PO-042 Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2

Introduction Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5 year survival rate of merely 8%. The tumour microenvironment is one of the factors contributing to PDAC chemoresistance. More specifically, the hypoxic tumour cores and the metabolic switch to aerobic glycolysis (e.g. the Warburg effect), contribute to the lack of drug response. Interestingly, two glycolysis components glucose transporter 1 (GLUT-1) and lactate dehydrogenase A (LDH-A) are overexpressed in PDAC. The latter, LDH-A, is also correlated with prognosis in metastatic PDAC. N-Hydroxyindole-based LDH-A inhibitors (NHI-1 and NHI-2) have shown a synergistic effect in hypoxic PDAC cells when combined with gemcitabine. A glucose conjugated NHI-Glc-2 was designed to exploit the GLUT-1 overexpression in PDAC cells and in the present study we evaluated whether this novel compound further improved the pharmacological effect of LDH-A inhibitors. Material and methods The effect of NHI-Glc-2 on cell growth is tested in our primary PDAC cancer cell cultures, characterised for their hypoxic signature and LDH-A/GLUT-1 expression levels by next-generation sequencing. Inhibition of cell and tumour growth was evaluated by the SRB assay, 3D spheroid-cultures and with an orthotopic bioluminescent in vivo model. Additionally, LDH-A enzyme activity inhibition and the effect on the glycolytic rate by NHI-Glc-2 were assessed by spectrophotometry and with the Seahorse XF analyzer, respectively. Results and discussions NHI-Glc-2 is capable of inhibiting PDAC cell growth in, especially in hypoxia, in nanomolar range and shows a synergistic effect with gemcitabine. In 3D cultures NHI-Glc-2 disrupts spheroid integrity, and preliminary in vivo studies show promising results. Conclusion Lactate dehydrogenase A is a viable target in PDAC, and the novel LDH-A inhibitor showed improved pharmacological effect in normoxic and hypoxic PDAC cells compared to NHI-1 and NHI-2. Moreover, this compound displays a synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours

Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes

Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes

(ADP-ribosyl)hydrolases: Structural basis for differential substrate recognition and inhibition

Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design