Novel high-throughput screening strategies in one-bead one-compound combinatorial methodology.

摘要

A method is described to immobilize and array large quantities of microbeads within microfabricated cassettes for subsequent high-throughput releasable solution-phase cell-based screening of combinatorial libraries. Jeffamine triamine derivative was sequentially coupled to the outer layer of topologically segregated TentaGel beads to increase the loading capacity, hydrophilicity, and porosity of the bead resin. This bead modification facilitates efficient release of the covalently attached surface compound. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and interior coding tags. Compound-beads were loaded into a Petri dish containing a 10,000-well polydimethylsiloxane (PDMS) microbead cassette such that over 90% of the microwells were each filled with only one bead. Matrigel with suspended Jurkat lymphoid cells was layered over the microbead cassette. Dithiothreitol was added to trigger the release of library compounds. MTT reporter assay was used to identify regions of reduced cell viability. From a total of approximately 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were re-synthesized and found to be cytotoxic against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line.;In one-bead-one-compound method, libraries are rapidly screened with enzyme-linked colorimetric, fluorescent, radiometric, or whole-cell binding assays. While fluorescence-based probes are powerful tools in OBOC screening, their utility is greatly limited by the intrinsic fluorescence of many commonly used solid supports, residual coupling reagents, and library compounds. Topologically partitioned TentaGel resin was derivatized with 3-nitro-tyrosine on the interior, followed by random peptide library construction. Spectral scans from a confocal microscope and fluorescence microscopy showed a dramatic decrease in the autofluorescence of blank beads and OBOC peptide libraries. Using a model ligand/receptor system, we demonstrated a marked increase in visibility of three commercially available fluorescent probes binding to quenched beads, and increased feasibility of bead sorting platform known as COPAS(TM). These data show that 3-nitro-tyrosine greatly enhances the compatibility of fluorescence-based OBOC combinatorial screening.