5-Amino-1MQ

Research Overview

5-Amino-1MQ serves as a valuable research tool for investigating nicotinamide N-methyltransferase (NNMT) biology and NAD+ metabolism regulation in laboratory settings. This small molecule compound represents a selective inhibitor of NNMT, an enzyme that methylates nicotinamide (a form of vitamin B3) to produce 1-methylnicotinamide (1-MNA). By inhibiting NNMT, 5-Amino-1MQ increases intracellular nicotinamide availability for NAD+ biosynthesis via the salvage pathway, thereby modulating cellular NAD+ levels and NAD+-dependent signaling processes.

Research interest in NNMT as a metabolic regulator emerged from observations that NNMT expression is elevated in obesity, diabetes, and certain cancers. The enzyme consumes both nicotinamide and S-adenosylmethionine (SAM, the universal methyl donor), potentially affecting both NAD+ metabolism and cellular methylation capacity. Laboratory studies investigate 5-Amino-1MQ’s effects on energy expenditure, mitochondrial function, adipose tissue biology, insulin sensitivity, and epigenetic regulation through modulation of NAD+ and methylation pathways.

5-Amino-1MQ research demonstrates the principle that targeting metabolic enzymes can modulate cellular energy status and metabolic phenotype. The compound’s NNMT inhibitory activity provides a tool for investigating whether reducing nicotinamide methylation and increasing NAD+ availability can enhance metabolic function, improve mitochondrial health, and affect body composition. This makes 5-Amino-1MQ valuable for studying metabolic regulation at the level of NAD+ metabolism rather than through hormone receptors.

Molecular Characteristics

Complete Specifications:

• Chemical Name: 5-Amino-1-methylquinolinium
• CAS Number: 42464-96-0
• Molecular Weight: 173.21 Da (cation)
• Molecular Formula: C₁₀H₁₁N₂⁺
• Structure: Quinolinium cation with amino group at position 5
• Chemical Classification: Quinolinium compound, NNMT inhibitor
• Appearance: Yellow to yellow-orange powder
• Solubility: Water-soluble (charged cation), DMSO
• Target Enzyme: Nicotinamide N-methyltransferase (NNMT)
• Selectivity: Selective for NNMT vs. other methyltransferases

The compound’s structure consists of a quinoline ring system with a quaternary nitrogen (N-methylated at position 1) and an amino group at position 5. This quinolinium cation structure enables selective recognition by the NNMT active site, where it acts as a competitive inhibitor with respect to nicotinamide substrate. The charged nature contributes to water solubility while the lipophilic quinoline ring enables cellular membrane penetration. Structure-activity relationship studies have identified the 5-amino position as critical for NNMT inhibitory potency.

Mechanism of Action and Target Biology

NNMT Enzyme and Function:

Nicotinamide N-methyltransferase (NNMT) catalyzes the SAM-dependent methylation of nicotinamide:

• Reaction: Nicotinamide + SAM → 1-methylnicotinamide (1-MNA) + S-adenosylhomocysteine (SAH)
• Expression: Liver, adipose tissue (particularly visceral), smooth muscle, some neurons
• Regulation: Increased expression in obesity, diabetes, aging, certain cancers
• Substrates consumed: Nicotinamide (NAD+ precursor) and SAM (methyl donor)

5-Amino-1MQ Inhibition Mechanism:

• Competitive inhibitor with respect to nicotinamide substrate
• IC₅₀: Submicromolar range for NNMT enzyme
• Selectivity: Minimal inhibition of other methyltransferases at relevant concentrations
• Result: Increased intracellular nicotinamide availability for NAD+ salvage pathway

Downstream Effects of NNMT Inhibition:
1. Increased NAD+ Levels: More nicotinamide available for NAD+ biosynthesis via NAMPT enzyme
2. Enhanced Sirtuin Activity: NAD+-dependent deacetylases (SIRT1, SIRT3) activated
3. Improved Mitochondrial Function: Enhanced oxidative phosphorylation and biogenesis
4. Altered Methylation: Preserved SAM for other methylation reactions
5. Metabolic Reprogramming: Enhanced fatty acid oxidation, improved insulin sensitivity

Pharmacokinetic Profile in Research Models

5-Amino-1MQ characterization in preclinical research:

Absorption and Distribution:

• Oral bioavailability: Moderate (30-50% in rodent models)
• Subcutaneous bioavailability: Higher (>70%)
• Tissue distribution: Liver, adipose tissue (high NNMT expression sites)
• Blood-brain barrier: Limited penetration (peripheral action)

Metabolism and Elimination:

• Plasma half-life: 4-6 hours (rodent models)
• Metabolism: Hepatic Phase II conjugation
• Elimination: Primarily renal
• Accumulation: Possible in tissues with active uptake

Pharmacodynamics:

• NNMT inhibition: Dose-dependent in liver and adipose
• NAD+ elevation: Detectable within hours, sustained with repeated dosing
• Sirtuin activation: Secondary to NAD+ increase
• Duration: Effects persist during chronic dosing

These properties inform research protocol design, particularly regarding dosing frequency, route selection, and timing of metabolic assessments.

