Chemical Tools to Study AP-Sites in DNA
Dr. Anton Granzan.
Apurinic/apyrimidinic(AP, or abasic) sites represent common DNA lesions that naturallyoccur through deglycosylation of DNA, or through glycosylase-catalyzed removalof damaged nucleobases.1 In the context of chemotherapyemploying DNA-alkylating drugs (e.g., temozolomide, or TMZ), the major DNAalkylation product, N7-methylguanine, undergoesspontaneous depurination leading to formation of AP sites, whichare instantly processed by the base excision DNA repair (BER) pathway thateliminates the drug-induced DNA damage and may lead to chemoresistance.Inhibition of BER can increase the sensitivity of cancer cells towardsDNA-alkylating drugs; consequently, significant efforts have been devoted to thedevelopment of inhibitors of AP endonuclease 1 (APE1), the key enzyme of theBER pathway that recognizes and cleaves AP sites.2 In analternative approach, we as well as others3,4 consideredindirect inhibition of BER using ligands that selectively bind to AP sites andmask them, rendering them unreactive towards APE1. Thus, we demonstrated thatcationic naphthalenophanes bind to abasic sites with high affinity andselectivity5 and inhibit the DNA-cleaving activity ofAPE1 in vitro with high efficiency, comparable to that of thebest catalytic APE1 inhibitors.6
Recently,we explored the interaction of a novel series of functionalizednaphthalenophanes with AP-sites. We demonstrated that most ligands strongly andselectively bind to AP-sites and inhibit APE1 activity in vitro,with inhibitory constants in the nanomolar range. Remarkably, the APE1inhibitory activity of ligands, as characterized by IC50 and/or Ki values,could be directly related to their affinity and selectivity to AP-sites,assessed from biophysical experiments. Moreover, some ligands demonstratedenzyme-independent, intrinsic AP-site cleavage activity due toligand-catalyzed β- and β,δ-elimination reactions at AP sites that could befine-tuned through molecular design of naphthalenophanes, as illustrated bycompounds either having an exceptionally high AP-site cleavage activity (e.g.,2,7-BisNP-S, 125-fold more efficacious than spermine) or totally devoid of thisactivity. In addition, we described an unprecedented covalent DNA–ligand adductthat was formed upon reaction of AP sites with one of the ligands.7 Finally,we elucidated the chemical structure of this unusual adduct using asmall-molecule mimic of the AP site and studied the selectivity of itsformation upon the reaction of bis-naphthalene ligands with AP site versus5-formyluracil, another aldehyde-containing nucleobase that is present in thegenome at a rate comparable with that of AP sites. On the basis of these data,we developed novel chemical tools towards selective detection of AP sites inDNA by tagging them with a fluorophore or an affinity tag.
1. P.S. Thompson, D. Cortez, DNA Repair 2020, 90,102866.
2. S.S. Laev, N. F. Salakhutdinov, O. V. Lavrik, Bioorg. Med. Chem. 2017, 25,2531.
3. J.Malina, P. Scott, V. Brabec, Nucleic Acids Res. 2015, 43,5297.
4. A.Ulhusna, A. Murata, K. Nakatani, Biochemistry 2022, inpress (10.1021/acs.biochem.2c00344)
5. N. Kotera, A.Granzhan, M.-P. Teulade-Fichou, Biochimie 2016, 128–129,133.
6. N. Kotera, F.Poyer, A. Granzhan, M.-P. Teulade-Fichou, Chem. Commun. 2015, 51,15948.
7. C. Caron, X. N. T.Duong, R. Guillot, S. Bombard, A. Granzhan, Chem. Eur. J. 2019, 25,1949.