A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets - PubMed (original) (raw)

A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets

J Huang et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein-protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein-protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These "nanodroplets" contain defined media, cells, and one or more beads containing approximately 100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein-protein interactions in living cells or organisms.

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Figures

Figure 1

Figure 1

General schematic of the inducible (small molecule) reverse two-hybrid system designed to detect small molecule inhibitors of protein–protein interactions. The expression of the interacting proteins is controlled by the GAL1 promoter, which is repressed in Glc. After Gal induction, the two interacting protein partners are synthesized, and their association in turn induces the synthesis of a toxic gene product, leading to death, unless an inhibitor of the protein–protein interaction is present in the cell. Only cells with this disruption should be selected.

Figure 2

Figure 2

(A) Reporter constructs used for homologous recombination. The LexAop_4–_SPO13

tata

–URA3 reporter construct contains four LexA operators upstream of a SPO13 promoter–URA3 fusion reporter gene. Nucleotide numbering is relative to the translation start codon ATG, where A is +1. Red numbering represents nucleotides from the URA3 gene, and green represents those from the SPO13 gene. (B) Schematic representation of reporter yeast strains.

Figure 3

Figure 3

Vectors for inducible two-hybrid protein expression. Expression of the two interacting proteins is under the control of the GAL1 promoter. Expression vectors contain antibiotic markers for selection. Both centromeric and 2-μ vectors were designed.

Figure 4

Figure 4

FK506 specifically interferes with the interaction between FKBP12 and the activin type I receptor R1 as detected in the small molecule reverse two-hybrid system. Sc* refers to synthetic complete (Sc) medium without any sugar; gal/raf, 2% galactose plus 1% raffinose; and glc, 2% glucose. (A) Phenotypes of R1C–FKBP12 interaction and its dissociation by FK506. Yeast transformants with both vectors appear within 24–36 h on Sc-H-W plates. Colonies were picked and patched on various selective plates for reported gene assays. Each pairwise transformation was analyzed by six independent transformants, three of which are shown here. (B) FK506-mediated 5-FOA resistance is specific to R1C–FKBP12 transformants. (C) Detection of the FK506 effect in liquid media using arrayed nanodroplets (≈200-nl vol) (36). The droplets containing yeast, medium, and FK506 beads were formed in polydimethylsiloxane plastic molded to contain wells 40 μm in depth, 1 mm in diameter, and 250 μm apart from each other.

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