Fully Synthetic Phage-Like System for Screening Mixtures of Small Molecules in Live Cells Gerardo Byk,* ,†,‡ Shirly Partouche, †,‡ Aryeh Weiss, § Shlomo Margel, † and Raz Khandadash †,‡ Deptartment of Chemistry, Laboratory of Nano-Biotechnology, and School of Engineering, Bar Ilan UniVersity, Ramat Gan 52900, Israel ReceiVed October 4, 2009 A synthetic “phage-like” system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 µm 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. Introduction High-throughput screening (HTS) of small molecules is mostly based on the panning of candidate molecules (organic small molecules, peptides) for binding to isolated target proteins (like enzymes or antibodies), usually anchored to a solid matrix that allows automation of the process, thus providing high throughput. HTS brought about an exponen- tial growth in the number of detected biologically relevant small molecules (hits). On the other hand, current HTS systems do not assay pharmacokinetic (PK) parameters such as bioavailability, stability and selectivity, which are essential for transforming the identified in vitro hits into in vivo leads. Therefore, it is of great importance to develop new screening techniques in live cells and in vivo aimed at facing and solving problems related to pharmacokinetic barriers. Panning of carrier-supported mixtures has been shown to be robust in two different and extensive approaches applied during the last twenty years: (a) Millions of molecules in “mix and split” combinatorial libraries (one bead one compound) were panned for binding to a variety of proteins, 1-5 antibodies in immunoassays, 6,7 or for catalysis of a variety of reactions in vitro. 8,9 (b) Phage display peptide libraries of millions of peptides, were panned in live cell 10 and in vivo assays 11 for binding of peptides to specific tissues or cell types without a particular known target. A common feature shared by these two techniques is that the molecules (mostly peptides) are linked to a carrier (microspheres of 100-500 µm diameter or phages of about 1 µm size). In both cases, binding of cells to the carrier is mediated by the specific molecule bound to its surface. Between these two approaches we were especially attracted by the phage display random peptide libraries screening method, which is uniquely available for liVe cells and in vivo HTS in mammalians. 11 This method can provide some of the PK parameters such as bioavailability: In an animal tumor model, molecules targeting to the tumor area were isolated from the tumor after IV administration of a phage display peptide library. Many targeting peptides have been identified by this method, and some of them have been used for drug targeting 11 and diagnostics. 12 However, this approach is limited to short natural peptides that bind to specific cells or organs. Recent studies 13 proposed the use of subcellular- sized particles for applications in live cells such as cargo delivery (streptavidine, 14 si-RNA 15 ) and sensing 16,17 (fluo- rescent dyes, 18 -galactosydase substrate 19 ). These systems * To whom correspondence should be addressed. E-mail: bykger@ mail.biu.ac.il. † Deptartment of Chemistry. ‡ Laboratory of Nano-Biotechnology. § School of Engineering. J. Comb. Chem. 2010, 12, 332–345 332 10.1021/cc900156z  2010 American Chemical Society Published on Web 03/26/2010