Evaluating the genotoxicity of topoisomerase-targeted antibiotics

Abstract

Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine139-phosphorylated histone H2AX (γH2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal γH2AX induction >1.4-fold (relative to controls) identified 22/27 NBTIs that induced >6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of γH2AX induction and G₂M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on γH2AX (≤1.4-fold) and also evoked G₁ arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold ‘cut offs’ were applied. These findings were corroborated through inspection of human TOPO IIα IC₅₀ data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the γH2AX by flow assay coupled with structure–activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules.

Introduction

Bacterial type II topoisomerases and types of inhibitors

The quest for new and effective antibiotics has again become very urgent because of concerns about rising bacterial resistance rates and the threat of (re-)emergent epidemics or pandemics, particularly given the paucity of novel anti-infective agents (1). Alongside many promising novel ‘druggable’ targets, bacterial type II topoisomerases remain tractable and clinically validated and thus represent a mainstay for many anti-infective discovery initiatives (2). Bacterial type II topoisomerases, i.e. DNA gyrase and topoisomerase IV, catalyse DNA cleavage reactions that regulate DNA topography and their function is linked inextricably to cell viability; inhibition, among other things, leads to interference in DNA replication, which ultimately results in bactericidality (3).

Since the discovery of DNA gyrase in Escherichia coli some 35 years ago, several chemical classes of bacterial type II topoisomerase inhibitors have been developed that exhibit potent antibacterial activity (4). Heterogeneity in the types of inhibition exists as multiple target sites are present on the topoisomerase enzyme and these respond to different types of agent. For example, fluoroquinolones (FQs; see Figure 1), a widely administered class of antibiotics for the treatment of both Gram-positive and Gram-negative infections, bind to the quinolone resistance-determining region of the catalytic domain and, importantly, immobilise the topoisomerase-DNA complex following DNA cleavage (a process known as topoisomerase ‘poisoning’) (5). Aminocoumarins and cyclothialidines on the other hand target the ATPase region and induce a type of ‘catalytic’ inhibition (6,7). In fact, chemical-mediated catalytic inhibition can occur as a result of interactions at several stages in the catalytic cycle of bacterial type II topoisomerases (8).

Fig. 1.

Exemplars of the bacterial type II topoisomerase drug class; NBTI GSK299423 and FQs ciprofloxacin and moxifloxacin.

Recently, GlaxoSmithKline (GSK) has reported the development of a class of novel bacterial type II topoisomerase inhibitors (NBTIs) whose broad spectrum antibacterial efficacy is underpinned by a unique topoisomerase binding mode (see Figure 1) (9). Rather than modulating type II topoisomerase activity via typical poisoning or catalytic inhibition, NBTIs mediate their bactericidality by stabilising the pre-cleavage topoisomerase-DNA complex prior to DNA cleavage (9). By targeting bacterial type II topoisomerases at a site distinct from other classes of antibiotics, such as FQs, it is thought that even drug-resistant pathogens may be susceptible to NBTIs (9). Thus, it is hoped that this new topoisomerase inhibitor class of antibiotics may go some way to addressing the growing medical concerns regarding the dearth of antibacterial agents with novel mechanisms of action (MoA) in clinical development (1).

