Characterization of a series of 4-aminoquinolines that stimulate caspase-7 mediated cleavage of TDP-43 and inhibit its function

Abstract

Dysfunction of the heterogeneous ribonucleoprotein TAR DNA binding protein 43 (TDP-43) is associated with neurodegeneration in diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here we examine the effects of a series of 4-aminoquinolines with affinity for TDP-43 upon caspase-7-induced cleavage of TDP-43 and TDP-43 cellular function. These compounds were mixed inhibitors of biotinylated TG6 binding to TDP-43, binding to both free and occupied TDP-43. Incubation of TDP-43 and caspase-7 in the presence of these compounds stimulated caspase-7 mediated cleavage of TDP-43. This effect was antagonized by the oligonucleotide TG12, prevented by denaturing TDP-43, and exhibited a similar relation of structure to function as for the displacement of bt-TG6 binding to TDP-43. In addition, the compounds did not affect caspase-7 enzyme activity. In human neuroglioma H4 cells, these compounds lowered levels of TDP-43 and increased TDP-43 C-terminal fragments via a caspase-dependent mechanism. Subsequent experiments demonstrated that this was due to induction of caspases 3 and 7 leading to increased PARP cleavage in H4 cells with similar rank order of the potency among the compounds tests for displacement of bt-TG6 binding. Exposure to these compounds also reduced HDAC6, ATG7, and increased LC3B, consistent with the effects of TDP-43 siRNA described by other investigators. These data suggest that such compounds may be useful biochemical probes to further understand both the normal and pathological functions of TDP-43, and its cleavage and metabolism promoted by caspases.

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

“…37 While we observed a substantial decrease in affinity for nucleic acids for TDP-43 upon UBQLN2 binding, the affinity of AAQ1 for TDP-43 was much less effected, being only 5-fold less potent for inhibition of UBQLN2 binding to TDP-43 compared to inhibition of nucleic acid binding to TDP-43. Further characterization of AAQ1 mediated inhibition of UBQLN2 binding indicated that AAQ1 was competitive with UBQLN2 binding, therefore suggesting that AAQ1 also binds to the C-terminal region of TDP-43.…”

“…Based on this biochemical assay using HTRF technology, UBQLN2 binds to both full length and a C-terminal fragment of TDP-43 (261-414 aa) with similar affinities, but not N-terminal TDP-43 (1-260 aa), suggesting that UBQLN2 interacts with TDP-43 at the C-terminus. Single stranded DNA oligonucleotides and 4-aminoquinolines which bind to TDP-43 37 , also inhibit UBQLN2 binding to TDP-43 with similar rank order affinities. Inhibitor characterization experiments demonstrated that the TDP-43 oligonucleotide TG12 and UBQLN2 interact noncompetitively with TDP-43, thereby supporting the hypothesis that UBQLN2 interacts with TDP-43 at a site distinct from oligonucleotide binding.…”

“…4-aminoquinolines: AAQ1 (3-{[2-(4-methoxyphenyl)quinolin-4-yl]amino}propyl)dimethylamine, compound 1 in ref. 37) and AAQ2 (N-[2-(dimethylamino)ethyl]-N-methyl-2-phenylquinolin-4-amine, compound 9 in ref. 37) were provided by Fox Chase Center for Chemical Diversity, Inc. (Doylestown, PA).…”

“…We have determined that UBQLN2 binds to the C-terminal region of TDP-43, which is consistent with the findings of other investigators who demonstrated co-localization of cytoplasmic C-terminal TDP-43 fragments and UBQLN2 18, 19 . Titrations of single stranded DNA oligonucleotides as well as 4-aminoquinlines (AAQs), a series of small molecules which we described previously as inhibitors of nucleic acid binding to TDP-43 37 , inhibited UBQLN2 binding to TDP-43 with the same rank order potency as inhibition of nucleic acid binding to TDP-43. In addition, we demonstrate that UBQLN2 is noncompetitive with oligonucleotide binding to TDP-43, while AAQ1 is competitive with UBQLN2 binding to TDP-43.…”

Staurosporine Induced Apoptosis Rapidly Downregulates TDP-43 in Glioma Cells

“…While caspase-3 is thought to be the major caspase for the cleavage of TDP-43, caspase 7 may be also involved as reported previously (Zhang et al, 2007;Cassel et al, 2012). Our experiment did not specify exactly which protease is the executer.…”