From recombinant proteins to cells: targeting TDP-43 in preclinical ALS and FTD therapeutic development

Abstract

The transactive response DNA-binding protein 43 kDa (TDP-43) is a DNA and RNA binding protein involved in RNA transcription and translation. Accumulation of intracellular TDP-43 inclusions is a pathological hallmark in some patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To date, there are no disease-modifying treatments for these diseases. The pathological overlap of TDP-43 in both diseases makes it an attractive target for therapeutic intervention and PET ligand development. This thesis aimed to develop methods to inform lead development of small molecules that target TDP‑43 proteinopathy or bind to TDP-43.

The use of cellular models is a common means to investigate potency of therapeutics in pre-clinical drug discovery. However, there is currently no consensus on which model most accurately replicates key aspects of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathology. By characterising two TDP-43 proteinopathy cellular models that were based on different aetiologies of disease, it was demonstrated that each cellular model captured different aspects of TDP-43, stress granule and ubiquitin pathology. When these two cellular models were exposed to small molecule chemical probes, different effects were observed across the two models. For example, a previously disclosed sulfonamide compound, 1, decreased cytoplasmic TDP-43 levels and increased soluble levels of stress granule marker TIA-1 in the cellular stress model without impacting these levels in a TDP-43 M337V mutant cell line. These evaluations highlight the challenges of using cellular models in lead development during drug discovery for ALS and FTD and reinforces the need to perform assessments of novel therapeutics across a variety of cell lines and aetiological models.

To assist in development of therapeutic compounds and PET ligands, several previous studies have attempted to purify full-length TDP-43 with a reasonable yield, but limitations in reproducibility have been reported. We aimed to develop a method of full‑length TDP-43 production using two different plasmid constructs encoding full-length TDP‑43 expressed using E. coli bacterial expression systems. A novel purification protocol was successfully developed for one of the protein constructs consisting of full‑length TDP-43 with a hexahistidine and ubiquitin fusion tag (His-Ub-TDP-43). This novel protocol resulted in the preparation of pure, and soluble TDP-43 protein that was characterised using electrophoresis and mass spectrometry. Protein folding was revealed by far-UV circular dichroism which showed mostly random coil with some α‑helical and β-sheet structure. Self‑assembly was monitored with a turbidity assay and a thioflavin T fluorescence assay which revealed the formation of aggregates with an amyloid structure.

In order to develop small molecule TDP-43 binders, the amplified luminescent proximity homogeneous assay (AlphaScreen™) was used to measure binding affinity against TDP-43. After assay optimisation, a library of twenty-one molecules were tested and three novel compounds were identified as TDP-43 binders. A novel chemotype 20 displayed partial inhibition of binding with low micromolar binding affinity. The compounds 24 and 25 exhibited novel two-site binding to TDP-43, with both compounds showing a high‑affinity site with nanomolar affinity and a low affinity site with micromolar binding affinity. The compounds 24 and 25 offer new opportunities as tools to further study the biological implications of compounds that occupy two TDP-43 binding sites. This work establishes a foundation for future iterative drug discovery, with the ultimate aim of producing therapeutics and PET ligands for use in ALS and FTD.