Identification of dual DNA-PK MDR1 inhibitors for the potentiation of cytotoxic drug activity Emily Mould a , Philip Berry a , David Jamieson a , Christopher Hill a , Celine Cano b , Niu Tan a , Sarah Elliott a , Barbara Durkacz a , David Newell a , Elaine Willmore a, * a Newcastle Cancer Centre at the Northern Institute for Cancer Research, Medical School, Newcastle University, Paul O’Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK b Newcastle Cancer Centre at the Northern Institute for Cancer Research, School of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne NE1 7RU, UK 1. Introduction DNA is the main target of many conventional anticancer agents and therefore the inhibition of DNA repair is an attractive approach to chemosensitisation. DNA double-strand breaks are the most lethal type of DNA damage and therefore two major pathways have evolved to repair these DNA lesions; homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA-PK is a serine/ threonine protein kinase and is a member of the phosphatidyli- nositol 3-kinase like kinase (PIKK) family. DNA-PK is essential for the NHEJ repair pathway [1], and has also been implicated in HR [2], H2AX phosphorylation [3], metabolic gene regulation [4], and in the inflammatory response through interactions with NFkB [5]. NU7441 (8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen- 4-one, Fig. 1(a)) is a potent ATP-competitive DNA-PK inhibitor (IC 50 = 14 nM) developed from the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 [6]. NU7441 has been shown to increase sensitivity to radiation and topoisomerase II poisons, and increase etoposide-induced tumour growth delay in mice bearing SW620 xenografts [7]. The mechanism by which this occurs has previously been demonstrated that NU7441 retards the repair of both IR- and etoposide-induced DNA double strand breaks and inhibits homologous recombination in a DNA-PK dependent manner [7,8]. NU7441 is around 20-fold more selective for cellular DNA- PK over PI3K inhibition [8]. NU7742 (8-dibenzothiophen-4-yl-2- piperidin-1-yl-chromen-4-one, Fig. 1(b)) is an inactive derivative of NU7441 (IC 50 > 10 mM) in which the morpholine oxygen has been replaced with a methylene group, resulting in the loss of sensitisation of cells to DNA-damaging agents [9]. The introduction of a methyl substituent at the 7-position of the chromen-4-one Biochemical Pharmacology 88 (2014) 58–65 A R T I C L E I N F O Article history: Received 24 October 2013 Accepted 3 January 2014 Available online 10 January 2014 Keywords: DNA-PK MDR1 NU7441 Cancer Multidrug resistance A B S T R A C T Inhibition of DNA repair is an attractive therapeutic approach to enhance the activity of DNA-damaging anticancer chemotherapeutic agents. Similarly, blockade of the multidrug-resistance protein 1 (MDR1) can overcome efflux-mediated resistance. DNA-dependent protein kinase (DNA-PK) is essential for the non-homologous end-joining DNA repair pathway. NU7441 is a potent DNA-PK inhibitor (IC 50 = 14 nM) that is used widely to study the effects of DNA-PK inhibition in vitro. In growth inhibition studies, 1 mM NU7441 sensitised vincristine-resistant CCRF-CEM VCR/R leukaemia cells (1200-fold resistant) to a range of MDR1 substrates, including doxorubicin (8-fold, p = 0.03), vincristine (14-fold, p = 0.01) and etoposide (63-fold, p = 0.02), compared with 1.4-fold (p = 0.02), 2.2-fold (p = 0.04) and 3.6-fold (p = 0.01) sensitisation, respectively, in parental CCRF-CEM cells. This difference in NU7441 sensitivity was confirmed in another two parental and MDR1-overexpressing cell line pairs. A doxorubicin fluorescence assay showed that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1 mM NU7441 increased doxorubicin nuclear fluorescence 16-fold. NU7441 and 3 structurally related compounds (NU7742 (an NU7441 analogue that does not inhibit DNA-PK – IC 50 > 10 mM), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative – IC 50 = 2 nM) and DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative - IC 50 = 7 mM)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC–MS. This paper demonstrates that NU7441 is a dual DNA-PK and MDR1 inhibitor, and this extends the therapeutic potential of the compound when used in combination with MDR substrates. ß 2014 Elsevier Inc. All rights reserved. * Corresponding author. E-mail addresses: e.v.a.mould@ncl.ac.uk (E. Mould), philip.berry@ncl.ac.uk (P. Berry), david.jamieson@ncl.ac.uk (D. Jamieson), chrishill86@gmail.com (C. Hill), celine.cano@ncl.ac.uk (C. Cano), niujinmy@gmail.com (N. Tan), sarahelliott80@hotmail.com (S. Elliott), barbara.durkacz@ncl.ac.uk (B. Durkacz), herbie.newell@ncl.ac.uk (D. Newell), elaine.willmore@ncl.ac.uk (E. Willmore). Contents lists available at ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m 0006-2952/$ – see front matter ß 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2014.01.001