In mammalian cells, the process of DNA ligation is necessary during DNA replication to create an intact "lagging" strand from a series of smaller Okazaki fragments and to repair DNA strand breaks that arise either due to the direct action of a DNA damaging agent or as a consequence of DNA damage excision during DNA repair. In humans, there are three genes that encode for members of the DNA ligase family (LIG1, LIG3 and LIG4) (Ellenberger and Tomkinson in Ann Rev Biochem 77:313-338. 2008). Although these genes code for polypeptides with overlapping functions in the nucleus, the only mitochondrial DNA ligase (DNA ligase IIIα), which is essential for mitochondrial genome maintenance, is encoded by the LIG3 gene (Lakshmipathy and Campbell in Mol Cell Biol 19:3869-3876, 1999; Zong et al. in Mol Cell 61:667-676, 2016) Because mitochondria play a central and multifunctional role in malignant tumor progression, there is emerging interest in targeting key mitochondrial proteins. Notably, there is evidence in pre-clinical models that inhibitors of DNA ligase IIIα, which is frequently up-regulated in cancer, preferentially target cancer cells via their effect on mitochondria (Zong et al. 2016). Since NMR spectroscopy provides unique capabilities for identifying small molecules that bind specifically to DNA ligase IIIα versus the other DNA ligases), the backbone