Genotoxicity of bacterial type II topoisomerase inhibitors

Although relatively diverse at the molecular level, bacterial type II topoisomerases and their eukaryotic counterpart topoisomerase II (TOPO II) demonstrate sufficient structural homology for potential ‘exaggerated pharmacology’ by bacterial topoisomerase inhibitor drugs to occur, particularly at high concentrations (10). Both poison- and catalytic-type inhibitors have measurable binding against mammalian TOPO II, albeit with affinity sometimes orders of magnitude below those for bacterial type II topoisomerases and, as a result, are known to induce mammalian cytotoxicity in vitro and in vivo (11). Importantly, evidence suggests that the mechanisms driving this cytotoxicity are distinct; poisons induce genotoxicity (as DNA double-strand breaks; DSBs) via the formation of covalent TOPO II-DNA cleavage complexes, whereas catalytic inhibitors affect DNA replication without damaging DNA (12,13). Clearly, both mechanisms may potentially result in adverse effects within the cell, especially if TOPO II-driven toxicities manifest at therapeutic concentrations. Among these is the risk of mammalian cell genotoxicity and the potential consequences for carcinogenesis. TOPO II-mediated genotoxicity, however, is considered to be pharmacologically driven and exhibits a thresholded MoA (see Committee on Mutagenicity 2010 guidance at http://iacom.org.uk/guidstate/documents/Thresholdstatementrevisedfeb2011.pdf). Indeed, there is experimental evidence for the occurrence of a threshold for micronucleus induction by a number of TOPO II inhibitors (14). The European Centre for Ecotoxicology and Toxicology of Chemicals defined an ‘absolute’ threshold as a ‘concentration below which a cell would not notice the presence of the chemical,’ i.e. a threshold assumes that a biological effect only occurs at concentrations above a certain threshold concentration of a chemical. In other words, ‘the chemical is present but does not affect the cellular target’. By implication, if a therapeutic dose is below the threshold for genotoxicity it may be possible to define conditions, e.g. when the affinity of the drug for bacterial type II topoisomerases is orders of magnitude higher that it is for TOPO II and identify a therapeutic window based on cautious safety margins, where the risk of genotoxicity is negligible (15).

Although NBTIs bind to bacterial type II topoisomerases in a unique mode, studies on prototype NBTIs demonstrated that they have the potential to induce DNA damage in mammalian L5178Y cells using the mouse lymphoma screen (MLS) just like FQs and other TOPO II-targeted drugs. At GSK, this assay was used to rank the genotoxicity of bacterial type II topoisomerases inhibitors including NBTIs as part of early drug development. Screening criteria were defined to exclude from further development those compounds that induced relative mutation frequency (MF) increases >6-fold at the limit of cytotoxicity, i.e. relative total growth (RTG) of 10–20%. This 6-fold ‘cut off’ does not represent a definitive assessment of genotoxicity per se but merely provides a means to prioritise compounds for development. It is derived from several years of genetic toxicology screening MLS data using FQs (both externally/internally produced) and NBTIs. This experience was used by the discovery team to progress candidate antibiotics through development with the knowledge that they were not overtly genotoxic like TOPO II-targeted anti-neoplastic agents like etoposide and amsacrine. However, because the MLS is a relatively low-throughput screen with a prolonged data turnaround time (2–3 weeks), an alternative approach was sought to screen NBTIs for genotoxic activity and improve the lead-optimisation process.

Early genotoxicity screening; γH2AX, an emerging biomarker of DNA damage

There are many benefits to promoting early attrition; implementation of higher throughput genotoxicity testing strategies in early phases of drug discovery can improve the prioritisation of compounds for advanced testing, compress lead-optimisation timelines and permit the redirection of resources to chemical series with superior safety profiles (16). In addition, such activities permit structure–activity relationship (SAR) rules to be refined, which in turn can improve rational drug development. We have recently described a novel genotoxicity assay based on the detection of increased serine139-phosphorylated histone H2AX (defined as γH2AX) using flow cytometry (17) and also proposed the use of the same assay as an early genotoxicity screen for new and existing classes of bacterial topoisomerase inhibitors (18). The assay is rooted in the cell’s fundamental response to DNA damage; γH2AX is formed upon the generation of nuclear DSBs and facilitates DNA repair by modulating chromatin structure to allow the retention of DNA repair factors at the site of damage (19,20). Since its discovery, γH2AX expression has been studied as a biomarker of genotoxicity, a biodosimeter of ionising radiation exposure, a surrogate for cell killing (apoptosis) and to assess clinical efficacy in cancer therapy (21–25). Importantly, the γH2AX by flow assay has previously been used to investigate DNA damage induced by topoisomerase I and TOPO II inhibitors, where the high-content nature of this technique also provides information on cell cycling status and apoptosis (21,26,27). As such, the γH2AX by flow assay can produce distinctive cellular response profiles defined as ‘signatures’ which, in addition to DNA damage assessment, can help elucidate chemical MoA (28). Other benefits include low compound requirements, rapidity of data generation and medium-throughput nature, all of which render it, in principle, amenable as a genotoxicity screen in an early drug discovery setting.

