Isolation of biologically active Topoisomerase I-binding peptides via phage display

Topoisomerase I (top1) is a critical component of replication, transcription, mRNA splicing, recombination and DNA repair machinery. Top1 is a kinase, a DNA unwinding enzyme and a transcription regulator. These activities transpire through discrete and not completely resolved covalent (with DNA) and noncovalent (with proteins, DNA and possibly RNA) interactions. Top1, through its DNA binding activity, can recognize and cleave damaged DNA and form 'cleavable complexes' with top1 poisons. This cleavable complex formation is lethal for certain human neoplasia. The top1poisons utilizing camptothecin pharmacophore are one of the most active classes of chemo-therapeutics for colo-rectal and ovarian cancers. Novel top1 poisons and suppressors are under development. New leads for top1modulators are in demand. Phage display is a decade old technique for finding novel peptide ligands with affinity for target molecules. A phage display library is a collection of tens of millions of clones of an M13 phage, each of which displays a particular random polypeptide sequence near the N-terminus of a coat protein. The power of the technique lies in the fact individual phage clones with high affinity for a target protein can be selected, amplified, assayed, and sequenced. The hypothesis to be tested was that top1-targeted phage display technology can be applied to find new ligands for top1 and that these binding peptides would affect the enzyme activity. In this work, full-length human top1 has been screened with a 15mer phage display library and top1 binding peptides discovered. Several novel peptides have been demonstrated to bind top1, alter top1 residence on DNA, and affect DNA directed top1 catalytic activity. Additionally one peptide has been discovered that potentiates 9-amino-camptothecin toxicity in cultured tumor cells and xenografted human tumors.