Uncompetitive, adduct-forming SARM1 inhibitors are neuroprotective in preclinical models of nerve injury and disease.

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.

Structural and Mechanistic Regulation of the Pro-degenerative NAD Hydrolase SARM1.

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.

Efficient determination of purine metabolites in brain tissue and serum by high-performance liquid chromatography with electrochemical and UV detection.

The purine metabolic pathway has been implicated in neurodegeneration and neuroprotection. High-performance liquid chromatography (HPLC) is widely used to determine purines and metabolites. However, methods for analysis of multiple purines in a single analysis have not been standardized, especially in brain tissue. We report the development and validation of a reversed-phase HPLC method combining electrochemical and UV detection after a short gradient run to measure seven purine metabolites (adenosine, guanosine, inosine, guanine, hypoxanthine, xanthine and urate) from the entire purine metabolic pathway. The limit of detection (LoD) for each analyte was determined. The LoD using UV absorption was 0.001 mg/dL for hypoxanthine (Hyp), inosine (Ino), guanosine (Guo) and adenosine (Ado), and those using coulometric electrodes were 0.001 mg/dL for guanine (Gua), 0.0001 mg/dL for urate (UA) and 0.0005 mg/dL for xanthine (Xan). The intra- and inter-day coefficient of variance was generally <8%. Using this method, we determined basal levels of these metabolites in mouse brain and serum, as well as in post-mortem human brain. Peak identities were confirmed by enzyme degradation. Spike recovery was performed to assess accuracy. All recoveries fell within 80-120%. Our HPLC method provides a sensitive, rapid, reproducible and low-cost method for determining multiple purine metabolites in a single analysis in serum and brain specimens.

Deletion of adenosine A1 or A(2A) receptors reduces L-3,4-dihydroxyphenylalanine-induced dyskinesia in a model of Parkinson's disease.

Adenosine A(2A) receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson's disease (PD). Clinical trials of A(2A) antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A1 receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A1 and/or A(2A) receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A1, A(2A) and double A1-A(2A) knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A1 and A(2A) receptors) or saline were treated daily for 18-21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A1 and A(2A) KOs, but not in A1-A(2A) KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A1 or A(2A) receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear.