An acridine derivative, [4,5-bis{(N-carboxy methyl imidazolium)methyl}acridine] dibromide, shows anti-TDP-43 aggregation effect in ALS disease models

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with aggregation of TAR DNA-binding protein-43 (TDP-43) in neuronal cells and manifests as motor neuron dysfunction & muscle atrophy. The carboxyl-terminal prion-like domain of TDP-43 can aggregate in vitro into toxic β-sheet rich amyloid-like structures. So far, treatment options for ALS are very limited and Riluzole, which targets glutamate receptors, is the only but highly ineffective drug. Therefore, great interest exists in developing molecules for ALS treatment. Here, we have examined certain derivatives of acridine containing same side chains at position 4 & 5, for inhibitory potential against TDP-43 aggregation. Among several acridine derivatives examined, AIM4, which contains polar carboxyl groups in the side arms, significantly reduces TDP-43-YFP aggregation in the powerful yeast model cell and also abolishes in vitro amyloid-like aggregation of carboxyl terminal domain of TDP-43, as observed by AFM imaging. Thus, AIM4 can be a lead molecule potentiating further therapeutic research for ALS.

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

“…In addition to the C-terminal region of TDP-43, the available structures of the tandem RRM domains (PDB ID: 4BS2, aa: 96-269) [77] and Nterminal domain (NTD) (PDB ID: 2N4P, aa: 1-89) [78] were also used for the docking studies. The overall domain architecture of TDP-43, also highlighting the amyloidogenic C-terminal region peptides, has been schematically depicted in Ligand structure AIM4 and other acridine-imidazolium derivatives previously examined in vitro for its propensity of anti-aggregation effects on wild-type TDP-43 2C , were used as test compounds for docking studies [71,72]. For comparison, certain other ligands such as dimebon and diphenhydramine hydrochloride (DPH) which have already been tested in cellular models for their inhibitory activity on TDP-43 aggregation were also used [70].…”

“…For recombinant protein expression, the pET15b-His-TDP-43 2C (A315T) plasmid constructed here as described above, was first transformed into the expression competent Rosetta 2 (DE3) E. coli cells (Novagen, USA). Expressions of the mutant TDP-43 2C -A315T protein and its affinity purification was carried out following prior published methods with minor modifications [30,71]. Briefly, the Figure S3).…”

“…The precipitate was then pelleted by centrifugation at 14000 x g for 10 min at 4 °C and the supernatant was discarded to remove GuHCl. The pellet was then re-suspended and dissolved in 4M urea in PBS pH 7.5 and used for the aggregation following the method as described previously [71]. nm upon excitation at 442 nm [11,71].…”

“…The pellet was then re-suspended and dissolved in 4M urea in PBS pH 7.5 and used for the aggregation following the method as described previously [71]. nm upon excitation at 442 nm [11,71]. For examining their inhibitory potential towards the aggregation of mutant TDP-43 2C -A315T, first 50 mM stock solutions of the acridine imidazolium derivative compounds AIM4 and AIM1 were prepared in 25% DMSO [71,72].…”

“…nm upon excitation at 442 nm [11,71]. For examining their inhibitory potential towards the aggregation of mutant TDP-43 2C -A315T, first 50 mM stock solutions of the acridine imidazolium derivative compounds AIM4 and AIM1 were prepared in 25% DMSO [71,72]. Then the compounds were added to the aggregation solution containing 400 µM of mutant TDP-43 2C -A315T, at a protein: AIM4/AIM1 ratio of 1:10, 1:15 or 1:20.…”