Jeffamine derivatized TentaGel beads and poly(dimethylsiloxane) microbead cassettes for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound combinatorial small molecule libraries

Abstract

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.

Figure 1

Immobilization of TG beads using photo-patterned microfabricated device. A) Si wafer substrate is coated with SU-8 2150 negative tone ultra thick photoresist and photo patterned. B) After developing, C) the device mold is coated with PDMS prepolymer D) to construct the reverse replica PDMS cast which can be loaded with polystyrene beads. E and F) PDMS cast consisting of arrayed “wells” after loading of 120 μm TG beads and swelled in 70% EtOH.

Figure 2

Synthesis of (Fmoc)₂-Jeffamine-acid linker. (a) Succinic anhydride (1 eq.), Dioxane, 0 °C; (b) Fmoc-OSu, Dioxane/H₂O, Na₂CO₃.

Figure 3

Synthesis of Jeffamine-derivatized TG beads displaying octa-arginine/FITC and LLP2A. Reagents: (a) TG resin was swollen in H₂O, 48 h; Fmoc-OSu, DCM/Ether, DIEA; Boc₂O, DIEA, DMF; (b) 20% Piperidine/DMF; Fmoc₂-JA-OH (1), HBTU, HOBt, DIEA, NMP; this step was repeated once (c) 20% Piperidine/DMF; a mixture of Fmoc-OSu and Alloc-OSu (1:1), DIEA, DMF; (d) 20% Piperidine/DMF; Fmoc-[S-S]-OH (2), HOBt, DIC, DMF; (e) 20% Piperidine/DMF; Fmoc-K(Dde)-OH, HOBt, DIC, DMF; (f) 20% Piperidine/DMF; Fmoc-R(Pbf)-OH, HOBt, DIC, DMF (repeated 7 more times); (g) 20% Piperidine/DMF; Boc₂O, DIEA, DMF; (h) Pd(PPh₃)₄, PhSiH₃, DCM; Fmoc-Ach-OH, HOBt, DIC, DMF; (i) 20% piperidine/DMF; Fmoc-Aad(OtBu), HOBt, DIC, DMF; (j) 20% piperidine/DMF; HOBt, DIC, DMF; Fmoc-K(Alloc); (k) 20% piperidine/DMF; 2-{3-[(2-toluidinocarbonyl)amino]phenyl} acetic acid, HOBt, DIC, DMF; (l) Pd(PPh₃)₄, PhSiH₃, DCM; (E)-3-(3-pyridinyl)-2-propenoic acid, HOBt, DIC, DMF; (m) 2% hydrazine/DMF; FITC, DIEA, NMP;TFA:Thioanisole:Phenol:H₂O:TIS (84:5:5:5:1) (v/v).

Figure 4

Release of octa-arginine-FITC from a bead displaying LLP2A, the lymphoma cell capturing ligand (see Figure 3). Compound release was triggered by addition of 0.1% DTT into the medium. (A, B) Molt-4 cells were first incubated and bound to the bead. Surface release of octa-arginine–FITC was facilitated by the addition of 0.1% DTT to the culture medium. (C, D) Molt4 cells were (C) suspended in 1% low melt agarose and coated over Petri dish and (D) seeded over the PDMS microbead cassette and coated with a thin layer of 1% low melt agarose. Fluorescent micrographs were obtained 10 minutes after the addition of DTT (488nm/520nm ex/em).

Figure 5

Synthesis of an encoded OBOC small molecule releasable library. Reagents: (a) (3), 20% Piperidine/DMF; Fmoc-[S-S]-OH (2), HOBt, DIC, DMF; (b) TFA/DCM (1:1); Fmoc-K(Dde)-OH, HOBt, DIC, DMF; (c) 20% Piperidine/DMF; Fmoc-X-OH, HOBt, DIC, DMF (repeated 1 more time); (d) 20% Piperidine/DMF; H₂O, 48 h; Fmoc-OSu, DCM, Ether DIEA; Boc₂O, DIEA, DMF; (e) 2% hydrazine/DMF; R-COOH, HOBt, DIC, DMF; (f) TFA:Thioanisole:Phenol:H₂O:TIS (82.5:5:5:5:2.5) (v/v).

Figure 6

In-situ release of surface bound compound from dipeptide library and resulting MTT cell viability assay. A) Jurkat cells were suspended in BD Matrigel™ and seeded over loaded bead cassette and cultured to desired confluency before compound release was initiated with the addition of 0.1% DTT. B-D) After 48 hr treatment period, cell viability was measured topologically throughout the cassette with the addition 0.05% MTT reagent (terazolium bromide). Beads with uniform regions of metabolically inactive cells were selected and sequenced.

Figure 7

Positive compounds identified from initial cell killing screen (LTS1, LTS2 and LTS3) were synthesized on TG resin with disulfide cleavable linker and Rink cleavable resin resulting in sulfhydryl (-SH) and amide (-CONH₂) forms of the three compounds, respectively.

Figure 8

Quantitative cell viability assays plotted as (y) difference in percent reduction of alamarBlue® substrate vs. (x) compound concentration using two cancer cell lines.