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European Pharmaceutical Students’ Association
IDENTIFYING NOVEL NATURALLY-OCCURING CHEMICAL SCAFFOLDS AS POTENTIAL INHIBITORS OF POLY(ADP-RIBOSE) POLYMERASE (PARP) Author: Zrinka Duvnjak, Maria Eznarriaga Gutierrez, Jack Greenhalgh Scientific Coordinator: Dr. Taufiq Rahman - university lecturer, group leader Institution:University of Cambridge, Department of Pharmacology
INTRODUCTION: Introduction: The poly(ADP-ribose) polymerase (PARP) proteins catalyze the polymerization of poly(ADP-ribose) (PAR) and their covalent attachment to proteins (PARylation), and as such play important role in base excision repair, homologous recombination, and nonhomologous end joining. The discovery that loss of PARP (PARP-1 in particular) activity triggers cytotoxicity in cells deficient in homologous recombination (for example, lacking BRCA1/2) has sparked a decade of translational research efforts that culminated in the FDA approval of an oral PARP1 inhibitor - Olaparib for clinical use in patients with ovarian cancer and defective homologous recombination. Historically, nature has always been a rich source of diverse chemical scaffolds, some of which have been used directly as drugs whilst most others serve as hits and leads for further development into modern medicines. AIM: Using published structures of inhibitorbound PARP1, we aimed to identify novel naturally-occurring small molecular scaffolds that could potentially inhibit PARP1. MATERIALS AND METHODS: Structurebased virtual screening of Molport Natural ProductTM library by FREDTM and then rescoring by GOLDTM gave 65 hits which were again rescored using AutoDockVinaTM and LeDockTM docking software. After additional in silico assessment of ADME properties, 9 hits were purchased and subjected to wet screening using In Cell WesternTM. Inhibition of PARP-1 is reflected by decreased levels of PARylation and levels of H202 induced PARylation in A549 cells were measured. Spectrally-distinct infrared dye conjugates were used to quantify PAR (at 680 nm) and PARP-1 (at 800 nm). PARylation signal was normalized to total PARP-1. Positive control was Olaparib (100% inhibition), and negative control was 0,5% DMSO (solvent). RESULTS: 2 out of 9 purchased hits seemed to reduce H2O2-induced PARylation (Caffeic acid phenethyl ester and Psoralidin). Possible
binding models with PARP-1 show the interactions of ligands with 3-4 amino acid residues which are in the interaction with registered PARP inhibitors too. CONCLUSION: Our in silico screening protocol has identified few natural products that appear to be significantly reducing PARylation in wet experimental validation. Future study should involve enzyme kinetic essay to prove impairment of catalytic function of recombinant PARP-1, biophysical assays to prove physical engagement of compounds and target, and further characterisations (selectivity for PARP-1 over PARP-2, effect on PARP trapping etc.).
