A Duplex Structure of SARM1 Octamers Induced by a New Inhibitor

Abstract

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1 by stabilizing the ARM-dependent inactive, compact octamer ring conformation, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a Calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also promotes the formation of a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 A resolution. In the duplex, each ARM domain protomer is not only engaged in lateral interactions with neighboring protomers but is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans

“…Weak activating mutations were recently discovered in ALS cohorts, 45 , 46 most of which are located in the ARM domain. We found that while some hSARM1 mutations are too strong and others too weak, one mutation, V112I 26 , 45 (although a significant activation by this mutation was not observed 46 ), seems to inflict similar cell death level as ceTIR-1 in HEK293F ( Figure 6A ).…”

“…NAM, a product of NADase activity ( Figure S2 ), inhibited ceTIR-1 ( Figure 3C ) as was previously demonstrated for hSARM1. 20 , 24 , 26 Notably, we found that the chemical structure of the reaction products ( Figure 3D ) is different between the two orthologs: hSARM1 generates about 90% ADPR and 10% cADPR, while ceTIR-1 yields nearly opposite ratios ( Figure 3D ). However, at high protein concentrations, the isolated TIR domain of ceTIR-1 (ceTIR-1 TIR ) ( Figure S7A ) can also produce substantial amounts of ADPR in addition to cADPR ( Figures 3D and S7B ).…”

“…NAM is produced at the first stage of NAD + catalysis, regardless of ADPR or cADPR production in the second stage ( Figure S2 ). It is an in vitro inhibitor of SARM1, with an IC 50 of 43–140 mM, 20 , 24 , 26 and based on its measured levels in brain extracts (46 ± 13 mM), 41 it is likely to contribute to SARM1 inhibition in vivo . However, it was recently suggested that what seems like competitive inhibition by NAM could actually be a result of a base-exchange reaction, in which NAM re-attaches to the newly formed ADPR-enzyme complex.…”

“… 19 The essential role for SARM1 in promoting WD has been demonstrated in knockout studies across different species and contexts. For example, SARM1 NADase loss of function by genetic 20 , 21 or pharmacological 22 – 26 means inhibits WD and even rescues the development and survival of axons deficient for NMNAT2, 27 while SARM1 gain of function induces WD. 28 …”

Local translation is engaged to sustain synaptic function in impaired Wallerian degeneration

“…They remain morphologically intact and physiologically active for months after injury (Bittner, 1988). Whether activated programmed axon degeneration i) fails to deplete NAD + as observed in C. elegans (Khazma et al, 2023), ii) is not engaged due to a tight posttranslational regulation (Murata et al, 2018), or iii) is absent remains to be determined. This long-term survival was first observed in crayfish motor axons (Hoy et al, 1967), and subsequently in various other nervous systems, including leeches (Muller & Carbonetto, 1979), earthworms (Birse & Bittner, 1976), crickets (Clark, 1976), crustaceans (Bittner, 1973), and mollusks (Murphy A.…”