In the present study, the γH2AX by flow assay was used to evaluate the genotoxicity of 63 NBTIs and four comparator compounds (the TOPO II inhibitors amsacrine and etoposide and the FQs ciprofloxacin and moxifloxacin) that had been previously characterised in MLS. The aim was to address the performance of the γH2AX by flow assay against the MLS with respect to a focussed NBTI drug discovery programme. Results of the γH2AX by flow assay are presented and discussed in terms of their concordance with MLS and human TOPO II isoform α (hTOPO IIα) IC₅₀ data.

Materials and methods

Cell culture and treatments

Mouse lymphoma L5178Y cells (tk^+/−) were maintained in exponential growth phase in RPMI 1640 medium (GIBCO, UK) supplemented with horse serum (10% v/v; Biosera, UK), penicillin (50 U/ml; GIBCO), streptomycin (50 μg/ml; GIBCO), L-glutamine (2 mM; GIBCO), sodium pyruvate (0.5 mM; GIBCO) and pluronic acid F68 (0.05% v/v; GIBCO). All 63 test NBTIs were dissolved in dimethyl sulphoxide (Sigma–Aldrich, UK). Cell cultures (1.5 × 10⁵ cells/ml) were exposed to either test compound or vehicle for 24 h at 37°C over a range of concentrations. For the γH2AX by flow assay, relative cell counts were immediately determined via Coulter counting to provide an index of cytotoxicity; cells that underwent 25, 50 and 75% cytotoxicity were selected and processed as per the ‘Nuclei isolation and detection of γH2AX’ section. MLS was conducted as per the ‘Mouse lymphoma screen’ section.

Test NBTI nomenclature was defined according to their structural core which has been coded for intellectual property protection purposes; A-J class-derived compounds were screened in this study. All NBTIs were of a similar molecular weight. The podophyllotoxin etoposide, acridine-derived amsacrine (both Sigma–Aldrich) and the FQ antibiotics ciprofloxacin (Sigma–Aldrich) and moxifloxacin (Sequoia Research Products, UK) were also included in the analysis as examples of known TOPO II poisons with varying potencies.

Nuclei isolation and detection of γH2AX

Cells were subjected to centrifugation (150 × g, 5 min) and the supernatant aspirated. Cells were resuspended in cold nuclei isolation buffer (320 mM sucrose, 10 mM Tris–HCl pH 8.0, 2.5 mM MgCl₂, 150 mM NaCl, 0.5% Triton X-100 and 1 mM PMSF; 1 ml per sample) and nuclei subsequently collected by centrifugation (1000 × g, 5 min) before being resuspended in PBS containing 4% anti-human γH2AX-FITC (Biolegend, USA) and 4% 7-AAD DNA counterstain (BD Biosciences, UK) (total volume 300 μl per sample) to form a single nuclei suspension. Visible cell debris/clumps were removed by pipetting. Samples were stored under subdued lighting for 15 min at room temperature before being analysed by flow cytometry.

Flow cytometric evaluation of γH2AX in isolated nuclei

Nuclear green (γH2AX) and red (genomic DNA) fluorescence and forward/side light scatter from 10⁴ nuclei were measured using a FACSCalibur flow cytometer (BD Biosciences) with standard emission filters, FL1 and FL3, respectively. Flow cytometric data were expressed as fold change in FL1 (γH2AX) relative to control levels. Gating analysis on FL3 (genomic DNA) histograms was also carried out to determine the fraction (%) of nuclei in each phase of the cell cycle, i.e. sub-G₁, G₁, S and G₂M.

Mouse lymphoma screen

The 96-well assay (in the absence of S9 metabolic activation) was carried out according to established methodology (29,30). Briefly, following treatment, cell cultures were washed in PBS and adjusted to 2 × 10⁵ cells/ml and adjusted again after a further 24 h. Cell viability, i.e. RTG, was assessed by plating ∼1.6 cells/well and scoring cell colonies 7 days later. To determine relative MF, 2 × 10³ cells/well were plated in media containing 4 μg/ml trifluorothymidine, and mutant cell colonies were scored 10 days later. Evaluation criteria were set around an MLA result of relative MF 6-fold @ RTG 10–20% and this was used as a cut off to rank NBTI genotoxic potential.

