Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor

Abstract

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing DNA ligase I were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.

DNA Ligase 1 is an essential mediator of sister chromatid telomere fusions in G2 cell cycle phase

“…To cross-validate these observations, we employed a LIG1-specific chemical inhibitor, L82 ( 23 , 46 ) (provided by Dr Alan Tomkinson, University of New Mexico, USA) to examine whether this would correspondingly prohibit sister chromatid telomere fusions. Treatment of WT and LIG3 −/− :LIG4 −/− cells with even moderate levels (5 μM compared with in vitro IC 50 of 12 ± 2 μM ( 23 )) of this inhibitor resulted in augmented proportions of G2/M phase cells (defined by pHH3 expression) compared with untreated cells assayed at 12 and 24 h time points ( Supplementary Figure S2F (i); central panels).…”

“…The LIG1 inhibitor, L82, was a kind gift of Dr Alan E. Tomkinson and Dr Tim Howes of the University of New Mexico, USA ( 23 , 46 ). L82 was reconstituted to 10 mM using DMSO and stored in aliquots at −80°C.…”

The oncogenic RNA-binding protein SRSF1 regulates LIG1 in non-small cell lung cancer

“…For this reason, DNA repair inhibition has emerged as a promising target for anticancer therapy [39]. Accordingly, DNA ligases have been suggested as therapeutic targets; in particular, specific LIG1 inhibitors as L82 and L82-G17 have been developed and applied to preclinical cell models in cancer, leading to a decrease in cell viability [40][41][42]. Of note, previous studies have pointed out that cells expressing LIG1 are more sensitive to L82-G17 inhibitor than isogenic LIG1 null cells [42].…”

“…Accordingly, DNA ligases have been suggested as therapeutic targets; in particular, specific LIG1 inhibitors as L82 and L82-G17 have been developed and applied to preclinical cell models in cancer, leading to a decrease in cell viability [40][41][42]. Of note, previous studies have pointed out that cells expressing LIG1 are more sensitive to L82-G17 inhibitor than isogenic LIG1 null cells [42]. These effects are in line with our findings, suggesting that further exploration of these molecules would help to determine their potential synergic use with the DNA damaging agents in the future.…”

Pol μ ribonucleotide insertion opposite 8-oxodG facilitates the ligation of premutagenic DNA repair intermediate

“…Protein purifications. Human DNA polymerases, namely, full-length (Met1-Ala494) pol μ, the catalytic domain (Pro132-Ala494) of pol μ, and full-length pol β, were purified as described 35,[46][47][48] . Briefly, the proteins were overexpressed in Rosetta2 (DE3) cells overnight at 16 °C.…”