Advanced microscopy solutions for monitoring the kinetics and dynamics of drug–DNA targeting in living cells

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Abstract

Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug–target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon ‘dark’ DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug–target interactions with cellular stress responses.

Introduction

DNA is a significant target for a wide range of pharmacologically active agents not least many of the drugs deployed in anticancer therapy. A wide range of molecular interactions and target modifications is possible with critical events often involving the minor groove. Indeed, drugs that bind within the DNA minor groove are of considerable interest for their antimicrobial and antitumor activities, providing an impetus to studies on the dynamics of interactions at the minor groove together with the development of tools to investigate critical events in the native environment of the living cell. There are three areas in which the study of minor groove binding (MGB) ligands (MGBLs) have raised considerable interest:

The original X-ray analysis studies of Kopka et al. [1] of the complex of netropsin with a B-DNA dodecamer revealed that the antitumor antibiotic was capable of binding within the minor groove, by displacing the water molecules of the spine of hydration with base specificity achieved not by hydrogen bonding but by close van der Waals contacts. Accordingly, polyamide drugs, such as netropsin, distamycin and their derivatives, can be inserted into a narrow B-DNA minor groove to form 1:1 complexes that can distinguish AT base pairs from GC [2]. Subsequent attempts to develop synthetic “lexitropsins”—molecules capable of reading any desired short sequence of DNA base pairs—have involved a wide range of compounds including epipodophyllotoxin, bithiazoles, acridines, anthraquinones, ellipticine, nitrosoureas, benzoyl mustards and nitrogen mustards [3]. In most of these cases, each of the individual components of the lexitropsin conjugate retain their modes of action as far as interaction with DNA, and therefore opening up the possibility of the minor groove interaction being a critical design feature in the development of drugs with modified sequence selectivity.

Understanding the mechanisms of action of MGBLs is important given the increasing knowledge of the role of DNA sequence and conformation recognition in the assembly of molecular complexes for replication, transcription, recombination and repair. Thus, DNA sequence-selective binding agents bearing conjugated effectors have potential applications in the diagnosis and treatment of cancers as well as providing probes for investigating the molecular biology of the cell [3]. An important step forward is the realization that a number of MGBLs interfere with the catalytic activities of the DNA topoisomerases, and there is evidence that in some cases, this may be a primary determinant of the cytotoxic action of the agent [4]. The type I and type II DNA topoisomerases are nuclear enzymes that regulate topological and conformational changes in DNA, critical to cellular processes such as replication, transcription, chromosome segregation and the efficient traverse of mitosis. For example, the identification of the MGB activity of the type I DNA topoisomerase poison class of camptothecins provides new insights into the mechanisms of enzyme trapping on DNA and the subsequent cytotoxic events as the ternary complex of DNA–drug–enzyme interacts with active DNA replication forks in S-phase of the cell cycle. Thus, there is considerable interest in topoisomerase I as a therapeutic target [5], not least due to the cell cycle specificity of the pharmacodynamic response and the potential for combination with other agents generating discrete effects in other cell cycle phases.

This overriding issue is a major problem limiting the effectiveness of initially active anticancer agents. Resistance can arise from cellular or subcellular pharmacokinetic reasons, changes in target sensitivity or availability and not least in the effector pathways for drug responses. Active drug efflux transporters of the ATP binding cassette (ABC)-superfamily of proteins have a major impact on the pharmacological behavior of most of the drugs in use today [6]. For example, the MDR1 (ABCB1) gene product, P-glycoprotein, is a membrane protein functioning as an ATP-dependent exporter of xenobiotics from cells. Its importance was first recognized because of its role in the development of multidrug resistance (MDR) of cultured tumor cells against various anticancer agents, but it also has critical function in normal tissues such as the brain, kidneys, liver and intestines. Early studies recognized the potential for MGBLs to act as substrates for drug efflux mechanisms [7], [8].

An MGBL target DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin and its derivatives specifically target DNA topoisomerase I, and several mutations, have been isolated that render the enzyme camptothecin-resistant [9]. Interestingly, there may be other discrete mechanisms of drug resistance associated with MGBL that have yet be fully characterized. The discovery of an enhanced process for the ejection of the MGBL, Hoechst 33342, from the DNA [10] of cells with selective resistance to MGBLs [11], [12] highlights the need to understand the dynamics of DNA–ligand interaction in live cells.

