A Minimal Two-State Allosteric Dimer Model for SARM1 Regulation and Paradoxical Activation by Orthosteric Inhibitors

Technical Summary: A Minimal Two-State Allosteric Dimer Model for SARM1 Regulation and Paradoxical Activation by Orthosteric Inhibitors

Problem Statement
Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is an octameric NAD⁺-consuming enzyme that drives programmed axonal degeneration. Its activity is regulated by the balance between NAD⁺ (which stabilizes an inactive state) and nicotinamide mononucleotide (NMN, which promotes activation). While orthosteric base-exchange inhibitors (BEIs) were developed to suppress SARM1, a paradoxical phenomenon has been observed where subinhibitory concentrations of these inhibitors activate the enzyme, leading to neurodegeneration. Existing kinetic models fail to unify three critical observations: (1) substrate inhibition at high NAD⁺ concentrations, (2) regulation by the NMN/NAD⁺ ratio, and (3) low-dose activation by orthosteric BEIs. There is a lack of a unifying kinetic framework that explains how concerted transitions in this oligomeric enzyme lead to these complex behaviors.

Methodology
The authors integrated multiple experimental and computational approaches to evaluate a two-state Monod-Wyman-Changeux (MWC) framework for SARM1:

• Structural Analysis: Cryogenic electron microscopy (cryo-EM) was used to classify the conformational states of ligand-free SARM1ΔMTS (lacking the mitochondrial targeting sequence) and SARM1 in the presence of NAD⁺, NMN, and orthosteric inhibitors.
• Biochemical Kinetics: Fluorescence-based assays (PC6 and 1a probes) measured initial reaction velocities across varying concentrations of NAD⁺, NMN, and BEIs (GNE-5152, NB-3, NB3-ADPR). Data were fitted to logistic and Hill equations to determine cooperativity.
• Binding Measurements: Surface plasmon resonance (SPR) quantified the affinities of NAD⁺ and NMN for the catalytic (TIR) and allosteric (ARM) sites on various SARM1 constructs (full-length, SAM-TIR, ARM-SAM) under conditions biased toward either the inactive (T) or active (R) states using tool compounds.
• Kinetic Modeling: The authors formalized a minimal two-state MWC model incorporating state-preferential ligand binding and an additional term for orthosteric inhibitor interaction. They fitted experimental velocity data to this model to derive parameters such as the allosteric constant ($L$) and the number of cooperative units ($n$).
• Cellular Assays: Live-cell imaging in SH-SY5Y neuroblastoma cells monitored SARM1 activity via fluorescent ADPR adduct formation in the presence of vacor and BEIs.
• Chemical Synthesis: The study involved the synthesis of specific tool compounds (Compound 1, an allosteric inhibitor) and the NB3-ADPR adduct to facilitate binding and kinetic studies.

Key Contributions and Results

1. Conformational Baseline: Cryo-EM analysis of apo-SARM1ΔMTS revealed that the enzyme exclusively adopts an inactive (T) conformation with ARM domains in the autoinhibited state and TIR domains attached. This confirms a strong intrinsic bias toward the inactive state in the absence of activating ligands.
2. State-Selective Binding: SPR data demonstrated that NAD⁺ acts as a dual-function ligand: it binds the catalytic site (TIR) with moderate affinity and the allosteric site (ARM) with high affinity in the inactive (T) state ($K_D \approx 9$ µM), but with significantly lower affinity in the active (R) state ($K_D \approx 94$ µM). Conversely, NMN binds preferentially to the active (R) state ($K_D \approx 0.24$ µM).
3. Cooperative Transition: Kinetic analysis revealed state-dependent cooperativity. Hill coefficients for NMN activation were approximately 1.4 globally, rising to >1.5 when the enzyme was biased toward the inactive state. Cryo-EM showed that in the presence of equimolar NAD⁺ and NMN, particles existed exclusively in either all-active or all-inactive conformations, supporting a concerted transition mechanism.
4. The Minimal Dimer Model: The authors propose that the minimal cooperative unit for SARM1 is a dimer ($n \approx 1.27$ fitted, rounded to 2). This dimeric unit generates two catalytic sites. While SARM1 is an octamer, the model suggests that higher-order oligomerization increases catalytic capacity additively rather than cooperatively.
5. Explanation of Paradoxical Activation: The two-state MWC model successfully explains the paradoxical activation of SARM1 by low concentrations of orthosteric BEIs. When the enzyme population is predominantly in the T state (high $L$), partial occupancy of the orthosteric site by a BEI stabilizes the R state, promoting the T→R transition and increasing activity. At higher concentrations, the BEI acts purely as an inhibitor by blocking the catalytic site. This effect is most pronounced when the enzyme is T-biased (e.g., low NMN/NAD⁺ ratio).
6. Kinetic Distinctness of BEIs: The study found that BEIs exhibit slow association and dissociation kinetics and are poor substrates for base-exchange conversion compared to NAD⁺. This slow kinetics explains the delayed onset of activation observed in progress curves and precludes rapid-equilibrium analysis for direct fitting, though the equilibrium model remains valid for interpreting the direction of the effect.

Significance
The paper claims that a minimal two-state MWC framework with a dimeric cooperative unit provides a unifying explanation for SARM1 regulation, resolving previously conflicting observations regarding substrate inhibition and paradoxical activation. The authors assert that this mechanism is not compound-specific but is an inherent property of oligomeric enzymes undergoing concerted transitions. Consequently, the study highlights a critical risk in drug discovery: for such enzymes, partial occupancy of the active site by orthosteric inhibitors can unintentionally promote activation rather than inhibition. The authors conclude that these principles must be incorporated early in the development of inhibitors for oligomeric enzymes to mitigate the risk of unintended activation. The work does not propose new therapeutic applications but rather establishes quantitative criteria for assessing the safety of targeting concerted oligomeric enzymes.