Utilization of a Peptidomimetic Library to Bind and Sort a Retinal Vasculature–Targeting Subpopulation of Adult Bone Marrow–Derived Cells

Abstract

Purpose: : A population of bone marrow derived cells highly expressing the hyaluronic acid receptor, CD44 (CD44^HI) has been found to possess vasculo– and neurotrophic rescue activity in mouse models of retinal degeneration. Currently CD44^HI –expressing cells are extracted from whole bone marrow via positive antibody–based fluorescent activated cell sorting (FACS). In the past, an antibody–based positive selection procedure has been used to isolate a functional subpopulation of bone marrow–derived cells useful for cell based therapeutic approaches to the treatment of retinal vascular and neuronal degenerative disorders. We are also exploring the potential utility of a small molecule–based approach.

Methods: : Using a pre–competitive cell based ELISA–like assay, we have screened a non–peptidic library of 60,000 compounds was screened to identify small molecules that bind efficiently to CD44^HI–expressing cells. To assess the small molecules ability to inhibit CD44 antibody binding to bone marrow–derived cells, CD44^HI–expressing cells were first incubated with the small molecule and horseradish peroxidase (HRP) labeled CD44 antibodies were then added. A colorimetric assay was then performed to determine the extent of antibody binding inhibition and the results were analyzed by a microplate reader.

Results: : Candidate molecules that inhibit binding of CD44 antibody to CD44^HI–expressing cells have been identified from a library of non–peptidic chemicals. Of 1000 candidate molecules, 17 exhibited the ability to block CD44 antibody binding to the bone marrow–derived cells. Of these 17 candidates, 6 candidates continued to display the sufficient inhibition of antibody binding. In order to determine potency, dose–response experiments were performed. Interestingly, the most promising candidates were structurally similar to each other in that they had side chains in cis–, rather than trans–oriented, positions.

Conclusions: : We have developed a small–molecule based selection procedure taking advantage of the availability of a library consisting of over 10,000 non–peptidic molecules with modifiable side chains. In hopes that, this would (1) eliminate the need to use expensive immunoglobulins and (2) provide a readily modifiable chemical structure that could, theoretically, be synthesized with higher binding affinity.

Keywords: vascular cells • retinal degenerations: cell biology • retinal neovascularization