoma cells and use them as targeted imaging and therapeutic agents for this deadly disease. METHODS: One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo and ex vivo experiments on U-87MG xenograft mouse model. RESULTS: A cyclic peptide, LXY1, was identified and shown to be binding to the alpha 3 integrin of U-87MG cells with moderately high affinity (K (d) = 0.5 +/- 0.1 microM) and high specificity. Biotinylated LXY1, when complexed with streptavidin-Cy5.5 (SA-Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1-biotin-SA-Cy5.5 complex when intravenously injecting the animals with anti-alpha 3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1-Cy5.5 conjugate (2,279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1-biotin-SA-Cy5.5 complex (approximately 64 kDa). CONCLUSIONS: Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma.

Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity.

The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.

Human serum albumin and p53-activating peptide fusion protein is able to promote apoptosis and deliver fatty acid-modified molecules.

Therapeutic peptides offer a high degree of specificity, potency, and low toxicity; making them promising candidates for cancer therapy. Despite these advantages, a number of hurdles, such as poor serum stability and inefficient cellular penetration, must be overcome. Fusing a therapeutic peptide to human serum albumin (HSA) is a common approach to extend the serum stability of a peptide that binds to extracellular receptors. However, no study has shown that this approach can be applied to target intracellular proteins. Here we demonstrate the feasibility of using a recombinant human serum albumin (rHSA) fusion protein to simultaneously deliver two types of molecules: a peptide capable of binding an intracellular target, as well as fatty acid (FA)-modified FITC (FA-FITC). Two peptides reported to disrupt the intracellular p53 and MDM2/MDMX interaction were fused to the C-terminal of HSA. Cellular and biochemical studies indicate that rHSA fusion proteins were efficiently taken up by SJSA-1 cells and retained MDM2- and MDMX-binding activity. By inducing the accumulation of p53, both fusion proteins promoted efficient cytotoxicity in SJSA-1 cells via caspase activation. Long chain fatty acid (LCFA) transportation is an essential endogenous function of HSA. This study also demonstrates that rHSA fusion proteins formed highly stable complexes with FA-FITC via non-covalent interactions. FA-FITC complexed with HSA could be internalized efficiently and rHSA-P53i and rHSA-PMI retained apoptotic activity as complex components. It is expected that such an approach can ultimately be used to facilitate intracellular delivery of two anticancer therapeutics, each with distinct but complimentary mechanisms, to achieve synergistic efficacy.

Novel flavonoids with antiproliferative activities against breast cancer cells.

A series of flavone analogues were synthesized and evaluated for their antiproliferation activity against breast cancer cells. The IC(50) of compound 10 and 24 were determined to be at 5 µM. These compounds were used as baits to screen breast cancer cDNA expression phage display proteome library. DNA sequencing of the binding phages suggests that eEF1A1 is a target protein for 10 and 24. Further optimization of these compounds led to the discovery of 39 with higher cytotoxic potency (IC(50) = 1 µM) and binding to eEF1A2. Biological and biochemical data suggest that eEF1A2 might be a therapeutic target and that 39 is an excellent lead compound for further development.

Facile synthesis of glycosylated Fmoc amino acid building blocks assisted by microwave irradiation.

The synthesis of glycosylated Fmoc amino acids by reaction of mono- and disaccharide peracetates with Fmoc amino acids having free carboxyl groups was rapidly promoted by Lewis acids (SnCl(4), BF(3)·Et(2)O) under microwave irradiation. The products are useful building blocks for the synthesis of glycopeptides.

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvß3 integrin and cancer targeting arginine-glycine-aspartic acid ligands with a built-in handle.

The αvß3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvß3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvß3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvß3 and showed comparable binding affinity (IC(50) = 0.68 ± 0.08 µmol/L) to some of the well-known RGD "head-to-tail" cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvß3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvß3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake.

Structure-activity relationship studies of targeting ligands against breast cancer cells.

A series of LXY3 (1) analogues were designed and synthesized. Their binding affinity was demonstrated using MDA-MB-231 breast cancer cells adherence inhibition assay. Further structure-activity relationship was obtained. Analogue 29 was discovered to have 3.5-fold increase of the binding affinity. Fluorescent microscopy and in vivo and ex vivo imaging studies demonstrated that 29 is an efficient in vivo targeting agent against alpha3 integrin of MDA-MB-231 breast tumor xenograft implant.

Discovery of targeting ligands for breast cancer cells using the one-bead one-compound combinatorial method.

