Small molecule inhibitors of DNA repair nuclease activities of APE1

Abstract

APE1 is a multifunctional protein that possesses several nuclease activities, including the ability to incise at apurinic/apyrimidinic (AP) sites in DNA or RNA, to excise 3'-blocking termini from DNA ends, and to cleave at certain oxidized base lesions in DNA. Pre-clinical and clinical data indicate a role for APE1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs, particularly monofunctional alkylators and antimetabolites. In an effort to improve the efficacy of therapeutic compounds, such as temozolomide, groups have begun to develop high-throughput screening assays and to identify small molecule inhibitors against APE1 repair nuclease activities. It is envisioned that such inhibitors will be used in combinatorial treatment paradigms to enhance the efficacy of DNA-interactive drugs that introduce relevant cytotoxic DNA lesions. In this review, we summarize the current state of the efforts to design potent and selective inhibitors against APE1 AP site incision activity.

Fig. 1

The base excision repair response. Left are the five major enzymatic steps of short-patch BER. Other sub-pathways of BER exist (e.g., long-patch), and are described in greater detail elsewhere [80, 81]. Typically, BER is initiated by removal of a damaged or inappropriate base (yellow star) through the action of a DNA glycosylase, which hydrolyzes the N-glycosidic bond that links the base to the sugar-phosphate backbone, generating an AP site. APE1, the major mammalian AP endonuclease, then cleaves the DNA backbone immediately 5′ to the abasic lesion. The subsequent steps of BER—gap-filling to replace the missing nucleotide, removal of the 5′-deoxyribose phosphate (dRP), and sealing of the final nick—are performed primarily by DNA polymerase β and a complex of XRCC1 and DNA ligase 3α, although back-up mechanisms exist [82]. As amplified within the red circle, the hydrolytic 2′-deoxyribose AP site exists primarily (at ~99%) in a ring-closed form (left), in one of two racemeric hemiacetal arrangements, α or β, which are in an equilibrium mixture. Reduction of the ring-closed AP site can produce a ring-opened aldehyde form (middle). This ring-opened form is susceptible to hydration, generating a hydrated aldehyde AP site (right) ([83] and references therein)

Fig. 2

APE1 primary sequence conservation and 3-dimensional structure. Top is a linear, schematic comparison of the human APE1 and E. coli exonuclease III (Xth) proteins showing the conserved catalytic residues: E96, D210, D283 and H309 in APE1. The nuclear targeting region (shaded portion, NLS), the acetylation sites (K6 and K7, dashed line), and a critical redox regulatory cysteine residue (C65) is denoted within the unique N-terminal portion of the human protein [15, 16]. The amino acid length of the two proteins is indicated to the right in italics. Below is a ribbon diagram of the human APE1 protein. β-strands are shown in cyan and α-helices in red, with the four-layered α/β sandwich fold apparent. The proposed major groove DNA recognition loop, α8, is denoted, as is the minor groove recognition loop, α11. Coordinates were from 1BIX [84]

Fig. 3

APE1 catalytic reaction mechanism and 3′-damage substrates. In the reaction shown (left), H309 acts as the general base and abstracts a proton from a water molecule to generate the active site nucleophile [85]. D283 forms a hydrogen bond with H309 to help stabilize the positive charge that develops upon proton abstraction. The metal ion bound by E96 interacts with the negatively charged phosphate group and aids in nucleophilic attack by the hydroxyl, and in one model also serves to stabilize the leaving group [84]. Alternatively, D210, whose pKa is potentially altered by extensive hydrogen bonding, plays the role of the Lewis base and protonates the 3′ leaving group [86]. Additional reaction mechanisms have been proposed, but are not depicted or described herein [, –89]. To the right are 3′-damage substrates of APE1. Oxidation of C-4′ results in fragmentation of the deoxyribose, causing strand breakage and the formation of a 3′-phosphoglycolate ester or a 3′-phosphate; 3′-phosphate groups can also be generated by bifunctional DNA glycosylases [90]. The α,β-unsaturated aldehyde is the β-elimination product of a bifunctional DNA glycosylase. Troxacitabine is an L-stereoisomeric, chain-terminating nucleoside analog that is a primary substrate for APE1 excision activity [27]

Fig. 4

APE1 screening assay principle. A deoxyoligonucleotide containing an internal tetrahydrofuran abasic site analog [91] and a 5′ fluorophore is annealed to a complementary strand with a 3′ quencher to create a double-stranded DNA substrate. The close proximity of the fluorophore and the quencher results in a dampened signal upon light excitation. Following DNA backbone cleavage by APE1, a short deoxyoligonucleotide fluorophore-labeled product (typically around 4–6 nucleotides) is spontaneously released from the remaining DNA fragment possessing the quencher, causing the fluorophore emission to increase. F can be any fluorophore, and Q any compatible quench moiety. The APE1 incision site is indicated by the arrow. The right side of the duplex is not complete, as denoted by the squiggly lines

Fig. 5

Identified inhibitors of APE1 endonuclease activity. Shown are the identifiers, chemical features, and concentrations of compound reported to inhibit 50% of APE1 incision activity in vitro (in parentheses). IC₅₀s were obtained from the following articles, [33, 34, 53, 57, 60, 66], and compounds 1, 17 and 21 are described in [57]