title: Identification and characterisation of novel, natural product inhibitors of the poly (ADP-ribose) polymerase (PARP) enzyme for anti-cancer and other therapeutic applications
Creators: name: Benson, S
Publisher: name: Liverpool John Moores University
Year: 2025

description: Interest in the enzyme poly (ADP-ribose) polymerase (PARP) has grown significantly in recent years, especially in relation to the potential to harness for therapeutics development its multiple roles in detecting and repairing DNA damage and in non-oncological scenarios such as its mediation of neuronal death in neurodegeneration. Human cancer cells with a defective homologous recombination pathway due to deleterious mutations in their tumour suppressor genes such as BRCA1 and BRCA2, and which often rely on PARP for their single-strand DNA repair, can be selectively targeted through pharmacological inhibition of PARP, which results in their death through synthetic lethality. Currently, most PARP inhibitors (PARPi) are synthetic and designed with the benzamide motif to mimic the nicotinamide moiety of NAD+, the substrate for PARP, but which is also a substrate for some related enzymes. Only four drugs are currently approved as PARPi and used in the clinic: olaparib, rucaparib, niraparib and talazoparib. They are competitive inhibitors of NAD+ and, while relatively safe, they have drawbacks, including high cost and the development of resistance to them by cancer cells. It is, therefore, important to identify novel PARPi scaffolds that can generate a new pipeline of PARPi drugs, especially those that are not competitive inhibitors of NAD+. As natural products are a proven, veritable source of new drugs, with significant diversity for exploring chemical space, the aim of this PhD project was to identify and characterise new, natural product PARPi. A cell-based model assay was optimised for screening and characterisation, in which PARP-mediated cell death was induced by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), such that an agent with the ability to protect against the PARP1-mediated cell death was considered a PARPi. Morphological changes following the treatments were also captured using brightfield microscopy. The model assay was validated by using it to assess the relative potencies of olaparib, a clinically approved PARPi, and its fluorescent derivative, PARPi-FL. The results showed that PARPi-FL (EC50 = 55.7 nM) was less potent than olaparib (EC50 = 23.1 nM) (as assessed by the MTT assay), which was corroborated by the results of a cell-free (biochemical) assay with IC50 values of 17.7 and 5.4 nM, respectively. The efficacies and potencies of the four clinically approved PARPi were also determined. The EC50 values (nM) revealed the following rank order of potencies: talazoparib (3.6) &gt; rucaparib (8.4) &gt; niraparib (17.1) &gt; olaparib (23.1), which was consistent with the potency rankings of these PARPi in previous studies, thus validating the assay. When employed in a time-course experiment, the assay uniquely revealed that olaparib and DPQ could continue to significantly protect against PARP1-mediated cell death even when added up to 12 h and 8 h, respectively, after initiation of the MNNG insult. The assay was utilised to screen a small commercial library of 502 natural compounds, as well as a small, customised set of compounds, for PARP-inhibiting activity. While the few original hits did not sustain activity in re-screening, the work inspired a recourse to 4′-methoxyflavone (4MF) and 3′,4′-dimethoxyflavone (DMF), two flavones that have been previously found to inhibit parthanatos. These two natural products were further validated in the model assay, and their potencies (EC50) were 6.3 and 5.9 µM, respectively, based on the MTT assay. A cell-free, PARP binding assay also confirmed that 4MF and DMF could directly inhibit the PARP1 enzyme, with IC50 values of 2.54 and 2.07 µM, respectively. A combination of 4MF or DMF with cisplatin or doxorubicin revealed that 4MF (5 and 10µM) could sensitise HeLa cells to the effect of cisplatin or doxorubicin. Structure-activity relationship (SAR) study of 4MF and DMF showed that they had weak antioxidant activity, based on the DPPH free radical-scavenging activity, whereas their hydroxy- derivatives 4′-hydroxyflavone (4HF) and 3′,4′-dihydroxyflavone (DHF) exhibited higher and significant antioxidant activity: 55.9% and 77.9% for 4HF and DHF, respectively, compared to 4MF (17.3%) and DMF (15.5%). However, a reactive oxygen species (ROS) assay that measures intracellular levels of ROS revealed that none of 4MF, DMF and 4HF altered hydrogen peroxide-induced intracellular ROS levels in HeLa cells while, on the other hand, DHF significantly decreased the ROS levels (p&lt; 0.001). The PARP-inhibitory activities of 4MF, DMF, 4HF and DHF at 20 µM in a cell-free assay were 21.4%, 17.5%, 35.2% and 24.3%, respectively. In contrast, in the cell-based assay, the EC50 values for their effects against MNNG-induced, PARP-mediated parthanatos were 6.3, 5.9, 24.8 and 62.9 µM, respectively. These findings suggest that 4HF and DHF are stronger antioxidants than 4MF and DMF, and that, conversely, 4MF and DMF are more potent PARP inhibitors than 4HF and DHF. This strongly suggests that a significant antioxidant property does not necessarily correlate directly with a significant PARP-inhibitory property, and vice-versa. The SAR study revealed the importance of the 3′ and 4′ positions on the B-ring of the flavone compared to substitution on the A-ring, especially on the C7 position. In silico docking simulation indicated that 4MF, DMF, 4HF and DHF could each bind effectively to PARP1 and PARP2 enzymes. Furthermore, the physicochemical properties of these four flavones fulfilled drug-likeness criteria according to the Lipinski’s rule of five. In conclusion, this study has successfully demonstrated that PARPi could be obtained from natural sources and 4MF, DMF, 4HF and DHF, in particular, as flavones, have mild-to-moderate PARP-inhibitory activity and thus represent chemical scaffolds that could inspire the development of novel PARPi for the treatment of cancers and non-oncological conditions, including neurological and neurodegenerative diseases.