Robust suppression of HIV replication by intracellularly expressed reverse transcriptase aptamers is independent of ribozyme processing.

RNA aptamers that bind human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) also inhibit viral replication, making them attractive as therapeutic candidates and potential tools for dissecting viral pathogenesis. However, it is not well understood how aptamer-expression context and cellular RNA pathways govern aptamer accumulation and net antiviral bioactivity. Using a previously-described expression cassette in which aptamers were flanked by two "minimal core" hammerhead ribozymes, we observed only weak suppression of pseudotyped HIV.

Is phage display technology on target for developing peptide-based cancer drugs?

New tumor targeting agents are required to advance cancer diagnosis and treatment. Bacteriophage (phage) display technology, a molecular genetic means of combinatorial drug discovery, is an emerging approach to identify and improve peptide molecules as pharmaceuticals. Peptides are thought to have clinically desirable benefits over currently used biomolecules, such as antibodies, because of their rapid blood clearance, increased diffusion and tissue penetration, non-immunogenic nature and ease of synthesis.

Peptides specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated cancer cell adhesion.

Intravascular cancer cell adhesion plays a significant role in the metastatic process. Studies indicate that galectin-3, a member of the galectin family of soluble animal lectins, is involved in carbohydrate-mediated metastatic cell heterotypic (between carcinoma cells and endothelium) and homotypic (between carcinoma cells) adhesion via interactions with the tumor-specific Thomsen-Friedenreich glycoantigen (TFAg). We hypothesized that blocking the galectin-3 carbohydrate recognition domain with synthetic peptides would significantly reduce metastasis-associated carcinoma cell adhesion.

Biodistribution of filamentous phage peptide libraries in mice.

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens.

High-throughput fluorescence spectroscopic analysis of affinity of peptides displayed on bacteriophage.

Fluorescence spectroscopy titrations, although widely used to analyze binding affinity, are not an efficient screening method for detecting high-affinity binding among a large number of available ligands, such as during bacteriophage display selections. We hypothesize that a miniaturized, high-throughput fluorescence spectroscopy assay can be used to efficiently analyze selection results by applying the Langmuir equation to the binding data to estimate affinity constants for a large number of ligands, either as synthesized molecules or as displayed on bacteriophage. Here, bacteriophage-display-derived peptides specific for the Thomsen-Friedenreich disaccharide are used to develop a high-throughput fluorescence spectroscopy screening method, which uses one binding partner labeled with a fluorescent dye and different concentrations of a second partner to analyze binding affinity in bacteriophage display selections.

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides.

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation.

Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene.

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence.

Combinatorial discovery of tumor targeting peptides using phage display.

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting.

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen.

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility.