Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development.

Targeting multiple chorismate-utilizing enzymes with a single inhibitor: validation of a three-stage design.

Chorismate-utilizing enzymes are attractive antimicrobial drug targets due to their absence in humans and their central role in bacterial survival and virulence. The structural and mechanistic homology of a group of these inspired the goal of discovering inhibitors that target multiple enzymes. Previously, we discovered seven inhibitors of 4-amino-4-deoxychorismate synthase (ADCS) in an on-bead, fluorescent-based screen of a 2304-member one-bead-one-compound combinatorial library. The inhibitors comprise PAYLOAD and COMBI stages, which interact with active site and surface residues, respectively, and are linked by a SPACER stage. These seven compounds, and six derivatives thereof, also inhibit two other enzymes in this family, isochorismate synthase (IS) and anthranilate synthase (AS). The best binding compound inhibits ADCS, IS, and AS with K(i) values of 720, 56, and 80 microM, respectively. Inhibitors with varying SPACER lengths show the original choice of lysine to be optimal. Lastly, inhibition data confirm the PAYLOAD stage directs the inhibitors to the ADCS active site.

A spiroisoxazolinoproline-based amino acid scaffold for solid phase and one-bead-one-compound library synthesis.

An efficient, multigram synthesis of a spiroisoxazolinoproline-based amino acid, 7, requiring minimal purification, delivering good cis:trans diastereoselectivity (approximately 1:4), and providing good yields is reported. Surface-bound studies of the reduction of an arylnitro group in the presence of an isoxazoline ring with tin(II) dichloride dihydrate were undertaken to confirm the stability of the isoxazoline ring. Full derivitization of this spiroisoxazolinoproline-based amino acid scaffold was performed during the synthesis of a sample library with high yields and high purity that validated the efficiency of the chemistry that was employed in resin-bound library synthesis. A 129,600 member one-bead-one-compound (OBOC) library based on the scaffold 7 was synthesized utilizing a dual amino acid encoding method and bifunctionalization of TentaGel resin.

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors.

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.

Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries.

One-bead one-compound combinatorial chemistry together with a high-throughput screen based on fluorescently labeled enzyme allowed the identification of slow binding inhibitors of human serine racemase (hSR). A peptide library of topographically segregated encoded resin beads was synthesized, and several hSR-binding compounds were isolated, identified, and resynthesized for further kinetic study. Of these, several showed inhibitory effects with moderate potency (high micromolar K(I)s) toward hSR. A clear structural motif was identified consisting of 3-phenylpropionic acid and histidine moieties. Importantly, the inhibitors identified showed no structural similarities to the natural substrate, L-serine. Detailed kinetic analyses of the properties of selected inhibitors show that the screening protocol used here selectively identifies slow binding inhibitors. They provide a pharmacophore for the future isolation of more potent ligands that may prove useful in probing and understanding the biological role of hSR.