Structure-Based Targeting of Tdp-43 to Further the Development of Novel Therapeutics for Neurodegenerative Diseases

Neurodegenerative diseases are incurable and debilitating conditions that result from gradu-al and progressive degeneration and death of neural cells, leading to nervous system dysfunction that causes problems with movement (called ataxias) and mental functions (called dementias). Neu-rodegenerative diseases already affect millions worldwide and cases are rapidly increasing with the accelerating population with no current efficacious therapies or cures. Mounting evidence supports toxic oligomerization from atypical protein folding and aggregation of hallmark proteins that are of therapeutic interest as means of intervention. TAR DNA-binding protein 43 (TDP-43) is one of these hallmark proteins indicated in several neurodegenerative diseases pathophysiology and has been largely implicated in both familial and sporadic cases of Amyotrophic Lateral Sclerosis (ALS). TDP-43 is an intrinsically disordered multidomain protein that consists of an N-terminal domain (NTD1-102), two tandem RNA recognition motifs (RRM1¬102-177 and RRM2191-260), and an intrinsically disordered glycine-rich C-terminal domain (CTD). Structural characterization of the prion-prone TDP-43 has been challenging since its intrin-sically disordered regions represent 15-30% of the total protein. In this study, we seek to define possible new targetable sites on the apo structure of TDP-43 RRM domains (TDP-43102-269). To do so, we utilized multi-dimensional NMR experiments to assign the backbone residues for TDP-43102-269 along with Molecular Dynamics (MD) simulations to investigate the structure and dynam-ics of TDP-43 RRM domains in the absence of RNA (apoTDP-43102-269). We compared apo TDP-43 structures obtained from MD, experimental NMR restraints, and de novo sequence to structure predictions using AlphaFold2 (AF2), RaptorX, and I-TASSER of TDP-43 RRM domains and found differences within structures and looped regions. A Sitemap analysis identified five drugga-ble sites for NMR structures both apo and RNA bound, while fewer sites were identified follow-ing MD simulations and AF2 predicted apo structures. The bulk of this research focuses on the development of a structure-based drug discovery pipeline targeting the structured domains of TDP-43, including the N-Terminal domain (NTD) and RNA recognition motifs (RRMs) domains. We hypothesized that by targeting the RRMs of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. In silico docking of 50k compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43’s RRM1 and RRM2 domains; (ii) partially disrupted TDP-43’s interaction with the hexanucleotide RNA repeat of the disease-linked c9orf72 gene, but not with (UG)6 canonical binding sequence of TDP-43; and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overex-pression of mutant TDP-43. Targeting the NTD yielded nTRD22 that caused allosteric modulation of the RRMs of TDP-43 resulting in decreased binding to RNA in vitro. Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a Drosophila model of Amyotrophic Lateral Sclerosis. Our work on the chemical modulation of TDP-43 identified allosteric changes in the protein which we attributed to possible interdomain interactions between the NTD and RRM domains. To investigate this, we compared 2D [1H, 15N] HSQC-NMR spectra of TDP-43102-260 (RRMs alone) against TDP-431-260 (NTD and RRMs) and discovered clustered shifts in the RNA binding sites of both RRM domains. Probing this idea, we modeled NTD-RRM interactions using protein-protein docking along with de novo sequence-to-structure predictions of TDP-43 that propose NTD stack-ing onto the RRM domains. Using surface plasmon resonance (SPR) and Carr-Purcell-Meiboom-Gill (CPMG)- relaxation dispersion NMR spectroscopy, we show evidence of a tight interaction between the NTD and RRMs with nanomolar affinity. Finally, using 2D HSQC-NMR, we detect changes in the exchange rate of short UG-rich RNA binding between RMMs alone and in the presence of the NTD, suggesting the NTD has a role with the RRM domains for RNA binding