An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

Potent and Selective Inhibitors of MTH1 Probe Its Role in Cancer Cell Survival.

Recent literature has claimed that inhibition of the enzyme MTH1 can eradicate cancer. MTH1 is one of the "housekeeping" enzymes that are responsible for hydrolyzing damaged nucleotides in cells and thus prevent them from being incorporated into DNA. We have developed orthogonal and chemically distinct tool compounds to those published in the literature to allow us to test the hypothesis that inhibition of MTH1 has wide applicability in the treatment of cancer. Here we present the work that led to the discovery of three structurally different series of MTH1 inhibitors with excellent potency, selectivity, and proven target engagement in cells. None of these compounds elicited the reported cellular phenotype, and additional siRNA and CRISPR experiments further support these observations. Critically, the difference between the responses of our highly selective inhibitors and published tool compounds suggests that the effect reported for the latter may be due to off-target cytotoxic effects. As a result, we conclude that the role of MTH1 in carcinogenesis and utility of its inhibition is yet to be established.

Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2.

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.

Enzymatic characterisation of USP7 deubiquitinating activity and inhibition.

USP7 (HAUSP) is a deubiquitinating enzyme, which plays a crucial role in regulating the levels of the p53 tumour suppressor protein, through its ability to prevent the proteasomal degradation of the Ubiquitin ligase for p53, Hdm2. Supporting evidence suggests that an inhibitor of USP7 would act to abrogate the action of Hdm2, and thereby elevate levels of the p53 protein, with associated therapeutic benefits in cancer and potentially other diseases. In this article, we describe the characterisation of differential enzyme activity of both the full length and putative catalytic domain of human USP7 expressed in both bacterial and insect cell expression systems. We also demonstrate the way in which variations in the reducing environment surrounding the enzyme can dramatically affect both the stability of the enzyme and the range of small molecules able to inhibit the catalytic activity of the enzyme. Furthermore, we describe the validation and use of this assay for a high-throughput screening approach, again highlighting the critical nature of the enzyme's environment. Taken together, these findings not only increase our understanding of the enzymatic activity of deubiquitinating enzymes, but also highlight several key considerations of importance in the development of therapeutic agents against this novel class of therapeutic targets.