3) DNA polymerases as drug targets 3.1) General
As foreshadowed in previous chapters, DNA replication serves not only as a target for answering chemical genetic issues, but has been tried and tested for decades as prominent target for life‐saving medicines. Numerous pathological states, like cancer, autoimmune disease, and many bacterial and viral infections can be traced back to uncontrolled DNA metabolism.15‐17,20,37,51,70,79
For that reason, it is one of modern medicine's top priorities to combat those diseases with novel and innovative drugs. If one wants to specifically interfere in DNA metabolism, DNA polymerases of humans, animals, viruses, and bacteria are potential key drug targets, which are responsible for the correct synthesis and repair of the genetic code (see also chapter 2).15‐17,37,41,53,57,80
Due to the fact, that DNA polymerases share a similar 3D‐structure, and reaction mechanism to synthesise DNA, it is very challenging to address a drug molecule for a particular enzyme of interest (see also chapter 2.2). However, if the drug has a poor selectivity in its mode of action, the therapy is limited and inevitably associated with severe side effects. Current medicines employed to target the metabolism of DNA often induce DNA damage (see also Figure 5, and chapter 2.3),70 influence the dNTP pool, that is provided for DNA synthesis by the cell, or inhibit the enzymatic synthesis of DNA.41 For the last‐mentioned therapeutic strategy, which is relevant for this work, nucleoside analogs are a frequently used type of drug. Nucleosides, that are able to enter a cell (Figure 6, see also chapter 5.1), function as so called “prodrugs”, which have to be transformed by specific cellular nucleoside kinases to the actual enzyme substrate analogue;
the nucleoside‐5`‐triphosphates.20,41 Nucleoside‐5`‐triphosphates derivatives that are not/hardly able to enter cells cause often complications. In order to be effective, they have to compete with the natural dNTP substrate pool for the active site of the DNA polymerase target.20,41 On this account, the respective nucleosides must be administered in high
concentrations to be effective, which can result in turn in selectivity and drug resistance problems.20,41 An additional disadvantage is, that nucleotide analogues and nucleoside‐
5`‐triphosphates can also be utilized, modified, or degraded by a multiplicity of cellular pathways or other enzymes.13,15,17,20,37,42,70,74,79,81
Thus, in developing nucleoside analogues, it is necessary to investigate not only the optimisation of their interaction with DNA polymerases, but also their ability to be transformed to the 5`‐triphosphates without being degraded or showing serious side effects.20
Figure 6. Chemical structures of approved modified nucleoside analogues. (A) Current antiviral drugs (see also chapter 5.1). (B) Currant anticancer drugs. Figure was adapted from literature 20,41.
To address these problems and other issues, it is an ongoing concern to develop innovative therapeutic approaches and novel drugs.88‐91 In the following, some for this work relevant examples and perspectives are given.
One current option to open up the way for novel drugs and nucleoside analogues is, for example, to study certain structural features of various known molecules. Afterwards, the interesting features are fused together in one novel drug‐like molecule, bearing all desired properties (see also chapter 5.1).
Other research approaches proceed towards the development of non nucleosidic small‐
molecule inhibitors, which act on the activity of a respective DNA polymerase. Thereby, the
small‐molecule could act directly on the active site of the enzyme, induce conformational changes in further protein domains (e.g. finger or thumb domain) to act indirectly on the enzymes` activity, or block protein‐protein interactions that are important for the processivity.12,13,20,41,92‐94
In general, small‐molecule DNA polymerase inhibitors have important advantages over substrate analogues. They are ideally suited to tune for a high target selectivity, they are capable to enter a cell and do not require intercellular activation.13,20,41
Many chemotherapeutic regimes in use depend at least in part on the artificial induction of DNA damage. The clinical efficacy of anticancer drugs is often reduced by cellular DNA repair mechanisms.15‐17,20,37,51,70,78,79 Consequently, specialized DNA polymerases are discussed as promising future drug targets, to reduce the dosage of DNA‐damaging agents while improving their activity via inhibition of their respective DNA repair pathways (see also chapter 4).17,37,51,70,78,79
3.2) Screening methods for DNA polymerase inhibitors
In the preceding chapters it was described in detail, that there is a great demand for novel DNA polymerases inhibitors in basic as well as in the applied sciences. The discovery process of novel agents against a chosen protein target usually involves high‐throughput screening (HTS) or high‐content screening (HCS) techniques, wherein large compound libraries are screened for the desired effect (see also chapter 1).95 Apart from emerging in silico based methods20,96‐101, one needs for such large‐scale screening campaigns automated, robust, reliable, and cheap in vitro assays.95 DNA polymerase inhibitors generally exert their effects through interference with the enzyme and/or cofactors, or the direct interaction with
DNA.102 While there is no simple way to study DNA replication in vitro, the world‐renowned
polymerase chain reaction (PCR) is an ideally suited enzyme assay, which involves a similar set of DNA replication transactions.103‐105 Classic PCR and the respective electrophoresis techniques104,105 have been applied previously for the in vitro discovery and characterisation of several inhibitors of thermostable DNA polymerases.103,106‐110 However, to screen thousands of molecules, the modern real‐time PCR is considerably more favourable, and hundreds of in vitro reactions could be screened in parallel.111,112 Real‐time PCR instruments are able to monitor the concentration of the arising dsDNA products in multi‐well formats by
measuring for example the emission of sequence‐specific fluorescent oligonucleotide probes or fluorogenic dsDNA binders like SYBR® Green I.111
Because the common mammalian, viral, and bacterial target DNA polymerases are thermolabile, these proteins are not applicable for modern PCR and other screening methods are required that can be performed at moderate temperatures.
