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A. J. Johnson, M. I. Dorell, A. Shagini, M. R. Ritter, W. Jin, S. Moreno, D. L. Boger, M. Friedlander; Identification of Small Mole. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4103.
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Bone marrow-derived progenitor cells (BMCs) have been shown to target sites of neovascularization in the retina and exhibit vasculo- and neurotrophic rescue in retinal degenerative mouse models (Otani et al., 2002; Otani et al., 2004). They have also demonstrated potent rescue functions in models of ischemic retinopathy (Ritter et al., 2006). BMCs have been shown to differentially express the hyaluronic acid receptor CD44. Current methods of enriching the functional subpopulation of BMCs utilize differential expression levels of CD44; the functional subpopulation expresses high levels of CD44 (CD44Hi) while low level expressing cells (CD44Lo) are non-functional. The CD44Hi population of cells consists mainly of myeloid progenitors, while the majority of non-functional CD44Lo cells are erythroid or lymphoid in origin (Ritter et al., 2006). Together, Ter119 and CD45RB220 identify the majority of non-functional cells, binding the erythroid and lymphoid cells respectively. Thus Ter119 and CD45RB220 depleted populations of cells would represent a functional population of cells free of bound purification agents such as antibodies or magnetic beads.
We have used an in vitro, cell based selection assay to screen a peptidomimetic library of 60,000 molecules with modifiable side chains (Boger et al., 2003) to identify small molecules that efficiently bind to CD44Lo -expressing cells, for use as a negative selection tool. We have screened this library and identified compounds that competitively inhibit the binding of anti-Ter119 or anti-CD45R-B220 to CD44 Lo BMCs.
Candidate molecules that inhibit binding of either Ter-119 or CD45R-B220 antibody to CD44Lo-expressing cells have been identified from a library of non-peptidic chemicals. Of 1000 candidate molecules screened, 35 blocked antibody binding to the BMCs. 6 of these displayed efficient dose-dependant inhibition of antibody binding. Interestingly, the most promising candidates exhibited R groups that were similar in both orientation and structure.
We have developed a small-molecule based selection procedure. From the initial screen, we have identified 6 candidates that exhibit reproducible, significant antibody inhibition in a dose-dependant fashion. With appropriate chemical modifications, this could (1) eliminate the need to use immunoglobulins and (2) provide a readily modifiable chemical structure that could negatively select subpopulations of BMCs with retinal vascular rescue potential.
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