Research Applications

NNMT Enzyme Biology and Inhibition Studies

5-Amino-1MQ serves as a tool for investigating NNMT function:

• Enzyme Kinetics: Examination of inhibition mechanism, IC₅₀ determination, selectivity profiling
• Cellular NNMT Activity: Measurement of 1-MNA production in cells/tissues with/without inhibitor
• NNMT Expression Regulation: Studies on factors regulating NNMT gene expression
• Tissue Distribution: Investigation of NNMT expression patterns across tissues and cell types
• Disease Context: Research on NNMT dysregulation in metabolic disease, cancer, other pathologies

Research protocols use biochemical assays, mass spectrometry for 1-MNA quantification, and gene expression analysis.

NAD+ Metabolism and Salvage Pathway Research

Laboratory studies investigate effects on cellular NAD+ levels:

• NAD+ Quantification: Measurement of total NAD+, NADH, NAD+/NADH ratio in cells/tissues
• Salvage Pathway Flux: Investigation of nicotinamide → NAD+ conversion via NAMPT
• NAD+ Precursor Effects: Comparison to nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) supplementation
• Tissue-Specific Effects: Examination of NAD+ changes in liver, adipose, muscle, other tissues
• Temporal Dynamics: Studies on time course of NAD+ elevation following NNMT inhibition

Research examines whether NNMT inhibition represents an alternative strategy for enhancing cellular NAD+ beyond direct precursor supplementation.

Sirtuin Activation and Signaling Research

Given NAD+ dependence of sirtuins, substantial research focuses on these effects:

• SIRT1 Activity: Investigation of nuclear SIRT1 deacetylase activity, target protein acetylation status
• SIRT3 Mitochondrial Effects: Research on mitochondrial protein acetylation, oxidative metabolism
• PGC-1α Activation: Studies on SIRT1-mediated PGC-1α deacetylation and transcriptional activity
• FOXO Activation: Examination of FOXO transcription factor deacetylation and target gene expression
• Metabolic Gene Expression: Investigation of genes regulated by NAD+-dependent pathways

Experimental models include sirtuin activity assays, acetylation-specific immunoblotting, and gene expression profiling.

Energy Expenditure and Thermogenesis Research

Research applications extend to metabolic rate investigation:

• Oxygen Consumption: Measurement of whole-body or tissue-specific respiration rates
• Mitochondrial Function: Studies on oxidative phosphorylation, ATP production, membrane potential
• Mitochondrial Biogenesis: Investigation of mitochondrial mass, mtDNA copy number, biogenesis gene expression
• Brown Adipose Tissue: Research on BAT activation, UCP1 expression, thermogenic capacity
• Physical Activity: Examination of spontaneous activity and exercise performance
• Substrate Oxidation: Studies on fatty acid vs. glucose oxidation preference

Laboratory protocols use metabolic caging systems, Seahorse respirometry, and enzyme activity assays.

Adipose Tissue Biology and Body Composition

Laboratory studies investigate effects on adipose tissue:

• Adipocyte Metabolism: Examination of lipolysis, lipogenesis, mitochondrial function in adipocytes
• White Adipose Browning: Research on beige adipocyte formation, UCP1 expression in white depots
• Adipose NNMT: Investigation of visceral adipose NNMT expression and inhibition effects
• Adipokine Secretion: Studies on leptin, adiponectin, inflammatory cytokine expression
• Body Composition: Examination of fat mass, lean mass changes with chronic NNMT inhibition
• Adipose Inflammation: Research on macrophage infiltration, inflammatory signaling

Experimental models include adipocyte cell cultures, adipose tissue explants, and body composition assessment in obesity models.

Insulin Sensitivity and Glucose Metabolism

Research investigates metabolic improvements:

• Glucose Tolerance: Examination of glucose clearance, insulin secretion, tissue glucose uptake
• Insulin Sensitivity: Studies on insulin receptor signaling, AKT activation, glucose transporter expression
• Hepatic Glucose Production: Investigation of gluconeogenesis, glycogen metabolism
• Skeletal Muscle Metabolism: Research on muscle glucose uptake, glycogen storage, insulin signaling
• Pancreatic Beta Cell Function: Studies on insulin secretion capacity and beta cell health

Laboratory protocols include glucose and insulin tolerance testing, hyperinsulinemic-euglycemic clamps, and molecular signaling analyses.

Epigenetic Regulation Through Methylation

5-Amino-1MQ enables investigation of NNMT’s role in methylation homeostasis:

• SAM/SAH Ratio: Measurement of methylation capacity through SAM and SAH quantification
• Global DNA Methylation: Studies on genomic methylation patterns
• Histone Methylation: Investigation of histone methyltransferase activity and histone marks
• Gene Expression: Examination of methylation-sensitive gene regulation
• Metabolic Reprogramming: Research on epigenetic contribution to metabolic phenotype

Research addresses whether NNMT competes for SAM with other critical methyltransferases.

