A set of small molecule

A set of small molecule LigI inhibitors were identified through an in silico structure-based screen, using the atomic resolution structure of LigI complexed with nicked DNA [[18], [26]]. This screen yielded inhibitors that were selective for LigI (L82), inhibited both LigI and LigIII (L67) and inhibited all three human DNA ligases (L189). As expected, subtoxic levels of the DNA ligase inhibitors enhanced the cytotoxicity of DNA damaging agents in cancer cell lines [18]. Surprisingly, non-malignant cell lines were not sensitized to DNA damage by the DNA ligase inhibitors under similar conditions, suggesting that there are alterations in genome maintenance pathways between non-malignant and cancer Cy5 Firefly Luciferase mRNA [18]. Further studies revealed that the repair of DNA double-strand breaks is abnormal in cancer cells with elevated levels of LigIIIα and PARP1 and that these cells are hypersensitive to inhibitors that target LigIIIα and PARP1 [[22], [23], [25]]. The DNA ligase inhibitors L82, L67 and L189 are similar in that they are each composed of two 6-member aromatic rings separated by different length linkers [[18], [26]]. Here we have examined a series of related compounds in an attempt to identify determinants of activity and selectivity for LigI and LigIIIα. One of the compounds analyzed, L82-G17, is a selective, uncompetitive inhibitor of LigI. Furthermore, the activity of this compound in cell culture assays with genetically-defined cell lines indicates that it inhibits LigI function in cells.
Results
Discussion There is emerging interest in the use of DNA repair inhibitors to exploit either cancer cell-specific alterations in genome maintenance pathways or increased dependence upon a specific DNA repair pathway [38]. The abnormal expression of DNA ligases in cancer cell lines and samples from cancer patients suggest that DNA ligase inhibitors may have utility as anti-cancer agents either alone or in combination with DNA damaging agents [[17], [18], [22], [23], [25]]. Following the determination of the atomic resolution structure of LigI complexed with nicked DNA, small molecule inhibitors with differing activities against three human DNA ligases were identified by computer-aided drug design [[18], [26]]. The DNA ligase inhibitors were separated into three groups, LigI-selective, LigI/III-selective and inhibitors of all three human DNA ligases with L82, L67 and L189, respectively, serving as the prototypical compound for each of the groups. Each of these initial compounds contained two aromatic rings but had linkers that differed in length and chemical composition. Here we examined the activity of compounds that are related to L82, L67 and L189 to gain insights into determinants of activity and selectivity. The majority of LigI-selective inhibitors had a 3-atom arylhydrazone linker. In addition, the presence of at least one polar group on the right-hand ring appeared to be required for activity. While none of compounds with the arylhydrazone linker had activity against LigIII, indicating the potential utility of this linker in the rational design of LigI-selective inhibitors, the hydrazone linkage is unlikely to be compatible with drug development as this functional group has been shown to be a promiscuous metal scavenger which can lead to toxicity issues [[39], [40], [41]]. Among the compounds related to L82, L67 and L189, we identified one compound, L82-G17 that exhibited increased activity against and increased selectivity for LigI compared with L82. Notably, it has the lowest molecular weight of all arylhydrazone inhibitors with activity against LigI, suggesting that it may represent the minimal requirements for this type of inhibitor. Kinetic analysis revealed that L82-G17 is an uncompetitive inhibitor whereas L82 appears to act by both competitive and uncompetitive mechanisms. As expected, both L82 and L82-G17 enhanced the formation of stable complexes of LigI with nicked DNA. Since L82-G17 inhibits phosphodiester bond formation but not formation of the DNA-adenylate, we conclude that L82-G17 is a step 3 inhibitor that stabilizes the complex formed by non-adenylated LigI with the DNA-adenylate reaction intermediate. Although L82-G17 acts by the same mechanism as topoisomerase (topo) inhibitors, such as camptothecin, and a subset of Poly(ADP-Ribose) Polymerase (PARP) inhibitors that trap topo I-DNA and PARP1-DNA complexes, respectively [[42], [43]], we did not observe an increase in the amount of LigI associated with chromatin in cells exposed to L82-G17 (data not shown). This may be because LigI, unlike PARP1, does not have robust DNA binding activity [[42], [44], [45], [46]] and is recruited to sites of DNA replication via an interaction with PCNA [[47], [48]]. The increased cytotoxicity of L82-G17 compared with L82 is consistent with the studies showing that PARP inhibitors that trap PARP1-DNA complexes are more cytotoxic than PARP inhibitors that do not [[42], [45]]. It is presumed that DNA-protein complexes, even when non-covalent, cause problems because of collisions with either the DNA replication or transcription machinery. In contrast to the trapped DNA-protein complexes formed by topoisomerases and PARPs, the replication machinery is unlikely to encounter trapped LigI-PCNA complexes as these will predominantly formed behind the fork on the lagging strand.