Dataset Information

High-Throughput Sorting and Placement of One-Bead-One-Compound (OBOC) Libraries from Bulk to Single Wells in Organic Solvent.

ABSTRACT: One-bead-one-compound (OBOC) solid-phase combinatorial chemistry has been used extensively in drug discovery. However, a major bottleneck has been the sorting of individual beads, while still swollen in organic solvent, into individual wells of a microwell plate. To solve this problem, we have constructed an automated bead sorting system with integrated quality control that is capable of sorting and placing large numbers of beads in bulk to single wells of a 384-well plate, all in an organic solvent. The bead sorter employs a unique, reciprocating fluidic design capable of depositing 1 bead every 1.5 s, with an average accuracy of 97%. We quantified the performance of this instrument by sorting over 8500 beads, followed by cleaving the conjugated compound and confirming the chemical identity of each by liquid chromatography/mass spectrometry (LC/MS). This instrument should enable more efficient screening of combinatorial small molecule libraries without the need to dry beads or otherwise change the chemical environment.

SUBMITTER: Bordo MW

PROVIDER: S-EPMC4428994 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Publications

High-Throughput Sorting and Placement of One-Bead-One-Compound (OBOC) Libraries from Bulk to Single Wells in Organic Solvent.

Bordo Mark W MW Oketokoun Rafiou R Cross Conor J CJ Bao Kai K Lee Jeong Heon JH Feygin Ilya I Chang Alex B AB Frangioni John V JV Choi Hak Soo HS

ACS combinatorial science 20150422 5

One-bead-one-compound (OBOC) solid-phase combinatorial chemistry has been used extensively in drug discovery. However, a major bottleneck has been the sorting of individual beads, while still swollen in organic solvent, into individual wells of a microwell plate. To solve this problem, we have constructed an automated bead sorting system with integrated quality control that is capable of sorting and placing large numbers of beads in bulk to single wells of a 384-well plate, all in an organic sol ...[more]

PMID: 25879633

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Project description:A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-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 trifunctional 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 ultrahigh-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 min) 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 h 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 resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Dataset Information

High-Throughput Sorting and Placement of One-Bead-One-Compound (OBOC) Libraries from Bulk to Single Wells in Organic Solvent.

Publications

High-Throughput Sorting and Placement of One-Bead-One-Compound (OBOC) Libraries from Bulk to Single Wells in Organic Solvent.

Similar Datasets

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