A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

Abstract

DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP-DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP-DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.

Psoralen Derivatives with Enhanced Potency

“…Dis-coverX's KINOMEscan ® or KiNativ TM in situ kinase profiling should be appealing, straightforward technologies to assess this theory (54)(55)(56). For a proteome-wide approach, immunoprecipitation or fluorescent labeling approaches followed by MS profiling should be capable of identifying additional protein targets of psoralens (57). Using such methods, compounds like 8F and others emerging from our screen may be promising leads to begin exploring these non-DNA-mediated mechanisms of action.…”

Physicochemical Investigation of Psoralen Binding to Double Stranded DNA through Electroanalytical and Cheminformatic Approaches

“…Nonetheless, furanocoumarin chemical structures are relatable to several small ligands, which may confer such a wide range of possible biological targets (Figure 1). Despite the acknowledged biological activities of furanocoumarin derivatives such as PSO, many reports attribute remarkable deoxyribonucleic acid (DNA)-binding properties to this compound, which may be involved in its therapeutic effects [11][12][13][14][15]. In this sense, several authors investigated PSO-DNA binding through spectrophotometric methods, as well as chemoinformatic approaches, resulting thence in the understanding that covalent linking is involved in PSO anchorage on DNA strands through PSO-Thymine adducts [11,13,15].…”

Chemical Analysis of DNA Damage

“…Some analytical methods were also developed for the investigation of repair processes or potential toxicity from exogenous exposures by characterizing ICLs or AP sites. Evison et al 129 prepared 8-propargyloxypsoralen (8-POP) that could efficiently generate DNA ICLs in cells upon UVA irradiation. 8-POP harbors an alkyne handle, which allows post-labeling with an azide-tagged fluorescent reporter via click chemistry.…”