Biology subjects

Dhakar, S. S.

Publications and source records attributed to Dhakar, S. S..

3 recordsLinked to original sources

bioRxiv2025

Automodification of N-terminal serine residues in PARP2 impacts PARP2 release from DNA damage sites

ADP-ribosyltransferases PARP1 and PARP2 are involved in DNA repair mechanisms and play a major role in detecting DNA damage. PARP1/2 enzymes transfer the ADP-ribosyl moiety from NAD+ to DNA damage proteins, histones and to itself, activating the DNA repair cascade. Recent studies have shown that Histone PARylation Factor (HPF1) forms a joint active site with the catalytic domain of PARP1/2 and alters their target modification site from glutamate/aspartate towards serine. In this work, we have identified key serine residues within the N-terminus of full-length PARP2 that are the main targets of PARP1/2 automodification. We demonstrated this using site-directed mutagenesis, gel-based PARylation assays of automodification reactions, and by measuring the release of PARP2 from the DNA damage site using a fluorescence polarization assay. We show that in the presence of HPF1, PARP2 serine 8 and serine 73 are predominantly ADP-ribosylated and serine 8, which is present in both human PARP2 isoforms, is the major site for PARP2 automodification. Our results provide insight into the mechanistic role of the N-terminus of PARP2 in the PARylation-dependent release of PARP2 from DNA damage sites.

biochemistry↗

bioRxiv2024

Substitutions at the C-8 position of quinazolin-4-ones improve the potency of nicotinamide site binding tankyrase inhibitors

Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and some small molecule inhibitors have already been developed. These inhibitors typically interact with the nicotinamide binding site and extend along the NAD+ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as promising scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both nitro- and diol-substituents exhibit intriguing inhibition of TNKS2, with compound 49 displaying an IC50 of 65 nM, while compound 40s IC50 value is 14 nM. Both analogues show efficacy in cell assays and attenuate the tankyrase-controlled Wnt/{beta}-catenin signaling with sub-micromolar IC50. When tested against a wider panel of enzymes, compound 40 displayed high selectivity towards tankyrases, whereas 49 also inhibited other PARPs. The results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.

biochemistry↗

bioRxiv2023

High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the automodification site from Aspartate, Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds, Dimethylacrylshikonin and Alkannin, with {micro}M inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.

biochemistry↗