Pharmacological SARM1 inhibition protects axon structure and function in paclitaxel-induced peripheral neuropathy

Abstract

Axonal degeneration is an early and ongoing event that causes disability and disease progression in many neurodegenerative disorders of the peripheral and central nervous systems. Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of morbidity and the main cause of dose reductions and discontinuations in cancer treatment. Preclinical evidence indicates that activation of the Wallerian-like degeneration pathway driven by sterile alpha and TIR motif containing 1 (SARM1) is responsible for axonopathy in CIPN. SARM1 is the central driver of an evolutionarily conserved programme of axonal degeneration downstream of chemical, inflammatory, mechanical or metabolic insults to the axon. SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treat neurodegeneration characterized by axonopathies of the peripheral and central nervous systems. Small molecule SARM1 inhibitors have the potential to prevent axonal degeneration in peripheral and central axonopathies and to provide a transformational disease-modifying treatment for these disorders. Using a biochemical assay for SARM1 NADase we identified a novel series of potent and selective irreversible isothiazole inhibitors of SARM1 enzymatic activity that protected rodent and human axons in vitro. In sciatic nerve axotomy, we observed that these irreversible SARM1 inhibitors decreased a rise in nerve cADPR and plasma neurofilament light chain released from injured sciatic nerves in vivo. In a mouse paclitaxel model of CIPN we determined that Sarm1 knockout mice prevented loss of axonal function, assessed by sensory nerve action potential amplitudes of the tail nerve, in a gene-dosage-dependent manner. In that CIPN model, the irreversible SARM1 inhibitors prevented loss of intraepidermal nerve fibres induced by paclitaxel and provided partial protection of axonal function assessed by sensory nerve action potential amplitude and mechanical allodynia.

Keywords: ALS; CIPN; axonal degeneration; multiple sclerosis; neurodegeneration.

Figure 1

Identification of isothiazole inhibitors of SARM1 NADase. (A) Structure of isothiazole hit compound 1 identified as a weak inhibitor of ADPR production by NAD⁺ hydrolysis. (B) Core isothiazole structure subjected to structure-activity relationship studies. (C) Structure-activity relationship of the isothiazoles.

Figure 2

Isothiazole SARM1 inhibitors protect injured axons in vitro. (A) DRG mouse cultures were treated for 2 h with compounds 4, 8, 9 and 10 and then subjected to axotomy. Whereas axons in untreated cultures fragmented completely, axons from cultures treated with isothiazole inhibitors were completely protected 16 h post-axotomy. Scale bar = 25 µm. (B) Quantification of fragmentation showed that axonal protection with isothiazoles was dose-dependent. Values represent mean ± SEM n = 4/dose. Representative of three independent experiments with similar results.

Figure 3

Isothiazoles are irreversible SARM1 inhibitors. (A) SAM-TIR protein immobilized to Strep-Tactin^®XT plates was treated with DSRM-3716, compound 4 or compound 9 for 2 h and enzymatic activity was measured either immediately (t = 0), or plates were rinsed and assayed after 10 min (t = 10) or 60 min (t = 60) post removal of the compounds. Substantial loss of inhibition was observed with the reversible SARM1 inhibitor DSRM-3716 (left). In contrast, inhibition with compound 4 (middle) and compound 9 (right) was completely maintained after 60 min in the absence of compound, consistent with irreversible inhibition of the enzyme. (B) DRG mouse cultures were treated for 3 h with 10 µM of the reversible SARM1 inhibitor DSRM-3716 or 10 µM each of compounds 4, 9 and 10. Compounds were removed from the cultures and a subset of cultures had DSRM-3716 replaced immediately after rinsing (continuous). Axons were subjected to axotomy and examined at the times indicated below the bars (in hours). Whereas protection by DSRM-3716 was completely lost by compound removal at the 16-h time point, compounds, 4, 9 and 10 maintained axonal protection at 72 h. ANOVA with Holm–Sidak post hoc F(11,36) = 10.49, P < 0.0001, mean ± SEM; control n = 4. ns, not significant; **P < 0.01; ****P < 0.0001. (C) DRG mouse cultures were exposed to a 3-h pulse of 10-µM compound 4 or 10-µM compound 9 and compounds were removed from the cultures. The interval between removal of the compound and axotomy defines the chase interval. At the completion of the chase interval indicated below the bars, axons were subjected to axotomy and axonal protection was examined after 16 h. Almost complete axonal protection was maintained after a chase period of 24 h. The extent of axonal protection progressively decreased with longer intervals between compound removal and axotomy. Two-way ANOVA with Holm–Sidak post hoc F(3,24) = 71.81, P < 0.0001, mean ± SEM; n = 4. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4

