Drug discovery with engineered zinc-finger proteins

Key Points C 2 H 2 zinc fingers are the most common DNA-binding motif found in the human genome. The Zif268–DNA crystal structure shows how zinc fingers interact with DNA. The fingers act as modular units (each contacting three to four base pairs of DNA) and the structure reveals which residues should be changed to alter the specificity. Researchers have engineered zinc finger proteins (ZFPs) to bind a diverse set of DNA sequences, and thereby target specific locations in the human genome, such as the promoters of therapeutically relevant genes. Exquisite specificity can be obtained with proteins that have six fingers. ZFP transcription factors (ZFP TFs) are made by combining the ZFPs with domains that either activate or repress genes. ZFP TFs are used in drug discovery to regulate genes for target validation, high-throughput screening and human therapeutics. ZFP-mediated regulation of endogenous genes could make it possible to use genes in a drug discovery application that would otherwise require securing intellectual property rights for a corresponding complementary DNA sequence. Recently, ZFP TFs have been used to promote angiogenesis in a mouse ear model, and are now undergoing further preclinical testing. Combining ZFPs with novel functional domains makes it possible to target DNA for chromatin and DNA modification, DNA cleavage and for targeted integration of exogenous DNA. Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9–18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.