Novel Class of Potent and Cellularly Active Inhibitors Devalidates

Jul 5, 2017 - Mechanisms of MTH1 inhibition-induced DNA strand breaks: The slippery slope from the oxidized nucleotide pool to genotoxic damage...
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Letter

Novel class of potent and cellularly active inhibitors de-validates MTH1 as broad-spectrum cancer target Manuel Ellermann, Ashley Eheim, Fredrik Rahm, Jenny Viklund, Judith Günther, Martin Andersson, Ulrika Ericsson, Rickard Forsblom, Tobias Ginman, Johan Linstrom, Camila Silvander, Lionel Tresaugues, Anja Giese, Stefanie Bunse, Roland Neuhaus, Joerg Weiske, Maria Quanz, Andrea Glasauer, Katrin Nowak-Reppel, Benjamin Bader, Horst Irlbacher, Hanna Meyer, Nina Queisser, Marcus Bauser, Andrea Haegebarth, and Matyas Gorjanacz ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00370 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017

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Novel class of potent and cellularly active inhibitors de-validates MTH1 as broad-spectrum cancer target

Manuel Ellermann,† Ashley Eheim,† Fredrik Rahm,‡ Jenny Viklund,‡ Judith Guenther,† Martin Andersson,‡ Ulrika Ericsson,‡ Rickard Forsblom,‡ Tobias Ginman,‡ Johan Lindström,‡ Camilla Silvander,‡ Lionel Trésaugues,‡ Anja Giese,† Stefanie Bunse,† Roland Neuhaus,† Jörg Weiske,† Maria Quanz,† Andrea Glasauer,† Katrin Nowak-Reppel,† Benjamin Bader,† Horst Irlbacher,† Hanna Meyer,† Nina Queisser,† Marcus Bauser,† Andrea Haegebarth,† and Mátyás Gorjánácz†,*

†Bayer AG, Berlin, Germany ‡Sprint Bioscience, Huddinge, Sweden

*Corresponding author: [email protected]

ABSTRACT

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anti-cancer efficacy either in mono- or in combination-therapies. Therefore we conclude that MTH1 is dispensable for cancer cell survival.

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INTRODUCTION

Reactive oxygen species (ROS) are formed as natural byproducts during normal cellular metabolic processes and are known to damage diverse intracellular molecules such as lipids, proteins and DNA (1). Deoxynucleoside triphosphates (dNTPs) are particularly susceptible to oxidative damage by ROS, with 8-oxo-2´-deoxyguanosine-5´-triphosphate (8-oxo-dGTP) and 2hydroxydeoxyadenosine-5´-triphosphate (2-OH-dATP) being the two most abundant oxidative nucleotide lesions. During replication damaged nucleotides are incorporated into DNA resulting in mutations and breaks, which ultimately may lead to cell death (2). MTH1 (MutT homolog 1, also known as NUDT1) is a member of Nudix phosphohydrolase superfamily able to convert oxidized nucleotide triphosphates 8-oxo-dGTP and 2-OH-dATP into corresponding monophosphate forms, thus preventing their incorporation into DNA and circumventing cell death (3). Under normal physiological conditions cells tightly regulate production and elimination of ROS; therefore the role of MTH1 is non-essential for normal cell survival (4). In contrast, cancer cells are often characterized by increased ROS burden. Hence, it was proposed that cancer cells may overexpress MTH1 as a pro-survival adaptation mechanism to sanitize damaged nucleotides and prevent their incorporation into DNA. Inhibition of MTH1 and induction of a lethal level of 8-oxo-dGTP and 2-OH-dATP was hypothesized to be an attractive broad-spectrum anti-cancer therapeutic approach allowing nontransformed cells to be unaffected (5-7). Attractive target rationale combined with previous success in identifying potent and cellularly active inhibitors prompted us to develop novel MTH1 matter. We developed structurally distinct, potent and selective MTH1 inhibitors with high solubility, metabolic stability, cell permeability and cellular target engagement. However, these properties did not translate into in vitro or in vivo anti-cancer efficacy either in mono- or in combination-therapies. Based on these observations we concluded MTH1 is not essential for cancer cell survival or for intracellular sanitization of damaged nucleotides and thus not a viable target to be exploited for drug development.