Isothiazoles inhibit SARM1 in vivo in SNA. (A) Mice were treated with isothiazole SARM1 inhibitors 4, 9 and 10 at the doses indicated and subjected to unilateral SNA 30 min after the first dose. For compounds 4 and  9, a second dose was administered 8 h after the first dose, and 15 h after SNA NfL levels were measured in plasma. Compounds 4 and 9 were dosed by intraperitoneal injection and compound 10 was dosed once orally. All three compounds prevented increases in plasma NfL in a dose-dependent manner. The magnitude of the effect, expressed as percentage decrease from vehicle is as indicated in the figures. Values represent mean ± SEM. Compound 4, ANOVA with Holm–Sidak post hoc F(2,20) = 22.48, P < 0.0001, n = 7–8/group; compound 9, ANOVA with Holm–Sidak post hoc F(2,21) = 19.38, P < 0.0001, n = 8 per group; compound 10, ANOVA with Holm–Sidak post hoc F(2,17) = 28.54, P < 0.0001, vehicle n = 4, treated groups n = 8. **P < 0.01; ****P < 0.0001. (B) Wild-type, Sarm1^+/− and Sarm1^−/− mice were subject to SNA. At 15 h and 1 day after axotomy cADPR was measured in the cut and uncut contralateral nerves. Wild-type mice were treated with compound 4 (30 mg/kg) 30 min before SNA and received a second dose 8 h after the first dose in the 15-h group, and a third dose 10 h after the second dose in the 1-day group (TID dosing). We found cADPR increased in cut nerves but not the contralateral uncut nerves in wild-type. In Sarm1 mutants, no cADPR increase was observed in Sarm1^−/− at any time point and values were intermediate in Sarm1^+/−. Levels of cADPR with compound 4 were reduced from vehicle to levels that approached Sarm1^+/−. Values were normalized to the respective SNA vehicle within each group and represent mean ± SEM. One-way ANOVA F(15,88) = 188.2, P < 0.00001; ns. not significant; **P < 0.01; ****P < 0.0001; n = 4–8/group.

Figure 5

Pharmacological SARM1 inhibitor protects axonal integrity and function in a model of CIPN. Mice were subjected to the paclitaxel CIPN model described in the 'Materials and methods' section and Supplementary Fig. 5, while treated orally with vehicle, or the isothiazole SARM1 inhibitor compound 10 at the doses indicated in the figure. (A) SNAP amplitudes from tail nerves measured at 9 and 15 days after the first dose of paclitaxel were partially protected at the highest dose, while also showing a non-statistically significant trend towards protection at the lower dose. There was a significant main effect between groups, two-way repeated measures ANOVA F(3,33) = 28.12, P < 0.0001; time F(2,66)  = 17.37, P < 0.0001; Time × Treatment group F(6,66) = 10.61, P < 0.0001; Holm–Sidak post hoc *P < 0.05; **P < 0.01; ****P < 0.0001; n = 7–10 per group. (B) Nerve conduction velocity was not affected by paclitaxel or compound treatment. There was no significant main effect by two-way ANOVA, treatment groups F(3,33) = 1.287, P = 0.2951 and Time × Treatment group F(6,66) = 1.146, P = 0.346; n = 7–10 per group. (C) Mechanical withdrawal threshold was significantly reduced by paclitaxel treatment and showed partial protection by treatment with compound 10. One-way ANOVA F(3,34) = 43.43, P < 0.0001, Holm–Sidak post hoc **P < 0.01; ****P < 0.0001; n = 7–10 per group. (D) IENFs were stained with the pan-axonal antibody PGP 9.5 and confocal microscopy images were quantified by a blind experimenter as described in the 'Materials and methods' section. Loss of IENF density induced by paclitaxel was significantly protected by SARM1 inhibitor. One-way ANOVA F(3,26) = 13.16, P < 0.0001; Holm–Sidak post hoc **P = 0.0091; ***P = 0.0003; n = 7–8 per group. (E) Representative images of IENFs in vehicle, paclitaxel and paclitaxel + 300 mg/kg compound 10. (F) Percentage protection of SNAP amplitudes in mice treated with paclitaxel and compound 10 at 9 and 15 days versus percentage protection achieved through genetic reduction of SARM1 in Sarm1 heterozygous (HET) and Sarm1 knockout (KO) at 15 days. The magnitude of protection with the highest dose of compound 10 approached that of Sarm1 heterozygous.