Selective inhibitors of SARM1 targeting an allosteric cysteine in the autoregulatory ARM domain

Abstract

The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.

Proteomic discovery of chemical probes that perturb protein complexes in human cells

“…Second, we believe that the DOS principles of constructing focused compound libraries bearing sp 3 -rich cores that are stereochemically defined, entropically constrained, and densely functionalized may further improve the probability of engaging PPI sites, at least when compared to more fragment-like and/or sp 2 -based small-molecule libraries. An additional advantage of DOS-constructed libraries is that they can furnish chemical probes paired with physicochemically matched, inactive enantiomeric control compounds for cell biological studies (Feldman et al, 2022; Kim et al, 2004; Vinogradova et al ., 2020). Finally, the function-first screening approach likely also facilitated the discovery of chemical probes by prioritizing follow-up studies on a subset of covalent liganding events that alter protein complexation states in cells.…”

Axonal degeneration in chemotherapy-induced peripheral neurotoxicity: clinical and experimental evidence

“…101 Of note, covalent inhibitors targeting cysteine 311 in the armadillo repeat domain of SARM1 prevent vincristine-mediated degeneration in cell culture models. 102 Dehydronitrosonisodipine also blocked SARM1 activation via modification of cysteine 311, inhibiting cADPR production and axonal degeneration after vincristine treatment. 103 Similarly, the adduct forming SARM1 inhibitor NB-3 supressed plasma NfL release, and prevented loss of intradepidermal nerve fibre loss and the development of mechanical allodynia in vincristine treated mice.…”

Assigning functionality to cysteines by base editing of cancer dependency genes

“…The liquid phase was then aspirated, and the pellet is frozen at -80°C for later use. The subsequent sample processing and LC-MS instrumentation is the same as the previously described protocol for cysteine ligandability profiling 17,29 . Briefly, the protein pellets were reduced by DTT, alkylated by iodoacetamide, and digested by trypsin overnight.…”

“…Advantages of this approach include the deployment of covalent chemistry 19,[21][22][23] , which can address shallow and dynamic pockets on protein surfaces that are less amenable to binding reversibly to small molecules, as well as provide a selectivity filter in the form of targeting isotype-restricted nucleophilic residues (e.g., cysteines) within paralogous proteins, as have been demonstrated by several recent cancer therapeutics (e.g., osimertinib for EGFR_C797 24,25 , sotorasib for KRAS_G12C 26,27 ). Additionally, by evaluating small molecules directly in native biological systems, chemical proteomics methods such as ABPP can identify cryptic ligandable pockets regulating aspects of protein function that are difficult to discern with purified proteins or protein domains 17,28,29 .…”