A phase transition enhances the catalytic activity of SARM1, an NAD+ glycohydrolase involved in neurodegeneration.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to stress. NAD+ depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.

Identification of the first noncompetitive SARM1 inhibitors.

Sterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) is a key therapeutic target for diseases that exhibit Wallerian-like degeneration; Wallerian degeneration is characterized by degeneration of the axon distal to the site of injury. These diseases include traumatic brain injury, peripheral neuropathy, and neurodegenerative diseases. SARM1 promotes neurodegeneration by catalyzing the hydrolysis of NAD+ to form a mixture of ADPR and cADPR. Notably, SARM1 knockdown prevents degeneration, indicating that SARM1 inhibitors will likely be efficacious in treating these diseases. Consistent with this hypothesis is the observation that NAD+ supplementation is axoprotective. To identify compounds that block the NAD+ hydrolase activity of SARM1, we developed and performed a high-throughput screen (HTS). This HTS assay exploits an NAD+ analog, etheno-NAD+ (ENAD) that fluoresces upon cleavage of the nicotinamide moiety. From this screen, we identified berberine chloride and zinc chloride as the first noncompetitive inhibitors of SARM1. Though modest in potency, the noncompetitive mode of inhibition, suggests the presence of an allosteric binding pocket on SARM1 that can be targeted for future therapeutic development. Additionally, zinc inhibition and site-directed mutagenesis reveals that cysteines 629 and 635 are critical for SARM1 catalysis, highlighting these sites for the design of inhibitors targeting SARM1.

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD+, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 ± 2000 M-1 s-1) is at least as active as the TIR domain alone (kcat/KM = 1500 ± 300 M-1 s-1). Finally, we show that the SARM1 hydrolyzes NAD+ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.

Emergence of SARM1 as a Potential Therapeutic Target for Wallerian-type Diseases.

Wallerian degeneration is a neuronal death pathway that is triggered in response to injury or disease. Death was thought to occur passively until the discovery of a mouse strain, i.e., Wallerian degeneration slow (WLDS), which was resistant to degeneration. Given that the WLDS mouse encodes a gain-of-function fusion protein, its relevance to human disease was limited. The later discovery that SARM1 (sterile alpha and toll/interleukin receptor [TIR] motif-containing protein 1) promotes Wallerian degeneration suggested the existence of a pathway that might be targeted therapeutically. More recently, SARM1 was found to execute degeneration by hydrolyzing NAD+. Notably, SARM1 knockdown or knockout prevents neuron degeneration in response to a range of insults that lead to peripheral neuropathy, traumatic brain injury, and neurodegenerative disease. Here, we discuss the role of SARM1 in Wallerian degeneration and the opportunities to target this enzyme therapeutically.

Kinetic Mechanism of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide, pyridine, and other structural analogues. Aberrantly increased NNMT activity results in the depletion of SAM, nicotinamide (NAM), and nicotinamide adenine dinucleotide (NAD+); NAM is required for NAD+ biosynthesis. SAM depletion impairs the methylation potential of the cell, resulting in hypomethylated histones and an altered epigenetic profile. In addition, decreased NAD+ levels negatively affect energy metabolism by disrupting oxidative phosphorylation. Because of its impact on epigenetic states and NAD+ levels, NNMT is implicated in cancer, neurodegenerative diseases, and metabolic diseases, making it an appealing target for therapeutic intervention. To gain insights that would guide the design of inhibitors and activity-based probes, we performed detailed kinetic studies of human NNMT. Herein, we report the kinetic mechanism of NNMT. Our initial velocity, product inhibition, and dead-end analogue inhibition studies collectively indicate that NNMT uses a rapid equilibrium ordered mechanism, where NNMT first binds SAM, which is followed by NAM. Methyl transfer occurs, and methylated NAM and S-adenosylhomocysteine are released consecutively.

Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans.

The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

Current status of gene therapy for α-1 antitrypsin deficiency.

INTRODUCTION: As a common monogenic disease, α-1 antitrypsin (AAT) deficiency has undergone thorough investigation for the development of gene therapy. The most common pathology associated with AAT deficiency occurs in the lung, where the loss of function due to impaired secretion of mutant AAT prevents the inhibition of neutrophil elastase and leads to loss of elastin content from the alveolar interstitium. AREAS COVERED: Current treatment in the USA consists of recurrent intravenous protein replacement therapy to augment serum AAT levels. In an attempt to replace recurring treatments with a single dose of gene therapy, recombinant adenovirus, plasmid, and recombinant adeno-associated virus (rAAV) vectors have been investigated as vectors for transgene delivery. EXPERT OPINION: Large strides in gene therapy for AAT deficiency lung disease have led to the development of rAAV1-AAT capable of producing sustained serum AAT levels in clinical trials after intramuscular administration in humans at 3% of the target level. Further increases in levels are anticipated as limb perfusion targets greater muscle mass. The future roles of intrapleural and airway delivery, miRNA-expressing vectors, iPS cell platforms, and genome editing are anticipated.