A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.

Novel therapeutic modalities to address nondrugable protein interaction targets.

Small molecule drugs are relatively effective in working on 'drugable' targets such as GPCRs, ion channels, kinases, proteases, etc but ineffective at blocking protein-protein interactions that represent an emerging class of 'nondrugable' central nervous system (CNS) targets. This article provides an overview of novel therapeutic modalities such as biologics (in particular antibodies) and emerging oligonucleotide therapeutics such as antisense, small-interfering RNA, and aptamers. Their key properties, overall strengths and limitations, and their utility as tools for target validation are presented.

SELEX with modified nucleotides.

Aptamers, a promising new class of therapeutics, are single-stranded oligonucleotides generated via an in vitro selection process that bind to and inhibit the activity of target proteins in a manner similar to therapeutic antibodies. In order to enhance the drug-like character of aptamers, oligonucleotide libraries containing modified nucleotides are increasingly being used for selection. Principally, the choice of modifications aims to increase aptamer potency by enhancing nuclease-resistance, or increasing target affinity by providing more target recognition functionality or generating more stable aptamer structures.

Complex target SELEX.

Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents.

2'-Deoxy purine, 2'-O-methyl pyrimidine (dRmY) aptamers as candidate therapeutics.

Aptamers are short oligonucleotides that fold into well-defined three-dimensional architectures thereby enabling specific binding to molecular targets such as proteins. To be successful as a novel therapeutic modality, it is important for aptamers to not only bind their targets with high specificity and affinity, but also to exhibit favorable properties with respect to in vivo stability, cost-effective synthesis, and tolerability (i.e.

ADP-specific sensors enable universal assay of protein kinase activity.

Two molecular sensors that specifically recognize ADP in a background of over 100-fold molar excess of ATP are described. These sensors are nucleic-acid based and comprise a general method for monitoring protein kinase activity. The ADP-aptamer scintillation proximity assay is configured in a single-step, homogeneous format while the allosteric ribozyme (RiboReporter) sensor generates a fluorescent signal upon ADP-dependent ribozyme self-cleavage.

A novel strategy for selection of allosteric ribozymes yields RiboReporter sensors for caffeine and aspartame.

We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter trade mark sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM.