In Vitro Interaction of a C-Terminal Fragment of TDP-43 Protein with Human Serum Albumin Modulates Its Aggregation.

An increased level of naturally occurring anti-TDP-43 antibodies was observed in the serum and cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis patients. Human serum albumin (HSA), the most abundant protein in blood plasma and CSF, is found to interact with pathological proteins like Aß and α-synuclein. Therefore, we examined the effect on the in vitro aggregation of a C-terminal fragment of TDP-43 in the presence of HSA. We found that the lag phase in TDP-432C aggregation is abrogated in the presence of HSA, but there is an overall decreased aggregation as examined by thioflavin-T fluorescence spectroscopy and microscopy. An early onset of TDP-432C oligomer formation in the presence of HSA was observed using atomic force microscopy and transmission electron microscopy. Also, a known chemical inhibitor of TDP-432Caggregation, AIM4, abolishes the HSA-induced early formation of TDP-432C oligomers. Notably, the aggregates of TDP-432C formed in the presence of HSA are more stable against sarkosyl detergent. Using affinity copurification, we observed that HSA can bind to TDP-432C, and biolayer interferometry further supported their physical interaction and suggested the binding affinity to be in sub-micromolar range. Taken together, the data support that HSA can interact with TDP-432C in vitro and affect its aggregation.

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.

TDP-43 is an RNA/DNA-binding protein which is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease. TDP-43's cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation, are proposedly important TDP-43-toxicity causing mechanisms. So far, therapeutic options for ALS are extremely ineffective hence, multi-faceted approaches such as targeting the oxidative stress and inhibiting the TDP-43's aggregation, are being actively pursued. Recently, we have identified an acridine derivative, AIM4, as an anti-TDP-43 aggregation molecule however, its mechanism is not deciphered. Here, we have utilized computational tools to examine binding site(s) of AIM4 in the TDP-43 structure and compared with other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic region (aa: 288-319), with Gly-288 & Phe-289 residues which are also important for TDP-43's LLPS. Importantly, alike to previously reported effects of RNA, AIM4 could also inhibit the in vitro LLPS of a C-terminal fragment TDP-432C bearing an A315T familial mutation. Furthermore, isothermal titration calorimetry (ITC) data also support the binding of AIM4 to TDP-432C-A315T. This antagonism of AIM4 towards TDP-43's LLPS and presence of binding site of AIM4 on TDP-43 support AIM4's potential to be an important molecule towards ALS therapeutic research.

Luminescent zinc(ii) selone macrocyclic ring.

The synthesis and photophysical properties of macrocyclic Zn(ii) selone molecule have been reported. The structural property of Zn(ii) selone was elucidated using single crystal X-ray diffraction study. The solid-state structure of zinc(ii) selone molecule exhibits a perfect zinc(ii) selone 28 membered ring system with tetra coordination geometry around zinc(ii) center. The zinc(ii) selone ring system can be considered as the largest zinc(ii) ring system known without any non-interacting centered guest moiety. Detailed trends in photophysical as well as thermal properties were probed. In photoluminescence study, the solid-state sample of zinc(ii) selone ring system emits the bluish-yellow color with considerable quantum yields, while the solution state sample of zinc(ii) selone ring system in DMSO emits bluish-yellow. The luminescence lifetime of zinc(ii) selone was measured using standard time-correlated single photon counting (TCSPC) technique.

Blue-emitting acridine-tagged silver(i)-bis-N-heterocyclic carbene.

Herein, the photophysical properties of an acridine derivative of a bis-N-heterocyclic carbene silver complex were investigated. The HOMO and LUMO energy differences between 9-[(N-methyl imidazol-2-ylidene)]acridine and 4,5-bis[(N-methyl-imidazol-2-ylidene)methyl]acridine were theoretically compared. Based on the calculation, the 4,5-bis N-heterocyclic carbene-tethered acridine type of ligand was found to be a potential source for tuning the fluorescent nature of the resultant metal derivatives. Thus, a 4,5-bis N-heterocyclic carbene (NHC)-tethered acridine silver(i) salt was synthesized, and its photophysical properties were investigated. The 4,5-bis[(N-isopropylimidazol-2-ylidene)methyl]acridine silver(i) hexafluorophosphate complex was obtained from the reaction between [4,5-bis{(N-isopropylimidazolium)methyl}acridine] hexafluorophosphate and Ag2O in very good yield; this molecule was characterized by elemental analysis and FTIR, multinuclear (1H and 13C) NMR, UV-Vis, and fluorescence spectroscopic techniques. The molecular structure has been confirmed by single-crystal X-ray diffraction analysis, which has revealed that the complex is a homoleptic mononuclear silver(i) cationic solid. The charge of the Ag(i)-NHC cation is balanced by the hexafluorophosphate anion. The cationic moieties are closely packed in the chair and inverted chair forms where silver(i) possesses a quasi-linear geometry. Moreover, the silver complex provided blue emission from all the three excitations with good fluorescence quantum yield. The fluorescence lifetime of the silver(i) complex has been determined using the time-correlated single photon counting technique. Interestingly, the fluorescence decay pattern and the fluorescence lifetimes of the silver complex are largely different from those of the parent ligand acridine imidazolium salt. Moreover, the theoretical predictions have been found to be in good agreement with the experimental results.

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

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 ß-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.