Structural basis for inhibition of the lysosomal two-pore channel TPC2 by a small molecule antagonist.

Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.

Expanding the Toolbox: Novel Modulators of Endolysosomal Cation Channels.

Functional characterization of endolysosomal ion channels is challenging due to their intracellular location. With recent advances in endolysosomal patch clamp technology, it has become possible to directly measure ion channel currents across endolysosomal membranes. Members of the transient receptor potential (TRP) cation channel family, namely the endolysosomal TRPML channels (TRPML1-3), also called mucolipins, as well as the distantly related two-pore channels (TPCs) have recently been characterized in more detail with endolysosomal patch clamp techniques. However, answers to many physiological questions require work in intact cells or animal models. One major obstacle thereby is that the known endogenous ligands of TRPMLs and TPCs are anionic in nature and thus impermeable for cell membranes. Microinjection, on the other hand, is technically demanding. There is also a risk of losing essential co-factors for channel activation or inhibition in isolated preparations. Therefore, lipophilic, membrane-permeable small-molecule activators and inhibitors for TRPMLs and TPCs are urgently needed. Here, we describe and discuss the currently available small-molecule modulators of TRPMLs and TPCs.

Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington's disease by using an automated filter retardation assay.

Preventing the formation of insoluble polyglutamine containing protein aggregates in neurons may represent an attractive therapeutic strategy to ameliorate Huntington's disease (HD). Therefore, the ability to screen for small molecules that suppress the self-assembly of huntingtin would have potential clinical and significant research applications. We have developed an automated filter retardation assay for the rapid identification of chemical compounds that prevent HD exon 1 protein aggregation in vitro. Using this method, a total of 25 benzothiazole derivatives that inhibit huntingtin fibrillogenesis in a dose-dependent manner were discovered from a library of approximately 184,000 small molecules. The results obtained by the filter assay were confirmed by immunoblotting, electron microscopy, and mass spectrometry. Furthermore, cell culture studies revealed that 2-amino-4,7-dimethyl-benzothiazol-6-ol, a chemical compound similar to riluzole, significantly inhibits HD exon 1 aggregation in vivo. These findings may provide the basis for a new therapeutic approach to prevent the accumulation of insoluble protein aggregates in Huntington's disease and related glutamine repeat disorders.