In this present study, we have developed and validated a novel, quantitative, nonradiometric assay for nucleic acid binding to TDP-43. We used this assay to characterize the binding of bt-TAR-32 and bt-TG6 and are the first to report association and dissociation rate constants for these interactions. The binding of both bt-TAR-32 and bt-TG6 to TDP-43 was saturable and of high affinity, with sub-nanomolar KD
values. The association rates for both bt-TAR-32 and bt-TG6 binding to TDP-43 were similar, whereas the dissociation rate of bt-TAR-32 was slower than that of bt-TG6. The observed association rates of both bt-TAR-32 and bt-TG6 increased linearly with ligand concentration, which is consistent with a lack of cooperativity between binding sites.21
Furthermore, the equilibrium dissociation constants as determined by the ratio of the dissociation and association rate constants were the same as that obtained from equilibrium binding experiments. These results suggest that the stoichiometry of binding of DNA oligonucleotides to TDP-43 is 1:1, with no apparent cooperativity, and that binding is consistent with the law of mass action.
The rank order affinities of unlabeled DNA and RNA oligonucleotides obtained in this study were consistent with pharmacology reported by other investigators for TDP-43.8
Similar to previous studies, the greater number of TG repeats resulted in increased affinity for TDP-43. It is important to note that the apparent affinities of both TG8 and TG12 were in the picomolar range and therefore may be an underestimation of their actual affinities because of significant ligand depletion, given the concentration of TDP-43 (40 pM) in these experiments. It is also interesting that the affinity of TG8 was more than 100-fold greater than TG6, and the affinity of TG6 was 30-fold greater than TG4, thereby indicating that TDP-43 is highly sensitive to the number of TG repeats in an oligonucleotide sequence. Although the affinities of RNA oligonucleotides were much less than those of DNA oligonucleotides, the rank order affinities were consistent with TDP-43 pharmacology.8
Although it has been shown that TDP-43 does not bind TAR RNA,2
it is capable of binding well to both TG DNA repeats and UG RNA repeats in electromobility shift assays.7,8
In our assay, we found that UG repeats were at least 3 orders of magnitude less potent than corresponding TG repeats. It should be noted that although there is an overall shift in potency between RNA and DNA, the weaker interactions with RNA are sub-micromolar and would yield signals in the UV-crossing electromobility shift assays used previously.8
Interestingly, investigators have reported that TDP-43 binding to DNA oligonucleotides resulted in multiple species of complexes, whereas those for RNA formed single complexes.8
It may therefore be that the potency shift for DNA relative to RNA in our quantitative assay is due to multiple noncooperative binding interactions between DNA and TDP-43 that do not occur with RNA.
The dissociation rates of both bt-TAR-32 and bt-TG6 were extremely slow, with half-lives of 750 min and 150 min, respectively, which are consistent with their high affinity for TDP-43. In addition, the affinities of TG12 and TG8 for TDP-43 would suggest that the dissociation rates of these oligonucleotides may be even slower than that of bt-TAR-32. This characteristic “tight-binding” property of these oligonucleotides is interesting in light of the putative role of TDP-43 as a transcription repressor. Theoretically, once TDP-43 is bound to these DNA sequences, the interaction is “pseudo-irreversible,” and a conformational change in the protein is necessary for dissociation and subsequent transcription to occur. Such a conformational change could occur through phosphorylation and/or allosteric binding of another factor to TDP-43. Investigators have shown that the glycine-rich C-terminal tail of TDP-43 is necessary for recruitment of other splicing inhibitory proteins in exon 9 skipping of the CFTR mRNA.1,8,22
Although a direct negative allosteric interaction with TDP-43 has not yet been described, our quantitative assay could be useful in identifying such interactions.
The assay we have developed using AlphaScreen®
technology is suitable for HTS. The assay performance was robust, as indicated by Z′ values greater than 0.5 and variability within acceptable limits.20
As a result of screening a small library of 7360 chemically diverse and drug-like compounds, we successfully identified a series of structurally similar compounds that disrupt bt-TAR-32 binding to TDP-43 with affinities ranging from 100 nM to 10 μM. These compounds had no effect in the TruHTS®
counterscreen assay, thereby indicating that the observed inhibition of these compounds is specific for the DNA oligonucleotide binding to TDP-43. However, it is important to note that this assay will identify not only compounds that inhibit the interaction by binding to TDP–43 but also compounds that bind to bt-TAR-32. Future studies will determine the mechanism by which these compounds disrupt the TDP-43–nucleic acid interaction and their utility in understanding TDP-43’s normal and pathological functions.
Inhibitors of TDP-43’s binding to nucleic acids have potential utility as biochemical tools and possibly novel therapeutics. Small-molecule inhibitors of TDP-43 nucleic acid binding may improve respiratory function in cystic fibrosis by restoring the production of functional CFTR protein because investigators have shown that depletion of TDP-43 results in increased inclusion of exon 9 in transcription of the CFTR gene3–5
As biochemical tools, TDP-43 inhibitors could be useful for further understanding the role of TDP-43 in neurodegenerative diseases such as ALS. For example, although TDP-43 knockout mice are embryonic lethal,23
a pharmacological inhibitor of TDP-43 might be more tolerated and produce ALS-like symptoms in mice such as those seen in Drosophila
In addition, fluorescently labeled TDP-43 binding ligands could be used to develop high-content screening assays to identify novel compounds that inhibit the cytoplasmic aggregation and restore TDP-43 to the nucleus.
In summary, we have characterized DNA oligonucleotide binding to TDP-43 using a robust, high-throughput assay based on AlphaScreen® technology, which is easily scalable to 1536-well plates for screening of large libraries. We have reported association and dissociation rates for TAR-32 and TG6 oligonucleotides as well as equilibrium dissociation constants and demonstrated that these oligonucleotides bind to a single population of noninteracting sites. From screening our diverse, small-member chemical library, we have discovered a set of structurally similar compounds with nascent SAR and sub-micromolar inhibition of the TDP-43–nucleic acid interaction. Finally, we have discussed the potential utility of such compounds as biochemical tools and possibly as novel therapeutics.