Generation of hTOPO IIα IC₅₀s

hTOPO IIα IC₅₀s were generated as per a similar methodology described in (9). Briefly, hTOPO IIα activity was measured by relaxation of supercoiled pBR322 (scpBR322) DNA (TopoGEN, USA). NBTIs in DMSO were pre-incubated with hTOPO IIα (30 nM) and scpBR322 (250 ng) for 15 min prior to the addition of a reaction buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM MgCl₂, 2 mM ATP, 0.5 mM DTT and 30 μg/ml BSA in a total reaction volume of 20 μl. Following incubation at room temperature for 15 min, reactions were stopped using a buffer containing 5% sarkosyl, 0.0025% bromophenol blue and 25% glycerol. Aliquots containing 100 ng DNA were then subjected to electrophoresis in a 1% agarose-TBE gel typically for 18 h at 16 V in TBE running buffer. The gel was stained in TBE buffer containing 0.5 μg/ml ethidium bromide for 20 min, destained twice in TBE buffer for 20 min and then visualized using a Gel Doc-It Imaging System (UVP Inc., USA). Vision Works LS software (UVP Inc.) was used to quantify the intensity of the supercoiled DNA band in each lane and these data were fitted to a generic 4-parameter logistical equation using Grafit software (Erythacus, USA), which permitted the calculation of hTOPO IIα IC₅₀s.

Data analysis

The level of maximal γH2AX induction (derived from nuclei that underwent either 25, 50 or 75% cytotoxicity) was correlated with MLS data obtained up to the limit of cytotoxicity, i.e. relative MF at RTG 10–20% (31), for NBTIs and FQs, or from published data for etoposide and amsacrine using TIBCO Spotfire® Professional software (TIBCO, USA). Other toxicity parameters such as cell cycle arrest information (inherent in γH2AX by flow assay data) and hTOPO IIα IC₅₀ data were also included in the analysis where appropriate.

Results and discussion

We recently described the performance of the γH2AX by flow assay with respect to the standard in vitro genotoxicity test battery, i.e. Ames test, mouse lymphoma and chromosome aberrations assays, in predicting the genotoxicity of 34 chemicals, 22 of which were derived from the European Centre for the Validation of Alternative Methods list (17). In the present study, we applied a similar but modified approach for evaluating the genotoxicity of NBTIs as part of a drug discovery lead-optimisation programme. NBTIs were selected on the basis that they had been characterised in MLS, although published evaluation criteria that define positive or negative responses were deviated from (30). Rather, a relative MF cut off of 6-fold was applied when ranking the genotoxic potential of compounds to help facilitate their prioritisation for further development. These data were critical to the development of any higher throughput alternatives to MLS, in this case the γH2AX by flow assay, as they were to be ‘benchmarked’ exclusively against this endpoint.

γH2AX by flow assay response signatures comprising of γH2AX levels and cell cycling status were elucidated for 63 NBTIs with known genotoxicity in MLS. Data were captured at 25, 50 and 75% cytotoxicity following 24-h treatment as a means to determine any concentration-related effects, however, the only parameters used in this analysis were MLS derived relative MF at RTG 10–20% and maximal γH2AX induction (see Figure 2). Generally, the two genotoxicity endpoints were positively correlated, i.e. as relative MF at RTG 10–20% increased so did maximal γH2AX induction, with relatively few exceptions. In a similar fashion to MLS, a cut off was sought to help stratify NBTIs based on their genotoxic potential; maximal γH2AX induction in excess of 1.4-fold was best correlated with relative MF >6-fold in MLS, and thus 1.4-fold was selected as the γH2AX by flow assay cut off in the context of NBTIs. In addition to γH2AX levels, NBTIs could also largely be differentiated according to the cell cycle phase that cells were arrested in following treatment. Cells arrested in G₂M phase were, in the main, the same cells that exhibited γH2AX levels >1.4-fold. This pattern reflects the classical cellular response to TOPO II poisons where cell cycle arrest occurs after DNA synthesis as the cell progresses towards mitosis and prior to the completion of DNA repair or initiation of apoptosis (32,33). Reassuringly, the comparator TOPO II poisons etoposide, amsacrine, ciprofloxacin and moxifloxacin all induced a maximal γH2AX level >1.4-fold and G₂M arrest, but, as expected, the anti-neoplastic agents which produce their cytotoxic effects via lethal DNA damage exhibited far greater potency and magnitude of response than the FQs, which were more NBTI-like in nature (34,35). On the other hand, NBTIs that induced a maximal γH2AX level at or below the cut off, i.e. ≤1.4-fold, predominately arrested cells in G₁ phase; signatures of this nature have rarely been described in the scientific literature, although some evidence suggests that TOPO II catalytic inhibitors and DNA intercalating agents can elicit such a response (36,37). From the present data, it is difficult to pinpoint the exact underlying MoA but further investigative work, e.g. X-ray crystallography studies, may shed light on this. Interestingly, five NBTIs were shown to induce S phase arrest at their maximal level of γH2AX induction, however, it is not clear if this particular signature was triggered by a different MoA or simply due to temporal differences in DNA damage induction.