RESULTS AND DISCUSION 2 ACS Paragon Plus Environment

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Identification of novel MTH1 inhibitors. Small molecule inhibitors of MTH1 have been previously reported (5, 6, 8-10) and among them TH588 (Figure 1B) is one of the most extensively characterized (5). Encouraged by the work done on MTH1, we pursued a structure-based optimization program originating from our fragment-based screen. This approach enabled us to identify BAY-707 (Figure 1A) as a selective MTH1 inhibitor with low nanomolar enzymatic activity (IC50= 2.3+/-0.8 nM, n=6). Details about the structure-based optimization process towards BAY-707 will be described elsewhere (manuscript in preparation). BAY-707 showed an overall favorable physicochemical profile and promising in vitro pharmacokinetic properties with high metabolic stability in both human microsomes and rat hepatocytes (Table S1). Cell permeability of BAY-707 measured in the Caco-2 assay was also high (Table S1). Moreover, BAY-707 demonstrated high selectivity in an in-house kinase panel. In order to investigate the binding mode of BAY-707, a crystal structure with MTH1 protein was solved and refined to a resolution of 1.72 Å (PDB id 5NHY). It revealed substratecompetitive binding to the active site of MTH1 (Figure 1C) through hydrogen bonds to Gly34, Asp119, and Asp120 and a π-stacking interaction to Trp117. Overlaying BAY-707 with previously reported crystal structures of 8-oxo-dGMP (PDB id 3ZR0) (Figure 1C) and TH588 (PDB id 4N1U) (Figure 1D) shows co-localization of all three ligands within the protein active site. Therefore BAY-707 displays an overall profile that is capable of exerting MTH1 inhibition in biological systems.

MTH1 is not required for cancer cell survival. To investigate on-target cellular activity of BAY-707, we utilized the cellular thermal shift assay (CETSA) (11). This assay relies on ligand-induced thermal stabilization of the target protein measured in a dose-response manner at a single melting temperature. TH588 increased the cellular thermal stabilization of MTH1 with EC50 of 133 nM, which was in line with its enzymatic activity (IC50= 12.6+/-3.0 nM, n=6). BAY-707 demonstrated a superior cellular target engagement with EC50 of 7.6 nM (Figure 2A), in agreement with its higher enzymatic potency (IC50= 2.3 nM). Next we determined CETSA EC50s for additional structurally related BAY MTH1 compounds and found a good correlation between their biochemical potency and cellular 3 ACS Paragon Plus Environment

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target engagement (Figure 2B). Therefore we concluded that BAY-707 is potent and cellularly active MTH1 inhibitor, suitable to validate the MTH1 dependency of cancer cells. Since initial RNAi and tool compound experiments proposed that MTH1 is essential for cancer cell survival (5), we analyzed the growth inhibitory effects of BAY-707 and TH588 on several human cancer cell lines. We confirmed the previously published weak cellular toxicity of TH588 (5) (Figure 2C); however, this MTH1 tool compound also demonstrated equipotent cytotoxicity on non-transformed mammary epithelial cells (Figure 2C, Table S2). In contrast, we were unable to observe any anti-proliferative effects with BAY-707 up to 30 µM (Figure 2C, Table S2), even though the biochemical potency and cellular target engagement of BAY-707 was superior to TH588 (Figure 2B). As this was unexpected, we further determined the GI50s of additional structurally related BAY MTH1 compounds on NCI-H358 lung cancer cells; however, we observed no major growth inhibition and no correlation between cytotoxicity and biochemical potency of our compound series (Figure 2D). Increased duration of compound treatments also failed to inhibit the growth and clonogenic capacity of cancer cells (Figure S1A, S1B). Next we wondered whether upregulation of base excision DNA repair enzymes during MTH1 inhibition might explain the lack of in vitro efficacy. However, neither OGG1 (12) nor MUTYH (13) were upregulated on mRNA (Figure S1E, S1F) or protein levels (Figure 2E) upon compound treatments. Furthermore, RNAi-mediated down-regulation of MTH1 also had no effects on their expression (Figure 2F, S1H, S1J, S1K), suggesting that they cannot explain the lack of growth inhibition. Functional redundancy could be also excluded, since MTH1 is the only known enzyme that can hydrolyze 8-oxo-dGTP and 2-OH-dATP under physiological conditions (7, 14) and its closest paralogue MTH2 was also not upregulated upon inhibition (Figure 2E, S1C, S1D) or downregulation of MTH1 (Figure 2F, S1H, S1I). Consistent with our findings, recently three independent laboratories disclosed structurally diverse MTH1 inhibitors, which despite of being potent and cellularly active, also failed to induce growth inhibition (9, 10, 15). Moreover, cytotoxicity induced by the only two anti-proliferative MTH1 inhibitors, TH588 and (S)-crizotinib, could not be rescued with an overexpression of the human MTH1 protein (6, 15). Consistently, efficient knockdown of MTH1 (Figure 2F, S1H) with siRNA oligo 3 showed no significant growth inhibition when compared to the control RNAi (Figure S1G), while the previously widely used MTH1 RNAi reagents (oligos 1 and 2 (5)) showed cytotoxicity despite of being even less efficacious in downregulating MTH1 4 ACS Paragon Plus Environment

