Identifying novel naturally

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). 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.).

RESULTS: 2 out of 9 purchased hits seemed to reduce H2O2-induced PARylation (Caffeic acid phenethyl ester and Psoralidin). Possible

Questions & answers

Please, tell us a little bit more about yourself. My name is Zrinka Duvnjak and I’m a fifthyear student from Zagreb, Croatia. You may know me as a former Twinnet Coordinator for Zagreb or a former Pharmaceutical Sciences Coordinator at IPSF. At the moment, I’m an intern at Xellia Pharmaceuticals (R&D), and next semester you can meet me in Helsinki where I’ll be doing my master’s thesis. In my free time, I’m playing modern board games, checking off my list of escape rooms in Zagreb, preparing myself for some running race (or being disappointed when it is canceled a few weeks before the race due to the pandemic), playing volleyball, scratching my world map, playing guitar, or just enjoying life with my friends and family.

Tell us a bit more about your research and its significance. My research has been done at the University of Cambridge during 2,5 months of my summer internship. It is actually a side project of one Ph.D. topic funded by Astra Zeneca. Existing PARP inhibitors showed promising in practice and they are broadening its indications, so why not to try to find a new one, since we will always need new, better and different anti-cancer drugs? Another indicator of importance of this research is that with this work I won the prize for the best poster presentation at the Pharmacology Away Day, University of Cambridge. What was the biggest challenge whilst carrying out the research and how did you overcome it? The first part of my research was computational chemistry – structure-based screening and docking. There is numerous software for this, and they all work in a different way - one software will prefer partial charges overlapping for a good score (result), other will suggest that you have the best overlapping of the structures when there is a pi-stacking present, etc. Different software will give you different results so I needed to use 4 different software and to look at all the results at the same time to choose which molecules we will buy for wet screening. Additionally, I used 3 different conformations of PARP protein for all the docking. To succeed not to mix up everything I needed to be very organized and careful with naming files and putting them in the right folder. Another thing you should keep in mind is that these software are not flawless – each result I needed to check by looking it in software for visualization.

In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? Writing for ESSP is a great way of practicing your abstracts writing skills because except from the feedback you get, you have an opportunity to present yourself and your work to other students from other countries interested in research.