RESEARCH INTERESTS

Mechanism and Control of the ADP-ribosylation Cycle    

Residu-specific ADP-ribosylation is a reversible post-translational modification that regulates DNA repair, gene expression, and cell fate. Targeting poly- (PAR) and mono-ADP-ribosylation (MAR) metabolism and signaling emerges as a promising strategy for tumor-specific therapy and precision cancer medicine. Research projects in our lab are focused on understanding the structure, mechanism, and function of ADP-ribosylation reversal enzymes and downstream signaling proteins/protein complexes. We are particularly interested in developing small-molecule modulators of those proteins as novel anti-cancer therapeutics. We use multi-disciplinary research tools, including X-ray crystallography, time-resolved fluorescence resonance energy transfer (TR-FRET), small-angle X-ray scattering (SAXS), high-throughput screening (HTS), various biochemical assays, and chemical synthesis, to investigate 1) how proteins and domains communicate for their specialized functions, and 2) how they specifically recognize small-molecule ligands and substrates.

1. Reversal of residue-specific ADP-ribosylations by ADP-ribosyl-acceptor hydrolases.

    In response to genotoxic stresses, PAR polymerase 1 (PARP1) PARylates itself and its target proteins. PARP inhibitors specifically kill BRCA-deficient breast and ovarian tumors. However, uncontrolled accumulation of PAR results in PAR-dependent cell death (also called parthanatos). In human system, ADP-ribosyl-acceptor hydrolase 3 (ARH3) and PAR glycohydrolase (PARG) function in tandem to reverse PARylation. Our research is focused on the mechanism and control of these two PAR turnover enzymes.

    In addition to PARylation, ARH3 specifically hydrolyzes the serine-specific mono(ADP-ribosyl)ation, the last step to completely reverse PARylation, and O-acetyl-ADP-ribose. Serine ADP-ribosylation emerges as the major modification upon DNA damage. Therefore, ARH3 is a di-Mg-containing multi-functional enzyme that regulates two major NAD+-dependent cellular signaling pathways. In collaboration with Joel Moss in NHLBI (NIH), we study how the bimetallic Mg center of ARH3 mediates specific substrate hydrolysis and how ARH3 activity is regulated (Pourfarjam et al., JBC 2018; Figure 1). We are collaborating with Joel Moss (Deputy Chief and Senior Scientist in NHLBI/NIH) and John Tanner (Director of Structural Biology and Chemistry in MD Anderson & LBNL), to better understand the structure, mechanism, and function of ARH3 during DNA damage responses.

Figure 1. Overall structure of human ARH3 bound to ADP-ribose (product) and magnesium. We identified a flexible Glu41-flap as a gate that controls substrate entrance to the bimetallic Mg center of ARH3 (Pourfarjam et al., 2018 & 2021, JBC).

2. Chemical modulators of the ADP-ribosylation cycle as new tumor-selective therapeutics.

    PAR turnover emerges as the promising drug target for cancer-selective therapy. We are highly interested in developing specific inhibitors of PAR turnover enzymes using our established high-throughput TR-FRET PAR turnover assays that quantitatively monitor PAR turnover activity in real time (Fig. 2).

    We have successfully identified specific and selective PARG inhibitors through chemical library screening using our high-throughput TR-FRET PARG assay assay. The novel methyl-xanthine derivative JA2131 shows extensive interactions with human PARG and causes replication fork stalling and sensitizes cancer sells (Fig. 3).

Figure 3. Identification of JA2131 as a selective and potent PARG inhibitor. (Houl et al., 2019, Nature Communications)

3. ADP-ribosylation-mediated DNA damage response and the crosstalk between ADP-ribosylation and ubiquitination.

    PARylation is a key regulator for early stage of DNA damage response. We are interested in how PAR-signaling proteins specifically recognize PAR chains to signal DNA damage, how scaffolding proteins coordinate DNA repair activities, and how PARylation crosstalks with other signaling pathways and post-translational modifications. We are currently working on biomedically important DNA repair enzyme complexes, including human XRCC1-DNA Ligase III complex (Figure 4). We are also working on the mechanisms of crosstalk between ADP-ribosylation and ubiquitination. In addition, we use a high-throughput screening and structure-based drug development strategy that are already established in the lab (Figures 2 and 3), to identify new small-molecule inhibitors targeting PAR-dependent DNA damage response pathway.

W© In-Kwon Kim, Department of Chemistry, University of Cincinnati 2016