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than oligo 3 (Figure S1H). These suggest that MTH1 is dispensable for the survival of cancer cells and that MTH1 inhibition or downregulation is unlikely to be the primary cause of cytotoxicity observed with tool compounds TH588 and (S)-crizotinib or with some RNAi reagents (also (9).

MTH1 is not essential for sanitization of oxidized nucleotides within the cell. In response to DNA damage, histone H2AX becomes phosphorylated at double strand DNA breaks (DSBs) and called γ-H2AX. This is the first step of the sequential recruitment of repair proteins to DSBs, thus it is an appropriate marker to visualize the 8-oxo-dG-induced DSBs (Figure 3A). We utilized this readout in a high content analysis (HCA) of NCI-H358 cells. While treatment with TH588 resulted in a weak induction of DSBs (EC50= 3.11 µM), we observed no such effects with BAY-707 up to the highest concentration tested (12.4 µM) (Figure 3B). Similarly, most other potent and cell permeable BAY MTH1 compounds also failed to induce DSBs and their γ-H2AX EC50s showed no correlation with biochemical potency (Figure 3B) or with cellular target engagement (Figure 3C). These data suggest that induction of DSBs observed with some MTH1 inhibitors is independent of their enzymatic activity and is foreseeably due to off-target effects. To directly visualize the genomic incorporation of 8-oxo-dG we performed quantitative imaging analysis of U2OS cells immunostained for this lesion (Figure 3D). BAY-707 treatment resulted in no effects on nuclear 8-oxo-dG level alone (Figure 3E) or in combination with prooxidant LCS1 (Figure S2A). Additional structurally related BAY MTH1 compounds also demonstrated no major effects on 8-oxo-dG levels and the cell viability measured by nuclei counts (Figure 3E, red dots). In contrast, TH588 treatment resulted in a 3-fold increase of nuclear 8-oxo-dG level over the DMSO control (Figure 3E). This effect was comparable to the effect seen with 600 nM LCS1 and is likely to be the maximal achievable effect, as higher concentrations of these two compounds were cytotoxic and strongly reduced the average cell number (Figure 3E, red dots). However, comparable increase of nuclear 8-oxo-dG was also achieved by independent cytotoxic drugs, which are not MTH1 inhibitors, such as pan-Aurora inhibitor and CDK2, 7, 9 inhibitor (Figure 3E) or PLK1 inhibitor and BETi (data not shown). These observations were further confirmed in comet assay (Figure S2C). Consistently, cytotoxic siRNAs targeting other proteins than MTH1 also increased the nuclear 8-oxo-dG level to the 5 ACS Paragon Plus Environment

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same extent as the cytotoxic MTH1 siRNAs (Figure S2B). Therefore these results strongly suggest that off-target cytotoxicity of TH588 and MTH1 siRNAs are the primary reason for the increased nuclear incorporation of 8-oxo-dGTP and that in living cells MTH1 is not essential for sanitization of oxidized nucleotides.