The primer extension reaction (PEX) is an effective and useful way to explore thermolabile and thermostable DNA polymerases in the laboratory.43,44,75,76,113‐119
To study the DNA template dependent DNA polymerization function, a short radioactive or fluorescence labeled DNA primer strand gets annealed to a longer DNA template strand, and gets elongated by enzymatic dNMP incorporation (Figure 7A). Noteworthy, to analyze TdT enzyme activities the reaction is performed without the template strand and is named single‐
stranded PEX.59,60,120 However, after a defined period of time, the PEX reactions are quenched and quantitatively analyzed via polyacrylamide gel electrophoresis (PAGE) (Figure 7A).
Figure 7. Generally applicable radioactive PEX assays to characterize and screen small‐molecule inhibitors of thermolabile DNA polymerases (A) Principle of the radioactive PEX assay to analyze the effect of an inhibitor on the DNA template‐dependent polymerisation function of a respective DNA polymerase.75,76 (B) Assay scheme for inhibitor screening via the scintillation method. DNA polymerase polymerizes isotopic phosphate
labelled dNMPs. The exact amount of formed radioactive DNA can be determined by scintillation measurements.
Another common technique to analyse the PEX reaction mixtures is the scintillation method.
Therefore, the reaction is performed with an unlabelled primer template‐complex and isotopic labelled dNTPs as enzyme substrates. After the reaction, the radioactivity of the novel synthesized DNA is quantitatively measured by scintillation (Figure 7B).120‐125
So far, both PEX methods were implemented for the screening and characterisation of inhibitors,75,76,100,120,124,126,127
but to screen huge compound libraries, PAGE analytics and accordingly the usage of radioactive reagents is not practicable. Therefore, novel automatable assay readouts and the respective tailor‐made reagents were evolved. As a first excellent example, Summerer et al. developed a Förster Resonance Energy Transfer (FRET)‐
based assay format that translates the proceeding DNA synthesis into a fluorescent signal in real‐time (Figure 8A).128,129 The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon, by extension of the primer strand.128,129 The resulting distance alteration is reported by FRET between two dyes introduced into the molecular beacon stem and enables the quantitative characterization of inhibitors.128,129 Recently, the elegant real‐time strategy was adopted from others130 and successfully utilised in a further developed fashion for screening campaigns of human DNA polymerases (Figure 8B).131‐133
Figure 8. Screening for inhibitors via real‐time FRET methods. (A) The template probe labelled with donor (grey) and acceptor (brown) has a hairpin extension in closed conformation before start of reaction. While extension proceeds, the DNA polymerase (blue) opens the stem and prevents re‐annealing by DNA duplex formation. The increase in the distance between the two labels is reported by restoration of donor emission.
Figure was adapted from literature 128,129. (B) Strand displacement DNA synthesis assay. DNA polymerase incorporates dNTP thereby extending the primer strand and displacing the downstream reporter strand labelled with a 3`‐fluorophore donor, leading to an increased fluorescence signal. Figure was adapted from literature 131‐133.
To access continuous FRET‐based PEX assays, some researchers focused on the enzymatic turnover of fluorescently labled dUTP substrates. For that reason, Cauchon et al. desined a primer‐template complex, that was labelled at the 5`‐template‐terminus with a donor fluorophore.134 Polymerisation mediated by incubation with a DNA polymerase, dNTPs, and an acceptor labeled dUTP juxtaposes the donor‐acceptor pairs, resulting in donor quenching (Figure 9A).134 On the other hand, Krebs et al. reported an FRET assay that quantifies the incorporation of complementary pairs of fluorescently labeled dUMP into the DNA product, and taking advantage, that the dye‐conjugated dNTP pairs in solution do not interact to produce a FRET signal (Figure 9B).135
Figure 9. Screening for inhibitors using fluorescently labled dUTP substrates. (A) The substrate is a short DNA/DNA primer/template. The template strand is labelled with a donor fluorophore (grey). Polymerization mediated by incubation with DNA polymerase (blue), dNTP, and an acceptor‐labelled dUTP (brown) juxtaposes the donor‐acceptor pairs, resulting in donor quenching. Figure was adapted from literature 134. (B) Dye‐
conjugated nucleotide pairs in solution do not interact to produce a FRET signal. DNA polymerase incorporates the dye‐nucleotides into the DNA. The close proximity of the two dyes in the polymer allows interaction between the dyes causing the generation of a FRET signal proportional to the amount of DNA produced in the sample. Figure was adapted from literature 135.
