Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

Abstract

Aims:Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.Methods:An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.Results:Several specific APE1 inhibitors were isolated by this approach. The IC50 for APE1 inhibition ranged between 30 n and 50 μ. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.Conclusions:Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

Human Apurinic/Apyrimidinic Endonuclease 1

“…The ''low affinity'' attribute may stem from the fact that small molecules are unable to replicate the ''high affinity'' binding contacts seen in the APE1/AP-DNA complex, which spans roughly 7-8 bp in length. Indeed, in an extensive structure-function activity relationship study, the introduced modifications only slightly increased the effectiveness of the analogs against (7,131,142,167), confirming that the nuclease function(s) of the protein plays a critical role in repairing alkylative DNA damage, presumably abasic sites. Future studies will need to examine the utility of the most promising compounds in both combinatorial and synthetic lethal treatment paradigms, and may need to employ covalently linked, small molecules that bind simultaneously multiple functional sites of APE1.…”

“…Some of the earliest work, published by Kelley and colleagues, revealed that APE1 provides resistance against not only MMS and hydrogen peroxide, but also the anti-cancer agents thiotepa, etoposide, and ionizing radiation (221). Through the efforts of many labs since, there is a general consensus emerging that indicates a role for APE1 in dictating cellular responsiveness to clinically relevant alkylators, most notably temozolomide (7,14,137,142,193), and a subset of chain-terminating nucleoside analogs, most prominently troxacitabine (114,137,180). Such a role is consistent with the biochemical activities of APE1, as alkylating agents, particularly mono-functional versions, induce a high number of abasic sites through both direct and indirect mechanisms, and certain anti-metabolites generate exonuclease 3¢-end substrates.…”

Human Apurinic/Apyrimidinic Endonuclease is a Novel Drug Target in Cancer

“…Interestingly, the inhibitory effect was significantly increased in the Asp148Glu APE1 polymorph, previously implicated in cancer predisposition (see Section 5.4. APE1 polymorphisms and cancer susceptibility) (Mohammed et al 2011). A number of other groups have been working on the development of APE1 DNA repair inhibitors.…”

Targeting DNA base excision repair: a new strategy for personalised cancer therapy

“…5 An alternative approach has utilised knowledge of the crystal structure of the APE1 active site to strategically design inhibitor templates that are applied in a virtual screen of much larger compound libraries. 6 Using these strategies, a number of potential inhibitors have been identified and are currently undergoing preclinical evaluation.…”