Rapid isolation of high affinity and specficity ligands for cell surface recepters

Cell surface receptors represent one of the largest categories of drug targets. Small molecule drugs generally target the intracellular active sites of these receptors (most are kinases that trigger signaling cascades). Alternatively, therapeutic monoclonal antibodies can be employed to target the extracellular domains of receptors so as to block their interaction with hormones. However, both approaches have important limitations. Small molecules targeting the kinase site generally suffer from a lack of specificity, resulting in problematic off-target effects. Antibodies are expensive to manufacture and, of course, are very large molecules that are excluded from some target sites accessible to small molecules. Therefore, we have been interested in developing methods to identify small molecules capable of binding to receptor extracellular domains with affinities and specificities similar to that of a monoclonal antibody, but which would have the more desirable pharmacokinetic properties of a small molecule.

Dr. Gomika Udugamasooriya and co-workers in the Center developed the screening technology illustrated in the figure above. A library of peptoids (N-substituted oligoglycines) was synthesized on hydrophilic beads, such that each bead displayed many copies of a single peptoid. This library was then incubated with two types of cells. These cells differed only in the presence or absence of the target receptor (in this case human VEGF Receptor 2). The cells lacking the receptor were stained green and the cells containing the receptor were stained red using quantum dots that were internalized via endocytosis. Beads that bound only red cells and not green cells were collected. It was assumed that these beads displayed peptoids that would bind only to the VEGFR2 and not any other molecule on the cell surface (since if they did, green cells would be retained as well) thus demanding high specificity for the target receptor. Five “hits” were obtained form a library of more than 300,000 compounds. The ability to screen such a large library quickly and cheaply attests to the power of this screening technology.

Subsequent studies found that all five hits were highly specific ligands for the VEGFR2 extracellular domain. Dimerization of one peptoid provided a high affinity ligand for the dimeric receptor, with a KD of approximately 25 nM (see below). This compound, when injected into a mouse, was a potent antagonist of VEGF-mediated angiogenesis.

In recent unpublished work, we have successfully applied this approach to the isolation of peptoids that bind other cell surface receptors. Therefore, it appears that this technology may represent a general solution to the isolation of high affinity and specificity receptor ligands. We anticipate that many of these molecules will be therapeutically interesting antagonists or agonists.

For details, please see:

Udugamasooriya, D.G., Dineen, S.P., Brekken, R.A. and Kodadek, T. (2008) “A peptoid “antibody surrogate” that antagonizes VEGF Receptor 2 activity” J. Amer. Chem. Soc. 130, 5744-5752.