UC San Diego

Holliday junctions (HJs) are critical intermediates in many recombination-dependent DNA repair pathways. Our lab has identified several hexameric peptides that target HJ intermediates formed in DNA recombination reactions. One of the most potent peptides, WRWYCR, is active as a homodimer and has shown bactericidal activity due in part to its ability to interfere with DNA repair proteins that act upon HJs. In order to increase the possibility of developing a therapeutic that targets DNA repair, we searched for small molecule inhibitors that were functional surrogates of the peptides. Initial screens of heterocyclic small molecule libraries resulted in the identification of several N-methyl aminocyclic thiourea inhibitors. Like the peptides, these inhibitors trapped HJs formed during recombination reactions in vitro, but were less potent than the peptides in biochemical assays and had little antibacterial activity. In this work we describe the development of functional analogs based on either WRWYCR or the thiourea inhibitors. Our focus was on symmetry in an effort to mimic the symmetry of a WRWYCR homodimer and its target, the HJ. A set of 36 pyrrolidine bis-cyclic guanidine inhibitors were synthesized, and purified candidates were found to bind protein-free HJs and to interfere with the processing of HJs by DNA repair enzymes, in vitro. In addition, they are potent inhibitors of Gram-negative and especially Gram-positive bacterial growth, in contrast to the previously identified thiourea inhibitors. We also developed a single-chain linear analog of WRWYCR that does not require homedimer formation for activity. That analog, WRWYRGGRYWRW, binds to protein-free HJs and inhibits their resolution in vitro, in addition to causing DNA damage in bacteria and inhibiting bacterial growth. Finally, we implemented a peptide labeling strategy and demonstrated the effective isotope enrichment of several peptides using 2D and 3D nuclear magnetic resonance experiments. Together these inhibitors and the labeling strategy add to a "molecular toolbox" for studying the diverse set of reactions involving HJs. Additionally, these molecules are proof-of-principle of two classes of compounds each with novel activities which may in the future be developed into a new antibiotics that will expand the available choices for therapy against drug resistant bacteria