Four "one-bead one-compound" (OBOC) combinatorial libraries were designed, synthesized, and screened against MDA-MB-231 breast cancer cells. A novel cyclic peptide 1 (LXY1) with high binding specificity to alpha3 integrin was identified. Molecular interactions between alpha3 integrin and 1 were characterized by using a series of K562 cells transfected with various mutant alpha3 integrins. Using analytic flow cytometry, the binding affinity (K(d)) of 1 to alpha3 integrin on MDA-MB-231 breast cancer cells was determined to be approximately 0.4 microM. Based on the established structure-activity relationship (SAR) study, two highly focused cyclic peptide libraries were further designed, synthesized, and screened against MDA-MB-231 breast cancer cells under stringent conditions. A novel cyclic peptide 2 (LXY3) with a high binding affinity (IC(50) = 57 nM) was identified. Moreover, the targeting efficiency and specificity of 2 to the breast adenocarcinoma tumors in mouse xenografts were further confirmed by in vivo and ex vivo near-infrared fluorescence optical imaging.

Discovery of high-affinity peptide ligands for vancomycin.

Vancomycin, an important antibiotic against medically relevant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, exerts its antibacterial effects by binding with moderate affinity to the C-terminal Lys-D-Ala-D-Ala motif (Kaa) of the bacterial cell wall peptide precursor. Essential for Kaa binding to vancomcyin is the free-carboxyl group on the terminal D-Ala in Kaa. In efforts to identify other Kaa-based peptides which bind vancomycin with higher affinity, we utilized our one-bead-one-compound (OBOC) combinatorial library approach, a method which has been widely used to discover highly specific ligands against various receptors. In standard OBOC peptide libraries, the C-terminal end of the synthesized peptide is tethered to a solid-support/resin, however, this study reports development of a synthetic strategy for generating OBOC peptide libraries with a free D-Ala-D-Ala carboxyl end. We screened these "OBOC inverted" peptide libraries against vancomycin, and discovered a series of peptide ligands with strong consensus, which bind vancomycin. To further optimize these ligands, two highly focused Kaa-containing OBOC combinatorial peptidomimetic libraries were designed, synthesized, and screened against vancomycin under more stringent conditions. Peptidomimetic ligands which bind vancomycin with higher affinity than Kaa were identified. The dissociation constant of one of these ligands, Lys(Ac)-HOCit-Glu-Cha-Lys(3,5-dihydroxybenzoyl)-D-Ala-D-Ala (9), as determined by surface plasmon resonance, was 1.03 microM, roughly a 50-fold improvement in affinity compared to Kaa (K(D) = 50 microM).

Synthesis of flavonoid analogues as scaffolds for natural product-based combinatorial libraries.

The design and synthesis of flavonoid analogues as combinatorial scaffolds is reported. Using commercially available materials, we synthesized chalcones with fluoro and carboxy groups. Nitration of these compounds generated highly functionalized flavonoid scaffolds with an o-fluoronitrobenzene template. Subsequent cyclizations of these chalcones resulted in the formation of several flavone and flavonone scaffolds. One of the flavonones was chosen as the scaffold to synthesize flavonoid derivatives on the solid phase. A series of flavonoid derivatives were obtained in high yields, which demonstrates that these highly functionalized scaffolds can be used in the synthesis of natural product-based combinatorial libraries for drug discovery.

A spiroisoxazolinoproline-based amino acid scaffold for solid phase and one-bead-one-compound library synthesis.

An efficient, multigram synthesis of a spiroisoxazolinoproline-based amino acid, 7, requiring minimal purification, delivering good cis:trans diastereoselectivity (approximately 1:4), and providing good yields is reported. Surface-bound studies of the reduction of an arylnitro group in the presence of an isoxazoline ring with tin(II) dichloride dihydrate were undertaken to confirm the stability of the isoxazoline ring. Full derivitization of this spiroisoxazolinoproline-based amino acid scaffold was performed during the synthesis of a sample library with high yields and high purity that validated the efficiency of the chemistry that was employed in resin-bound library synthesis. A 129,600 member one-bead-one-compound (OBOC) library based on the scaffold 7 was synthesized utilizing a dual amino acid encoding method and bifunctionalization of TentaGel resin.

Conformational studies of resin-bound vancomycin and the complex of vancomycin and Ac2-L-Lys-D-Ala-D-Ala.

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.

Solid-phase synthesis and antibacterial evaluations of N-demethylvancomycin derivatives.

Twenty-five N-demethylvancomycin derivatives were synthesized on solid-support and their structures were determined by LC-MS/MS. Biological evaluation of these compounds indicated that bulky hydrophobic substituent on vancosamine of N-demethylvancomycin can increase antibacterial activity against vancomycin-resistant Enterococcus faecalis.

Solid-phase synthesis of O-glycosylated Nalpha-Fmoc amino acids and analysis by high-resolution magic angle spinning NMR.

Direct O-glycosylation of amino acids bound to TentaGel resin with a number of glycosyl trichloroacetimidate donors results in high yields. The glycosylation reaction can be easily monitored by analyzing the bead-bound amino acids with high-resolution magic angle spinning (HR-MAS) NMR. These studies pave a new way for the construction of "one-bead one-compound" O-glycopeptide libraries with standard amino acid building blocks and appropriate glycosyl trichloroacetimidate donors.