A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.

Herein we present a new application of a recently demonstrated fully synthetic "phage-like" system for screening of combinatorial mixtures in a live cell assay. The new application includes the direct synthesis of peptides and combinatorial libraries on 2 µm cross-linked mono-dispersed microspheres bearing a panel of fluorescence tags. Their characterization using classical chemical analysis as well as biological recognition of the synthesized sequences on the microspheres by specific antibodies, demonstrate the robustness of the system. Two biased positional combinatorial peptide libraries derived from peptide DUP-1 were synthesized and screened for affinity to prostate cancer PC-3 cell line as compared to DUP-1, a peptide with good affinity for this cell line. The best sub-library was deconvoluted and mixtures of deconvoluted peptides on microspheres were screened for identifying the sequences with the best affinity for cells. The best peptide, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala with high affinity for PC-3 cells, was individually synthesized and validated in an independent binding assay. Finally, the cellular fate of DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala in PC-3 cell line is demonstrated using confocal laser scanning microscopy. Overall, these results demonstrate that the synthetic phage-like system recently assessed for screening mixtures of small organic molecules, can be also used for synthesis and screening of combinatorial libraries of peptides in live cell assays.