A novel approach to restore functional TDP43 for the treatment of FTD and ALS

ABSTRACT TDP43 is considered a key target for drug discovery in frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and other TDP43 proteinopathies, including Alzheimer’s disease and related dementias (ADRD). The pathophysiology of TDP43 proteinopathies involves a loss of native function (RNA binding and splicing regulation) and a toxic gain of function (cytoplasmic mislocalization and toxic aggregate formation). Despite years of research, current small molecule discovery efforts targeting TDP43 dysfunction have not yielded strong inhibitors or successful clinical translations. Historically, TDP43 therapeutic strategies have focused on reducing its toxic gain of function. Our proposal, in contrast, aims to prevent the loss of TDP43’s native function. Recent high-impact publications have provided critical new insight into how we might accomplish this. It is now understood that TDP43’s RNA binding requires N-terminal domain (NTD) multimerization, and that pathological loss of NTD-NTD interactions is the key step preceding cytoplasmic mislocalization and aggregation. The goal of this proposal is, therefore, to identify novel small molecules that stabilize functional, NTD-dependent TDP43 multimers to restore TDP43 function and rescue TDP43-associated pathologies. Our high-throughput screening pipeline, using engineered fluorescence lifetime (FLT)-based FRET biosensors, specifically monitors NTD-dependent TDP43 multimerization in living cells. We present strong and extensive preliminary data to support our proposal. A pilot screen of the 2,684 compound Selleck library identified multiple promising hits previously known to bind and effect TDP43. We demonstrate the full pipeline on one of those hits, which stabilizes nuclear TDP43 multimers, rescues TDP43 mislocalization and aggregation, improves neuronal cell health and partially rescues motor function in a C. elegans model of TDP43 proteinopathy. These data strongly support the premise of our targeting strategy, the robustness of our HTS pipeline, and instill confidence in the promise of our proposed campaign. We will screen the 50,000-compound CNS-SetTM library, curated for BBB penetration, in an inducible neuronal cell line. Using a range of cell-based and biophysical assays we will identify two kinds of promising small molecules that stabilize NTD-dependent multimers: 1) compounds that directly bind to the TDP43 NTD; or 2) compounds that act through an indirect mechanism of action. To enrich the pool of direct acting compounds, we will also perform in silico docking screens of over 500,000 compounds targeting the NTD-NTD dimeric interface. Hit compounds from both screens will advance through the pipeline, which includes functional assays to monitor TDP43 splicing regulation, TDP43 mislocalization and aggregation, neurite length (a measure of cell health) in both 2D neuronal culture and 3D neurospheres, and a C. elegans movement assay. Lastly, we will engineer iPSC-derived motor neurons to express the TDP43 biosensors, providing an improved physiological model of disease for future refinement of the most promising leads.