Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.

Computational chemistry has now been widely accepted as a useful tool for shortening lead times in early drug discovery. When selecting new potential drug targets, it is important to assess the likelihood of finding suitable starting points for lead generation before pursuing costly high-throughput screening campaigns. By exploiting available high-resolution crystal structures, an in silico druggability assessment can facilitate the decision of whether, and in cases where several protein family members exist, which of these to pursue experimentally. Many of the algorithms and software suites commonly applied for in silico druggability assessment are complex, technically challenging and not always user-friendly. Here we applied the intuitive open access servers of DoGSite, FTMap and CryptoSite to comprehensively predict ligand binding pockets, druggability scores and conformationally active regions of the NUDIX protein family. In parallel we analyzed potential ligand binding sites, their druggability and pocket parameter using Schrödinger's SiteMap. Then an in silico docking cascade of a subset of the ZINC FragNow library using the Glide docking program was performed to assess identified pockets for large-scale small-molecule binding. Subsequently, this initial dual ranking of druggable sites within the NUDIX protein family was benchmarked against experimental hit rates obtained both in-house and by others from traditional biochemical and fragment screening campaigns. The observed correlation suggests that the presented user-friendly workflow of a dual parallel in silico druggability assessment is applicable as a standalone method for decision on target prioritization and exclusion in future screening campaigns.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging ‘open’ principles to develop a pharmacological tool for every human protein.

8‐oxoGuanine DNA glycosylase 1 (OGG1) is the first known target of organocatalytic switches (ORCAs), which rewrite the biochemical function of the enzyme through redirection of its preferred substrate from 8‐oxoG to AP sites. Previously, different ORCA chemotypes were shown to enhance the operational pH window for OGG1, possibly through direct involvement in proton transfer events during DNA strand cleavage. Accordingly, compound pK_a is a crucial and necessary consideration for the identification and application of future OGG1 ORCAs. Here, we identify a minimal structure of organocatalytic switches–4‐anilino pyridines and 6‐anilino pyrimidines–which are dimethyl‐amino‐pyridine (DMAP)‐type Brønsted bases binding the active site of OGG1. Systematic interrogation of compound basicity through modulation of electron‐withdrawing (EWG) and electron‐donating (EDG) substituents reveals that a pK_a less or equal to the assay pH is a viable parameter for prediction of compound activity. The lead structure (AC₅₀ 13 nM, pK_a 7.0) was then identified as a potent scaffold from a screen in a patient‐derived 3D model of metabolic dysfunction‐associated steatohepatitis (MASH), where it reduced hepatic fibrosis by 35%. Collectively, these findings deepen the knowledge of this novel modulator class, with important implications for future enzyme targets and probe development.