Other interesting examples amenable to automation are fluorometric PEX techniques that monitor the concentration of the arising dsDNA products.75,136‐138 The increase of the fluorescence signal caused by PicoGreen™136 or SYBR® Green I75,137,138 emitting upon binding to dsDNA was investigated as a fast readout for DNA polymerase activity (Figure 10). PEX resulted in high concentrations of dsDNA when the respective DNA polymerase was not inhibited.75,137,138 On the contrary, when the enzyme was inhibited, the primer was not extended and the fluorescence signal was low in relation to control reactions.75 Using that simple and economical method, diverse compound libraries were screened and potential
inhibitors of bacterial139 and human75 DNA polymerases were identified (Figure 10, see also chapter 4.1). Recently, Dallmann et al. extended the scope of this readout and established an in vitro assay for the parallel multiplicative target HTS against divergent bacterial replicases.140
Figure 10. Principle of the SYBR® Green I (stars) ‐ based HCS assay.75
Of note, a possible approach could also be the measurement of the arising pyrophosphates ions (PPi) that are produced by enzymatic dNTP consumtion (see also Figure 2). Reportedly, PPi release of DNA polymerases was investigated in real‐time for instance by different colorimetric and/or enzyme coupled assays.141‐143
In the recent years, DNA arrayed ultra‐HTS formats for PEX reactions were developed.
Interestingly, these systems are time and cost efficient, and require only minimal amounts of reagents. DNA arrays are based on the spatial separation of on a surfaces immobilized or covalently bound primers.20,53,119,144 The enzymes, templates, screening compounds, buffers, and natural as well as labelled (e.g. radioactive or fluorescent) dNTP substrates can also be applied with spatial separation to perform the PEX reactions.53,119,144 After the reaction, the surfaces are washed several times ‐ whereas the primers remain ‐ and can be analysed by phosphor imaging144 or fluorescents measurements53,119, (Figure 11). By employment of this time and cost efficient concepts, Boudsocq et al. could identify a variety of natural products that inhibit the BER enzyme pol β.53
Figure 11. Principle of DNA arrayed HTS format ‐ using fluorescently labelled dNTP substrates.
As one can see in this overview, many automatable techniques to screen DNA polymerase inhibitors are established and are ready for their application to screen the chemical space.
These methods have indeed the potential to discover novel interesting molecules that not only might be of great value for basic science but also may open up novel avenues for the treatment of diseases related to genome integrity.
Concepts and objectives
The survival and development of each organism relies on the equal distribution of its genome during cell division. DNA polymerases are key enzymes to pass the exact genomic information down generations.15,17,20,22
In the last decades several novel DNA polymerases were discovered, and so, at least 15 different human DNA polymerases are known today.15,17,20,22
Features of some of these enzymes are known, but to understand in depth the task of a particular enzyme stills await clarification in the majority of the cases. For that reason, there is a great demand for appropriate methods and molecular tools to dissect the respective biological functions of DNA polymerases.
The aim of this work (chapter 4) deals with the discovery and development of tailored small‐
molecule probes in order to gain insights into the functions of human pol λ and β. In previous surveys several small‐molecule inhibitors of pol λ and few moderate inhibitors of pol β were discovered.75,76 Importantly, the rhodanine‐based compounds were the most active inhibitor class and some of them could even discriminate between the highly homologous pol λ and β.75,76 Due to this facts, the rhodanine‐based small‐molecules were seen as an appropriate starting point for the development of molecular probes to specifically investigate the biological functions of pol λ and β.75,76 For this purpose, systematic synthetic optimization should be undertaken in order to further expand the chemical diversity and to find novel and more potent small‐molecule inhibitors of pol λ and β. The most promising inhibitors of the first and second generation should be further investigated enzymatically and should be evaluated in comparison to reference inhibitors. Out of the generated in virto data extensive SAR should be established and discussed in detail. With the aim to further develop the molecular probes in a cellular context, the probes should be explored on different human cell lines. Afterwards, the suited molecular probes should be investigated in proof‐of‐concept studies to specifically target the pol λ and β in their respective biological pathways.
In addition, DNA polymerases serve not only as a target for molecular probes to dissect their biological functions, but have been tried and tested for decades as prominent target for life‐
saving medicines.15,17,20,22,37,41
In the last decades, 4’‐C‐modified nucleoside analogues aroused scientific interest, because a couple of derivatives of this interesting compound class showed antiviral activity145‐153, even against multi‐drug resistant pathogens.146,154 Because
there is a great demand for the development of drugs and consequently also for novel nucleoside analogues, novel potentially antiviral 4’‐C‐modified nucleoside analogues should be designed and developed in the second part of this work (chapter 5). Therefore, the molecular features of 4’‐C‐modified nucleosides and other pharmacologically interesting nucleoside analogues should be fused together into one inventive small‐molecule.
Afterwards, the synthetic route for these innovative analogues and their versatile synthetic building blocks should be developed and performed.