Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One

Abstract

Induction of protein--protein interactions is a daunting challenge, but recent studies show promise for small molecules that specifically bring two or more protein molecules together for enhanced or novel biological effect. The first such bifunctional molecules were the rapamycin- and FK506-based "chemical inducers of dimerization", but the field has since expanded with new molecules and new applications in chemical genetics and cell biology. Examples include coumermycin-mediated gyrase B dimerization, proteolysis targeting chimeric molecules (PROTACs), drug hybrids, and strategies for exploiting multivalency in toxin binding and antibody recruitment. This Review discusses these and other advances in the design and use of bifunctional small molecules and potential strategies for future systems.

Figure 1

A generic bifunctional molecule brings two protein molecules together to induce a biological effect.

Figure 2

Overview of the yeast three-hybrid technique. A DNA-binding domain (DBD) is fused to Protein X. A heterobifunctional small molecule binds to Protein X, linking it to Protein Y, which in turn is fused to a transcriptional activation domain (AD). The presence of the small molecule brings the two protein fusions together, activating transcription of a reporter gene.

Figure 3

Bifunctional molecules used in cell-free studies. Colored shapes represent the regions of each small molecule that bind to the proteins listed. DHFR, dihydrofolate reductase; PLA2, phospholipase A2; SAP, serum amyloid P.

Figure 4

Bifunctional molecules used in techniques requiring genetic manipulation. Colored shapes represent the regions of each small molecule that bind to the fusion proteins listed. GFP, green fluorescent protein; ER, estrogen receptor; DHFR, dihydrofolate reductase; GR, glucocorticoid receptor.

Figure 5

Bifunctional molecules used in techniques that do not require genetic manipulation. Colored shapes represent the regions of each small molecule that bind to the proteins listed. ER, estrogen receptor; MetAP-2, methionine aminopeptidase 2; GFP-AR, green fluorescent protein-androgen receptor; VHL, von Hippel Lindau; AHR, aryl hydrocarbon receptor.

Figure 6

Overview of the PROTACS system. A PROTAC molecule brings a target protein into contact with an E3 ubiquitin ligase, prompting transfer of ubiquitin (Ub) from an E2 ubiquitin conjugating enzyme, leading to polyubiquitination of the target protein and degradation by the 26S proteasome.

Figure 7

Harnessing immune recognition with small molecules. A small molecule ligand for the target protein (brown), known to be expressed on the cells of interest, is linked to an epitope that is recognized by endogenous antibodies (purple).

Figure 8

Drug hybrids combine two pharmacophores in a single molecule. Functional moieties are indicated by colored shapes. MAO, monoamine oxidase; GABA, γ-aminobutyric acid.