Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 ( ∼ 440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. 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 coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 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 (IC50 50-150 μ M) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultra high-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays. from these beads and the uptake of the fluorescent cell-penetrating peptide into adjacent living cells. An encoded OBOC combinatorial small molecule library was also constructed on these beads; and when used in conjunction with the highly efficient microbead cassette as the cell-based screening platform, we were able to identify potent cytotoxic compounds against malignant T-lymphoid cells. mol), 5 NMP to the resin beads. The reaction mixture agitated the ninhydrin test negative. resin three times each and then followed by Fmoc This step was repeated once. A of Alloc-OSu mol), DIEA 216 mol) and DMF added to resin beads and the reaction mixture was agitated 30 minutes. The obtained and Alloc inside-Boc-trifunctional beads washed with DCM mL × DMF mL × DCM mL each, followed Fmoc A mixture Fmoc-disulfide linker mg, 162 mol), 162 mol), mol), and 5 mL DMF was added to the resin beads. The reaction mixture was agitated until the ninhydrin test negative. The DMF mL and DCM mL by Fmoc A Fmoc-K