Fig. 2.

Induction of genotoxicity by 63 NBTIs and 4 comparator TOPO II poisons. Compounds that induced relative MF at RTG 10–20% cytotoxicity >6-fold (6-fold threshold indicated by small dashed line) were more likely to induce γH2AX levels >1.4-fold (1.4-fold threshold indicated by large dashed line). In addition, NBTIs that exceeded both genotoxicity cut offs mainly induced G2M arrest, whereas less genotoxic NBTIs generally evoked G1 arrest. Circles represent G2M phase-arrested nuclei; squares, G1 arrest; triangles, S arrest; star, sub-G1 DNA.

Figure 3 illustrates the same genotoxicity data reported in Figure 2, although here NBTIs were ‘binned’ according to the cut offs used for the γH2AX by flow assay and MLS, i.e. 1.4- and 6-fold, respectively, in order to compare the performance between assays. NBTIs (22/27) were co-binned as >1.4- and >6-fold and 32/36 were co-binned as ≤1.4- and ≤6-fold, which equated to 86% (54/63) overall concordance when using these genotoxicity cut offs for both assays. Thus, only 14% (9/63) of NBTIs produced discrepant findings. Furthermore, cell cycle arrest data confirmed that 86% (19/22) of NBTIs that breached both genotoxicity cut offs also triggered G₂M arrest which is indicative of a TOPO II poison-like MoA, whereas, 81% (26/32) of NBTIs that exceeded cuts offs in neither assay induced G₁ phase arrest.

Fig. 3.

NBTIs (63) binned according to their effects in γH2AX (1.4-fold threshold) and MLS (6-fold threshold). Expression of data in this manner illustrates the high concordance (86%; 54/63) and low incidence (14%; 9/63) of false predictions (five false negatives and four false positives) produced in the γH2AX by flow assay with respect to MLS. The number in the right-hand corner of each pie chart represents the total number of compounds in each bin. The number of NBTIs that induced G1, S, G2M and sub-G1 phase arrest in each bin is represented as both an integer and %.

The ever-expanding chemical space of NBTIs led to no less than 10 chemical series undergoing genotoxicity assessment in the present study (see Figure 4). NBTI structural core information was overlaid on genotoxicity data to reveal a number of interesting SARs. H class-derived NBTIs accounted for the majority of NBTIs tested and were, on the whole, responsible for inducing genotoxicity above both cut offs, as well as TOPO II poison-like G₂M arrest. A small number of H class NBTIs also elicited the S and G₁ arrest-associated response signatures. J, I and G class-derived NBTIs primarily induced TOPO II poison-like signatures although the magnitude of genotoxic response was variable. NBTIs from the A class were all determined to induce the ‘lack of DNA damage response/G₁ arrest’ signature described above; these data suggest that A class NBTIs may exert their cytotoxicity via another MoA(s) rather than through TOPO II poisoning. Similarly, E, C and the two F and D class-derived NBTIs tested were also shown to exhibit low propensities to damage DNA. B class-derived NBTIs yielded particularly interesting SAR, with extreme responses being observed. B class NBTIs were split as either ≤1.4- and ≤6-fold or >1.4- and >6-fold, however, these responses appeared to be dependent on the configuration of the B class moiety. ‘NHCH₂-type’ B class induced genotoxicity below both cut offs, whereas, a slight chemical modification of the structural core to produce an ‘NHCO-type’ yielded a TOPO II poison-like NBTI that surpassed both cut offs, findings that have been corroborated by γH2AX assessment of other close analogues (data not shown). These SAR data provide clear evidence of how γH2AX by flow technology, if implemented as a frontline screen, could be used by medicinal chemists in the design of less genotoxic NBTIs. For example, providing that other developability properties were similar, greater emphasis could have been placed on optimising A or E class-derived NBTIs earlier rather than H class NBTIs, which appear to carry more genotoxic liability in vitro.

