In summary, the NAD+ analogues described in here have proven their potential for deciphering important questions in PAR research by offering the opportunity to monitor the polymer in real-time and in its natural environment with the help of fluorescence microscopy.
Although all compounds synthesised in here were obtained in reasonable yields, the NAD+ synthesis route offers still some possibilities for optimisations. In case of compounds 9, 10 and 11, the introduction of a different linker could be beneficial to avoid the formation of an unreactive side product during TFA deprotection. For instance, diaminoalkyl spacers resulting in compound 83 would be a possible option as they are known to be stable under the chosen deprotection conditions. Moreover, the reporter groups in this project were mostly introduced on the level of adenosine or adenosine monophosphate. Using a suitable protection group and mild cleavage conditions, the functionalisation at the level of NAD+ would become possible and thus creating a more flexible synthesis route and easier access to derivatives (Scheme 17).
Scheme 17. Possible NAD+ analogue 83 and more flexible synthesis strategy. PG… protecting group, NHS…
N-hydroxysuccinimide ester.
During the last years, research groups also reported on different strategies to from the phosphate anhydride linkage.[170] Methods involving P(III)-P(IV) couplings[171] were found to be extremely fast (minute scale) and high yielding (>90%). As the synthesis method applied in here takes four days for anhydride bond formation, these new chemistries should also be advantageous for synthesising NAD+ derivatives.
To achieve a higher applicability, milder delivery methods to transfer the compounds across the cell membrane should be explored. One possibility provides a new technique called cell squeezing.[172-176] Here, cells are pushed through a microfluidic chip with multiple channels, that feature constriction sites in the dimension range of the cells. Accordingly, the chip dimensions must be optimised for the applied cell line and it has been shown, that even sensitive and hard to handle cell lines can be transfected in an efficient manner. While passing them, cells are mechanically deformed and the cell membrane is temporally disrupted. Now, the surrounding medium can diffuse inside the cytoplasm, while the membrane recovers. It this way, small molecules,[172] peptides, proteins, oligonucleotides and antibodies[173] have already been transferred into cells in a mild way and making further biological manipulations possible.
Another possibility is to chemically modify the NAD+ molecule to obtain a cell-permeable nucleotide. Cell-permeability has already been achieved for other phosphates such as ATP[177-179] or inositol-pyrophosphates.[180-181]
In principle, there are three strategies imaginable (Figure 35). First, nucleotides can be complexed with suitable agents and the formed complex is then able to cross the cell-membrane. The carrier-peptide described in chapter 5.4 follows this strategy. However, there are a range of other molecular transporters[182] available, which usually contain poly-arginines or guanidinium-rich motifs. For instance, the transporter depicted in Figure 35 has been applied for the cellular delivery[180] of inositol-pyrophosphates.
A second promising approach is the so-called covalent ‘caging’ of the negative charge with positively charged substituents. A spermine-derived polyamine-linker has successfully been used to obtain a cell-permeable ATP analogue,[177] which could also be helpful in case of NAD+. However, the negative charge of PAR is crucial for its binding properties and a permanent positive charge on the PAR molecule might be contraproductive. Thus, a positively charged, traceless linker that can be cleaved off after cell entry,[182] e.g. by intracellular thiols as depicted in Figure 35, might also be effective.
Third, it might be sufficient to increase the overall lipophilicity of the NAD+ molecule. This could be achieved by either using longer alkyl-linkers in position 2, or by attaching lipophilic anchors via ester bond formation at the riboses or phosphates. These kinds of anchors are known to be cleaved by intracellular lipases. In case of ATP, the phosphate linker depicted has shown immense potential to transfer bioactive triphosphates as pro-drugs[178-179] across the cell-membrane.
Figure 35. Chemical Strategies to render NAD+ cell-permeable.
Another important area of research is the deciphering of the so-called ‘PARylome’. The identification and validation of covalent and non-covalent interaction partners help to understand the effects and consequences of PARylation. Progress has been achieved within the last years using 6-modified NAD+ analogues and mass spectrometric analyses[37, 68-69, 183-184] of pull-downs in cell-extracts. After finding that at least for ARTD1 and ARTD2 the 2-biotin-modified analogue 84 (Figure 36) might be a better substrate, new interaction partners
Outlook and Future Research Directions
that are usually not heavily PARylated might be unravelled. Moreover, the developed FLIM-FRET approach can then help to validate these interactions inside of living cells. In principle, it would be even possible to monitor the protein-specific PARylation in real-time. For this, the available FLIM-FRET microscope should be equipped with the femtosecond fibre laser source and the associated control instruments.
Figure 36. Possible NAD+ analogues to improve pull-down probes.
Moreover, non-covalent PAR interaction partners could be identified in pull-down experiment using a photocrosslinking approach. Earlier in PAR research, 2-azido-NAD+ has already been used for crosslinking.[185-186] Due to the azido/tetrazole-tautomerism, the crosslinking efficiency might be increased by applying different photocrosslinkers in adenine position 2 such as diaziridines, benzylazides or benzophenones (Figure 36). A powerful new NAD+ analogue would than contain both biotin and the photocrosslinker in one molecule.
Considering that the negative charge of PAR mediates protein binding, one could also try to attach the photocrosslinker closer to the phosphate backbone, for instance at the ribose ring position 4’ or directly at the phosphate. Of course, these analogues need to prove their substrate properties beforehand.
Furthermore, one could envision to create NAD+ hybrid molecules, that contain additionally peptide sequences targeting specific cellular compartments, such as mitochondria.[187] After reaching their desired localisation, they are cleaved for instance by specific proteases and will be available for ADP-ribosylation, photocrosslinking and pull-down of proteins in this area. This approach would be beneficial to unravel and study non-DNA-repair related functions of ARTD1, as usually the massive signal originating from the nucleus superimposes the others.
Finally, the tools presented in here can be very helpful to develop and establish new and advanced ADP-ribosylation assays. Since fluorescence lifetimes provide a fast read-out of ARTD1 activity in real-time, this can be exploited for a new in vitro ARTD1 activity assay that is more robust towards changes in probe concentration, fluctuations in excitation intensity, photobleaching, and other factors that limit intensity-based steady-state measurements.[100]
Moreover, new cellular inhibitor screenings can be envisioned. Combining the FRET approach with flow cytometry (FRET-FACS)[188-189] would even allow to design an assay, where the inhibition of DNA damage induced PARylation of ARTD1 by test substances could be assessed in cells and with statistical significance. This would enable to find and evaluate ARTD1 inhibitors in a more natural and meaningful setting.
Chemical Synthesis