Laboratory Handling and Storage Protocols

Powder Storage:

• Store at room temperature (15-25°C) or 4°C for extended stability
• Protect from light (amber vial or foil wrapping)
• Protect from moisture (desiccated environment)
• Stability: 24+ months under proper conditions
• Do not freeze (may affect solubility)

Preparation of Stock Solutions:

• Solvent selection: Sterile water (best), DMSO (if water-insoluble formulation)
• Water: Directly dissolve at desired concentration (highly soluble)
• DMSO: Use if preparing concentrated stocks; dilute in aqueous buffer for experiments
• pH consideration: Slightly acidic to neutral pH optimal
• Filter sterilization: 0.22μm filter if sterility required

Stock Solution Storage:

• Aqueous solutions: 4°C for short-term (1 week), -20°C for longer (1 month)
• DMSO stocks: -20°C in aliquots (stable for months)
• Avoid repeated freeze-thaw cycles
• Protect from light during storage
• Check for precipitation before use

Administration in Research Protocols:

• Oral gavage: Dissolve in water or saline
• Subcutaneous injection: Aqueous solution, well-tolerated
• In vitro: Add directly to culture medium (consider DMSO concentration if used)
• Dose optimization: Titrate based on research objectives and NNMT inhibition verification

Quality Assurance and Analytical Testing

Purity Analysis:

• HPLC: ≥98% purity
• UV detection: Characteristic absorption due to quinolinium structure
• Related substances: <2% total impurities

Structural Verification:

• ESI-MS or APCI-MS: Confirms MW 173.21 Da (M⁺ cation)
• NMR spectroscopy: ¹H and ¹³C NMR confirms structure
• Melting point: Characteristic range verification

Contaminant Testing:

• Heavy metals: <10 ppm per USP standards
• Residual solvents: Per ICH Q3C guidelines
• Water content: Karl Fischer (if lyophilized form)

Biological Activity:

• NNMT inhibition assay: IC₅₀ determination
• Comparison to reference standard
• Cellular 1-MNA production assay

Documentation:

• Certificate of Analysis with complete data
• NMR spectra available upon request
• Batch traceability by lot number
• Stability data for storage conditions

Research Considerations

Experimental Design:

1. Dose Selection: In vitro: 1-100 μM typical range. In vivo: 50-100 mg/kg (rodents) for robust NNMT inhibition

2. Verification of Target Engagement:

• Measure 1-MNA levels (should decrease)
• Measure NAD+ levels (should increase)
• Both confirm on-target NNMT inhibition

3. Temporal Considerations:

• Acute effects (hours-days): NAD+ elevation, initial sirtuin activation
• Chronic effects (weeks): Mitochondrial biogenesis, metabolic improvements, body composition

4. Model Selection:

• In vitro: Primary hepatocytes, adipocytes, cells with high NNMT expression
• In vivo: DIO models, aging models, genetic obesity models
• Consider NNMT expression levels in chosen model

5. Control Groups:

• Vehicle control
• NAD+ precursor supplementation (NR, NMN) for comparison
• NNMT overexpression models (opposite phenotype)

Mechanism Investigation:

Key research questions:

• Are effects mediated primarily through NAD+ elevation?
• What is the relative contribution of preserved SAM vs. increased NAD+?
• Which sirtuins are most critical for observed effects?
• Do effects require functional mitochondria?
• Are benefits additive with NAD+ precursor supplementation?

Combination Studies:

5-Amino-1MQ investigated in combination with:

• NAD+ precursors (NR, NMN): Potentially synergistic for NAD+ elevation
• Metabolic peptides (GLP-1 agonists): Appetite suppression + metabolic rate enhancement
• Exercise training: Enhanced mitochondrial adaptations
• Caloric restriction: Complementary NAD+ boosting approaches

Compliance and Safety Information

Regulatory Status:
5-Amino-1MQ is provided as a research chemical for in-vitro laboratory studies and preclinical research only. Not approved for human use, therapeutic applications, or dietary supplementation.

Intended Use:

• In-vitro enzyme inhibition studies
• Cell culture research on NAD+ metabolism
• In-vivo preclinical research with IACUC approval
• NNMT biology investigation
• Academic and institutional research only

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or metabolic enhancement
• Athletic performance enhancement
• Veterinary therapeutic use
• Non-research applications

Safety Protocols:

• Standard laboratory PPE (lab coat, gloves, safety glasses)
• Handle in well-ventilated areas
• Avoid dust inhalation (use fume hood for weighing)
• Follow institutional chemical hygiene guidelines
• Proper waste disposal per regulations
• Consult SDS for detailed safety information

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