Advanced microscopy solutions in the study of the spatial and temporal aspects of the drug–target interactions in live cells can address issues in each of the interest areas discussed above. Analysis at the single-cell level addresses the problems of inherent heterogeneity observed in many biological systems. Early studies on MGBL or anticancer drug–DNA interactions often exploited the convenient fluorescent signatures and spectral characteristics of the agents [13], [14], [15].

Section snippets

Minor groove binders with fluorescent signatures

Many bioactive molecules, particularly those comprising linked ring structures, have chromophores capable of fluorescence excitation and therefore offer the possibility for tracking target interactions through methods such as monitoring steady-state fluorescence intensity, fluorescence quenching and fluorescence lifetime measurements. The bis-benzimide dyes or Hoechst probes, for example, have been extensively used to determine DNA content and nuclear morphology in fixed cell preparations [16].

Mapping subcellular localization of bioactive drugs

Pharmacokinetic maps reflecting inherent cell–cell heterogeneity for TPT drug delivery within a population of human breast tumour MCF-7 cells were acquired using TPLSM. To confirm previous spectroscopy studies that TPT can be detected using two-photon excitation, a concentration of 10 μM of free TPT was added to cultures seeded in a coverslip observation chamber, and single optical sections collected using TPLSM at 790 nm and fluorescence emission collected between 460 and 630 nm. Initial

Single-cell time-lapse microscopy

Multicompartmental tracking of TPT uptake and washout kinetics in single cells provides a route for screening the dynamic process of drug delivery. The TPT uptake and delivery to the three cellular compartments (medium, cytoplasm and nucleus) was monitored using time-lapse TPLSM. Optical sections were acquired at an interval rate of 4.5 s (Fig. 2A). The presence of TPT fluorescence in the extracellular medium immediately became detectable post-addition and the cells appeared negatively stained.

Two-photon ‘dark’ DNA binding probes as quenching tools

Pharmacokinetic characterization of an MGBL requires assays which incorporate informative molecular tools and conceptually the strategy for drug discovery and fluorescent probe discovery are the same. Therefore, a molecular modeling approach can be implemented to search for MGBL quenching agents with defined DNA binding and spectral properties. Quenching assays provide a unique means for dissecting subresolution molecular interactions.

Fluorescence lifetime imaging microscopy (FLIM)

FLIM is a direct approach to monitoring all processes involving energy transfer between the fluorophore and the local environment [38], such as that which occurs when a fluorescent drug tethers to its DNA target [38], [39]. Any energy transfer between the excited molecule and its environment changes the fluorescence lifetime in a predictable way.

Linking multiscale responses

A significant challenge for the advancement of drug screening and evaluation is to link the biochemical and behavioural responses of genetically profiled cells with the initial events of drug-–target interaction. The efficiency and consequences of drug–target interaction can clearly be affected by pharmacokinetic factors but are also driven by parallel cellular events that are required to elicit the sought pharmacodynamic responses in a single cell. In understanding the pathways that determine

High-throughput screening for enhancing the drug discovery process

In order to meet the challenge of a rapidly increasing library of compounds within a drug discovery setting, and our growing understanding of emerging new cellular targets, the advanced assays described in this review need to be both sensitive and fast to work on high-throughput screening (HTS) imaging platforms. HTS calls for rigorous demands with respect to assay robustness and statistical accuracy. The current cellular imaging platforms commercially available are appropriate for both

Conclusion

Drug design, discovery and deployment paradigms must progress from a situation where the cell system is an ill-defined and often homogenous “black-box” to an approach where the critical targets and molecular events within the cells become well-defined and provide spatially and temporally rich information. This ensures that drugs are not being rejected from a study because the signal-to-noise of a heterogeneous population is low, while in fact, the specificity or targeting of a drug is actually

Acknowledgements

The work was funded by grants awarded to PJS and RJE by UK Research Councils (GR/s23483), BBSRC (75/E19292), AICR (00-292) and SBRI (19666).

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