Fig. 4.

SAR with respect to genotoxicity for 63 NBTIs and 4 comparator TOPO II poisons. H class-derived NBTIs were determined to be TOPO II poison-like compounds with a greater propensity to induce genotoxicity, with A and E class-derived NBTIs exhibiting less genotoxicity via a non-TOPO II poison MoA. Circles represent G2M phase-arrested nuclei; squares, G1 arrest; triangles, S arrest; star, sub-G1 DNA.

The capacity of a chemical to inhibit TOPO II activity, i.e. its IC₅₀, has previously been causally linked with its ability to induce DNA damage (38). Many anti-neoplastic agents including etoposide and amsacrine mediate their cell-killing effects through strong, targeted inhibition of the hTOPO IIα isoform, which is the variant primarily involved in DNA replication (39–41). For bacterial type II topoisomerase inhibitors that poison TOPO II, this association appears to hold true; four NBTIs that induced TOPO II poison-like responses, i.e. exceeded γH2AX and MLS cut offs, and G₂M arrest had hTOPO IIα IC₅₀s ≤5 μg/ml (see Figure 5). Other NBTIs that induced comparable levels of genotoxicity and that were similarly defined as TOPO II poisons due to their signature responses returned hTOPO IIα IC₅₀s between 26 and 50 μg/ml, however, somewhat paradoxically, several NBTIs with equivalent inhibitory capacity were found to be less genotoxic. These IC₅₀ data, in combination with information derived from response signatures, provide robust evidence for the existence of at least two distinct mechanisms for NBTI-mediated TOPO II inhibition. Importantly, although mouse and hTOPO IIα counterparts are highly homologous, subtle structural variations may also contribute towards the differences observed between these two in vitro systems (42).

Fig. 5.

Effect of hTOPO IIα inhibition on genotoxicity for 26 NBTIs. Strong inhibition of hTOPO IIα led to increased genotoxicity for some NBTIs, however, in some cases no or only weak genotoxicity was observed, suggesting the existence of at least two MoA. Spheres represent G2M phase-arrested nuclei; cubes, G1 arrest; cone, S arrest; star, sub-G1 DNA. It should be noted that markers are ‘jittered’ for clarity, however, because of this their position within the graph may not be entirely accurate.

Concluding remarks

The γH2AX by flow assay represents a rapid, medium-throughput and high-content genotoxicity assay that can be used to evaluate the propensity for compounds to induce DSBs in mammalian cells and also provide insight into MoA. Compounds which induce high levels of γH2AX are likely to generate positive results in the standard regulatory genotoxicity test battery and, therefore, the γH2AX by flow assay can be used to rank molecules according to genotoxic potential. Moreover, its high-content nature provides a rich source of SAR for bioinformatics assessment. Thus, when deployed in an early screening capacity, the γH2AX by flow assay coupled with SAR evaluation can positively impact medicinal chemistry efforts, which may ultimately lead to the production of inherently safer molecules. This conclusion is exemplified by the findings of the present study using bacterial type II topoisomerase inhibitors, where implementation of the γH2AX by flow assay alongside SAR assessment was used to develop NBTIs with superior genotoxicity profiles. Clearly, genotoxicity screening in this manner need not be limited to TOPO II-targeted drugs, as a similar strategy could be exploited in those scenarios where a potential genotoxic liability has been identified, e.g. based on target pharmacology or propensity for ‘off target’ effects (43).

Funding

Wellcome Trust Seeding Drug Discovery Initiative and Transformational Medical Technologies program contract HDTRA1-07-9-0002 from the Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA).