An Allosteric Modulator of RNA Binding Targeting the N-Terminal Domain of TDP-43 Yields Neuroprotective Properties

Abstract

In this study, we targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. In silico docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) 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 RNA 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 findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain.

Molecular Dynamics simulation of TDP-43 RRM in the presence and absence of RNA

“…Our interest has been to define druggable sites in TDP-43, either in the RRM or the NTD domain 7,8 . We hypothesized that the RNA-free RRM domains of TDP-43 might exhibit pockets distinct from RNA-bound TDP-43, which could lead to new targeting opportunities.…”

“…However, the localization – NTD, RRM or CTD – and the precise “form” or “state” of TDP-43 – RNA bound, RNA free, monomer, condensates, or aggregates – to be targeted to modulate TDP-43 cytotoxicity remain to be determined. We have developed small molecules targeting TDP-43 NTD as well as the RNA-bound RRM domains 7, 8 ; interestingly both molecules reduce RNA binding to various extent and mitigate ALS phenotypes in Drosophila, although further characterization of the compounds are needed. Defining small molecules that can distinguish and target RNA-bound vs RNA-free forms of TDP-43 of the RRM domain, would help in evaluating through chemical biology which form of TDP-43 can be targeted and more importantly which form reduces neurotoxicity.…”

Direct targeting of TDP-43, from small molecules to biologics: the therapeutic landscape

“…Despite its lack of traditional targetable pockets, we and others have shown the ability of small molecules to bind TDP-43, albeit with a rather low affinity 47,49,53 . SAR studies focusing on improving the affinity of existing compounds will reveal how effective these strategies are by increasing potency without increasing off-targeting.…”

“…Another molecule we recently discovered is nTRD22 (Figure 3C), found using the same pipeline as previously described, but targeting the N-terminal domain (NTD) 49 . The NTD is implicated in oligomer formation and support of nucleotide binding [50][51][52] , among other functions.…”

“…3C ), found using the same pipeline as previously described, but targeting the N-terminal domain (NTD). 49 The NTD is implicated in oligomer formation and support of nucleotide binding, 50–52 among other functions. Interestingly, nTRD22 was shown to bind specifically to the NTD, with an affinity of 145 ± 3 μM, but was also observed to (i) shift residues in the RRM domains, as demonstrated by HSQC-NMR and (ii) displace canonical UG 6 binding, unlike rTRD01 that directly targeted the RNA binding domains.…”