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Rational identification of a dual inhibitor of PARP-1 and ATM kinase


Type

Thesis

Change log

Authors

Eznarriaga, Maria 

Abstract

The DNA damage response (DDR) is composed of a complex network of genes responsible for detecting and responding to DNA damage. Defects in the DDR (DNA damage response) give rise to genomic instability which promote cancer initiation and progression. These defects also provide vulnerabilities that are targetable and are specific to cancer cells, which can be exploited by DDR inhibitors.

PARP inhibitors are used to exploit the synthetic lethality between pharmacological inhibition of PARP (poly-ADP ribose polymerase) and BRCA (breast cancer gene) defects leading to successful patient treatment in ovarian, breast and prostate cancers. However, their use is limited to patients that harbour these BRCA1/2 defects. Beyond PARP inhibitors there are some DDR inhibitors currently in various phases of clinical trials, targeting: ATR (Ataxia-telangiectasia mutated and Rad3-related), CHK1/2 (checkpoint kinase 1/2), DNA-PK (DNA-dependent protein kinase) and WEE1 (WEE1 G2 Checkpoint Kinase) proteins. These compounds are being developed for use in patients with specific genetic markers or as combination therapies with chemotherapy or radiation. PARP inhibitor monotherapy treatment can lead to resistance mechanism which highlights the need for novel therapies or combination treatments. By targeting multiple members of the DDR with a dual inhibitor, it can be possible to overcome resistance and limit overlapping toxicities whilst expanding the use of current DDR targeting drugs, like PARP inhibitors. The latter therefore presents an exciting opportunity for identifying other DDR targets that when inhibited in combination with PARP lead to synthetic lethality and provide new avenues for cancer therapy.

I carried out a phenotypic cytotoxicity screen in a panel of cancer cell lines in search of a secondary target to inhibit in combination to PARP, which would become the targets of a novel dual inhibitor. ATM was selected and proven to have a synthetic lethality interaction with PARP-1 in Glioblastoma, lung adenocarcinoma and osteosarcoma cell lines. Further characterisation revealed that the combination of PARP and ATM inhibition enhanced cytotoxicity in patient derived Glioblastoma stem cells and 3D spheroids. I also performed target validation experiments looking at the interplay between both targets, the role they play in the DDR and the possible mechanism(s) behind their synthetic lethality.

An effort was undertaken to identify a PARP-ATM dual inhibitor. An in silico and wet screening cascade were set up to search for novel compounds that could inhibit both targets. Several rounds of virtual screening, molecular docking and analysis of interactions were performed in combination with relevant cellular and biochemical assays and DNA damage assays to identify and profile the best hits. During this campaign, no dual inhibitor of PARP and ATM could be identified but several novel PARP inhibitory scaffolds were found. The most successful compound was crystalised with one of the targets as proof of concept.

In addition, I also performed a drug repurposing exercise with the aim to identify novel PARP-1 inhibitor scaffolds with the aid of in silico tools. Two novel structures were identified with alternative modes of action and interesting cytotoxic profiles.

Taken together the work presented in this thesis offers fundamental evidence behind targeting the DDR as a cancer strategy and how targeting multiple proteins like PARP- 1 and ATM can be advantageous and improve current small molecule treatments and hopefully benefit a larger group of patients in the future.

Description

Date

2023-03-30

Advisors

Rahman, Taufiq

Keywords

ATM, DNA damage, In silico, PARP-1

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
AstraZeneca