MTH1 inhibition in mono- or combination-therapies has no effect on in vivo tumor growth. To investigate the efficacy of MTH1 inhibition in vivo, both TH588 and BAY-707 were investigated in terms of tolerability, overall exposure profile and anti-tumor efficacy. TH588 was formulated as described by Gad et al. (5) and administered to mice to observe overall tolerability with a pharmacokinetics analysis performed at study end. With similar experimental conditions the published exposure profile of TH588 was not reproducible (data not shown), which was reported to cover the IC50 for approximately 30 minutes (5). In accordance with published data, animals treated with TH588 displayed minimal body weight loss; however dosing schemes were limited due to i.p. application route and poor solubility of the compound. In attempts to further replicate reported anti-tumor effects of TH588, we once again administered this compound to tumor bearing animals (NCI-H460, SW480 or A549 xenografts), but failed to observe any significant reduction in tumor burden of the animals (data not shown). BAY-707 has an overall superior physicochemical profile as well as improved metabolic stability. Therefore, to continue to determine if inhibition of MTH1 affects tumorigenesis in any capacity, this compound was further profiled in vivo. Due to enhanced solubility, BAY-707 was formulated for oral dosing and it was well-tolerated in nude mice (Figure 4A). Following a 7 day treatment period, body weight loss did not exceed 10% with doses up to 250 mg/kg QD. Exposure of BAY-707 was also superior to TH588 as evidenced by significantly enhanced coverage over biochemical and cellular IC50ss (Figure 4B). Two distinct tumor models were selected to explore potential antitumor activity of MTH1 inhibition; the syngeneic CT26 colon adenocarcinoma model (Figure 4C, 4D) and the NCI-H460 non-small cell lung cancer xenograft (Figure 4E, 4F). In the CT26 animal model, following formation of palpable tumors, mice were randomized and treated with 100 mg/kg BAY-707 (a dose known to provide significant IC50 coverage), radiation (indicationspecific standard of care) or a combination of both therapies. While radiation therapy provided the anticipated suppression of tumor growth, BAY-707 failed to provide significant anti-tumor activity in monotherapy or additive effects when combined with radiation treatment. Similar 6 ACS Paragon Plus Environment

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results were obtained testing our potent MTH1 inhibitor in the NCI-H460 model (Figure 4E, 4F). Following the same randomization scheme, mice were treated with BAY-707 alone or in combination with a variety of standard of cares (cisplatin, radiation, and doxorubicin). Once again, BAY-707 failed to provide in vivo anti-tumor activity as evidenced by no significant decrease in tumor volume or tumor weight. Mice genetically deficient in MTH1 are viable and fertile, with no significant phenotype reported (4). Results above obtained from xenograft models support lessons learned from knockout mice and confirm previous reports that MTH1 is not required for cancer cell survival in vitro or in vivo. Identifying the right targets is of utmost importance for drug discovery in the era of targeted therapies (16). It relies on the quality of chemical probes, knockdown and knockout reagents with minimized off-target activities. In this study we describe a novel class of highly potent MTH1 inhibitors, which despite of superior biochemical potency, cellular target engagement and pharmacokinetic profile to other MTH1 tool compounds, exert no in vitro or in vivo anti-cancer efficacy either in mono- or in combination-therapies. With BAY-707, a representative member of this compound class, we de-validate MTH1 as a broad-spectrum nononcogenic cancer dependency and provide the scientific community with a chemical probe having no off-target-related cytotoxicity, to elucidate the biology of MTH1 in cell cultures and living organisms.

METHODS

Details of experimental procedures are provided in the Supporting Information.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

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F. Rahm, J. Viklund, M. Andersson, U. Ericsson, R. Forsblom, T. Ginman, J. Lindström, C. Silvander and L. Trésaugues are employees of Sprint Bioscience. M. Ellermann, A. Eheim, J. Guenther, A. Giese, S. Bunse, R. Neuhaus, J. Weiske, M. Quanz, A. Glasauer, K. NowakReppel, B. Bader, H. Irlbacher, H. Meyer, N. Queisser, M. Bauser, A. Haegebarth and Mátyás Gorjánácz are employees of Bayer AG.

ASSOCIATED CONTENT

Supporting Information Additional figures, tables, experimental procedures and references are provided in the Supporting Information. The Supporting Information is available for free of charges via the Internet at http://pubs.asc.org.

Accession Codes The Structure of BAY-707 in complex with MTH1 has been deposited at the Protein Data Bank under accession code 5NHY and will be released upon publication.

ACKNOWLEDGEMENTS

We thank F. Hübner, M. Geyer, O. Gernetzki and M. Jarzombek for their technical assistance and A. Talagas for help with graphical abstract. Assistance with tables and figures was provided by Bernard Kerr, Scion (London, UK), and was funded by Bayer AG.

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FIGURE LEGENDS

Figure 1. BAY-707 binds into the substrate pocket of MTH1. (A) BAY-707 and (B) TH588 compound structures. (C-D) Superposition of complex between MTH1 and BAY-707 (green) with MTH1/8-oxo-dGMP (C, cyan) and MTH1/TH588 (D, magenta). BAY-707 and interacting residues are shown as sticks. Water molecules present in BAY-707 binding site and hydrogen bonds between MTH1 and BAY-707 are depicted as red spheres and yellow dashes, respectively. BAY-707 is displayed in the 2mFo-DFc electron density map contoured at 1σ (resolution: 1.72 Å). 8-oxo-dGMP and TH588 are shown as thin sticks compared to the representation used for the complex between MTH1 and BAY-707. The PDB accession code for structures of MTH1/BAY707, MTH1/8-oxo-dGMP and MTH1/TH588 complexes are 5NHY, 3ZR0 and 4N1U, respectively. Pictures were produced using Pymol (The PyMOL Molecular Graphics System, Version 1.7.2.1 Schrödinger, LLC.).

Figure 2. BAY-707 is a potent cellularly active MTH1 inhibitor demonstrating dispensability of MTH1 for cancer cell survival. (A) BAY-707 increased the cellular thermal stabilization of MTH1. Loading control was Vinculin. (B) Good correlation between the biochemical potency and cellular target engagement of structurally related BAY MTH1 inhibitors. (C) In contrast to TH588, BAY-707 displayed no cytotoxicity. (D) No correlation between cytotoxicity in NCIH358 cells and biochemical potency of BAY MTH1 inhibitors was observed. GI50 values were determined after 6 days long treatments. (E) Treatment of HeLa cells with TH588 and BAY-707 had no effect on the expression of MTH2, OGG1 and MUTYH. HSP90 was used as a loading control. Note, there was a slight increase in MTH1 protein levels after both compound

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treatments. (F) siRNA-mediated downregulation of MTH1 in HeLa cells had no effects on the expression of MTH2, OGG1 and MUTYH. HSP90 was used as a loading control.

Figure 3. MTH1 is not essential for sanitization of 8-oxo-dGTP in cells. (A) NCI-H358 cells were treated with DMSO and TH588 and immunostained to visualize γH2AX and DNA in HCA. Bars: 100 µm. (B) No correlation was observed between the biochemical potency of BAY MTH1 inhibitors and induction of DSBs. (C) No correlation was observed between the cellular target engagement of BAY MTH1 inhibitors and induction of DSBs. (D) U2OS cells were treated with DMSO and TH588 and immunostained to visualize 8-oxo-dG and DNA in HCA. Bars: 20 µm (E) Concomitant measurement of average nuclear 8-oxo-dG intensity (columns) and average nuclear count (red dots) in HCA. 8-oxo-dG signal of DMSO (gray); BAY-707 and related BAY MTH1 inhibitors (green); TH588 (red); LCS1 (SOD1 inhibitor), VX-680 (panAurora inhibitor) and SNS-032 (CDK2, 7, 9 inhibitor) (blue) treated cells. Drugs were used at 10 µM and 30 µM or at around their GI50 and GI90 concentration. Figure 4. BAY-707 treatment is well-tolerated in mice, but inhibition of MTH1 does not translate to antitumor efficacy in vivo. (A) Treatment of female NMRI nude mice with BAY-707 did not result in significant body-weight loss, overt signs of toxicity, or increased mortality. BAY-707 was administered p.o. (vehicle-PEG/Water [80:20]) with step-wise escalation of doses of 25–250 mg/kg over a period of 7 days. Body weight measurements and the general observation period were extended for an additional 7 days to assess putative latent toxicity. (B) Significant plasma exposure was observed with BAY-707 at 25 and 100 mg/kg p.o. (n=3 per timepoint). As 100 mg/kg dose was well-tolerated with significant coverage of biochemical IC50 and CETSA EC50 values for approximately 24 hours, this dose was selected for in vivo efficacy experiments. (C, D) In vivo efficacy of BAY-707 in an immunocompetent syngeneic tumor model. Female BALB/c mice bearing subcutaneous CT26 tumors were treated with BAY-707 or vehicle p.o. as monotherapy or in combination with focal radiation for the indicated number of days. (E, F) In vivo efficacy of BAY-707 in a model of non-small-cell lung cancer. BAY-707 was administered to NCI-H460-tumor-bearing mice alone or in combination with the indicated therapy for 10 days. (C–F) Focal radiation, 2.5 Gy QW; Cisplatin, 3 mg/kg i.p. Q3D; Doxorubicin, 10 mg/kg i.v. Q2W. Tumor volume was determined by digital caliper and 11 ACS Paragon Plus Environment

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expressed as mean tumor volume plus SEM; n=8–12 mice per group. Following study termination, tumors were excised and weighed as an additional parameter of antitumor efficacy.

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Figure 1 139x141mm (300 x 300 DPI)

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Figure 2 139x134mm (300 x 300 DPI)

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Figure 4 144x162mm (300 x 300 DPI)

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