5-Amino-1MQ (also written as 5A1MQ or 5-AMQ in the research) is not a peptide - it's a small manufactured molecule, though it's commonly sold next to peptides for body-composition research. It was developed by academic researchers as a way to block an enzyme called NNMT, which is overactive in fat tissue, aging muscle, and several diseases. Since its introduction in 2018, it has been used mainly as a research tool to study obesity, blood sugar, muscle aging, and even certain cancers in mice and rats. No human clinical trial of this compound has ever been published.
How strong is the evidence?
Every study that actually dosed animals or cells with this compound (or its close chemical cousins) was done in mice, rats, or lab dishes - there are no human trials. The most consistent finding, repeated across several independent studies, is that blocking NNMT helps obese mice lose fat and improve blood sugar control without obvious harm. There's also a solid single study showing it speeds up muscle recovery in old mice, and early cancer research showing it can slow tumor growth when combined with immunotherapy. The rest of the 35 papers on file are mostly about NNMT biology in general - cancer, kidney disease, liver disease, heart failure - using different NNMT-blocking chemicals, not 5-Amino-1MQ itself. That broader research explains why scientists are interested in this enzyme, but it isn't direct evidence for this specific compound.
Uses
What people use it for
Fat loss and body composition research
Animal / labThis is the main reason people look into 5-Amino-1MQ. It was originally built to fight obesity, and the strongest data behind it comes from studies in overweight mice, not people.
Blood sugar and metabolic health research
Animal / labResearchers have also tested it for insulin sensitivity and blood sugar control in obese mice, alongside its fat-loss effects.
Muscle recovery in aging
Animal / labOne research group tested it in old mice to see if it could restore the muscle's own repair cells after injury, with encouraging results.
Early-stage cancer and fibrosis research
Animal / labBecause the enzyme it blocks (NNMT) is overactive in some tumors and scarred organs, scientists are testing NNMT-blocking compounds - including this one, in at least one bladder cancer study - as an add-on to cancer immunotherapy. This is lab and animal research, not a personal-use case.
Potential benefits
What it may help with
Fat and body weight reduction (in obese mice)
Animal / labThe founding study showed that this class of compound reversed diet-induced obesity in mice - lower body weight, less white fat, smaller fat cells, and lower cholesterol - without reducing how much the mice ate. A later study using the compound by name (5A1MQ) found that it dose-dependently limited body weight and fat gains in obese mice - it slowed the weight and fat they packed on, rather than reversing existing weight - and a third study paired it with a reduced-calorie diet to produce dramatic fat loss in obese mice.
Better blood sugar control and insulin sensitivity
Animal / labIn obese mice, daily dosing improved how well the body handled a glucose challenge and made the animals more sensitive to their own insulin, while lowering excess insulin levels in the blood.
Studies:39161060Less fat buildup in the liver
Animal / labThe same obese-mice study found less fatty liver disease on tissue exams, smaller and lighter livers, lower liver triglycerides, and normalized liver enzyme levels in the blood.
Studies:39161060Faster, stronger muscle recovery after injury (in old mice)
Animal / labIn 24-month-old mice (elderly, in mouse terms), daily treatment after a muscle injury woke up dormant muscle repair cells, produced muscle fibers nearly twice as large during healing, and increased peak muscle strength by about 70% compared to untreated mice.
Studies:30753815Slower tumor growth in an early cancer-immunotherapy study
Animal / labIn mice with bladder cancer, this exact compound slowed tumor growth and made an immunotherapy drug (an anti-PD-L1 antibody) work better. This is very early, disease-specific research and not a reason for a healthy person to use it.
Studies:39067875
What to watch for
Side effects & risks
- Mild
No adverse effects reported in the core animal study
In the foundational obesity study, treated mice showed no drop in food intake and no observable adverse effects at the doses tested. That's reassuring, but it's one study in mice over a matter of weeks, not a safety study in people.
- Moderate
Dosing
Dosing — what studies used
There is no established human dose for 5-Amino-1MQ - it has never been given to a person in a published study, so anyone using it is essentially guessing. Even the animal research rarely states an exact milligram dose in the published summaries. The clearest documented example used 5 or 10 milligrams per kilogram of body weight, given daily, in mice. Other studies dosed mice once a day for about 4 weeks by mouth, under the skin, or into a vein, and one pharmacokinetic study in rats measured how long it stays in the bloodstream - but none of this translates into a known safe or effective human dose.
Muscle injury recovery in aged mice
Animal study5 mg/kg (low dose) or 10 mg/kg (high dose) body weight
Daily · 1 week (both doses) or 3 weeks (high dose only) after injury · Not specified in the abstract
This is the clearest numeric dose reported in the literature on file, but it's a mouse injury-recovery study, not a human protocol.
Diet-induced obese mice - fat loss and metabolic effects
Animal studyNot stated in the published abstract; described only as dose-dependent
Once daily · 28 days · Subcutaneous (main efficacy dosing); oral, IV, and subcutaneous all tested for absorption
Tracked body weight, fat mass, blood sugar, insulin, and liver fat over 4 weeks in mice.
Pharmacokinetics (how the compound moves through the body)
Animal studyNot stated in the published abstract
Single dose, comparing routes · Single-dose study, blood sampled over several hours · Intravenous and oral, in rats
Oral bioavailability was about 38%. The drug left the bloodstream with a half-life of roughly 4-7 hours - useful for understanding how often it would need to be dosed, but this is rat data, not human data.
Sold online as a 'research chemical,' not an approved drug or regulated supplement. There is no dosing guidance from any regulator or clinical trial - treat any specific human dose you see elsewhere (forums, vendor sites) as an unverified guess, not science.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
Every cell has a helper enzyme called NNMT. Its job is to grab a B vitamin (vitamin B3) and a molecule called SAM - think of SAM as the cell's supply of tiny chemical tags it uses to switch genes and proteins on and off - and stick them together. That uses up both ingredients. In fat cells, aging muscle, scarred organs, and many tumors, NNMT runs in overdrive, and that drains the cell's SAM along with NAD+, a molecule cells need to produce energy. Low SAM and NAD+ seem to push fat cells toward storing more fat instead of burning it, slow down muscle repair, and help tumors and scar tissue thrive. 5-Amino-1MQ blocks NNMT. With the enzyme switched off, SAM and NAD+ stay higher inside the cell, energy-burning genes switch on, and - in the animal studies done so far - fat tissue shrinks, blood sugar control improves, and aging muscle heals faster.
Who should avoid it
- Anyone who is pregnant or breastfeeding - there is zero safety testing in these groups
- Anyone with a current or past cancer diagnosis - NNMT plays a complicated, cancer-type-specific role, and self-experimenting with it outside medical supervision could do more harm than good
- Anyone with kidney or liver disease, given how closely this pathway is tied to both organs in the research, with no human data to guide dosing
- Anyone looking for a well-tested, doctor-approved option - this compound has not reached that stage
Interactions to know
- No interaction studies exist in humans - it has never been tested alongside any medication or supplement in people.
- Because it works on the same NAD+ and methylation pathways as niacin, NMN, NR, and SAMe supplements, combining them could theoretically change how each one behaves - but this has not been studied and is a mechanistic guess, not a documented finding.
The papers that matter most
Key studies
The foundational paper behind this whole compound class: methylquinolinium NNMT inhibitors reduced body weight, fat mass, and cholesterol in obese mice with no observable adverse effects and no change in food intake.
Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice
Testing 5A1MQ by name: dose-dependent fat loss, better glucose tolerance and insulin sensitivity, and reduced fatty liver in obese mice over 28 days, alongside pharmacokinetic data.
Nicotinamide N-methyltransferase inhibition mitigates obesity-related metabolic dysfunction
In 24-month-old mice, daily NNMT-inhibitor dosing (5-10 mg/kg) after a muscle injury woke up dormant muscle repair cells and increased peak muscle strength by about 70% versus untreated controls.
Small molecule nicotinamide N-methyltransferase inhibitor activates senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle
Established how the compound behaves in the body: about 38% oral bioavailability and a half-life of roughly 4-7 hours in rats - basic pharmacology, not efficacy or safety data.
Development & validation of LC-MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies
5-Amino-1-methylquinolinium reduced tumor growth and improved response to immunotherapy in mouse models of bladder cancer - an early signal for a very different, disease-specific use case.
NAD+ metabolism enzyme NNMT in cancer-associated fibroblasts drives tumor progression and resistance to immunotherapy by modulating macrophages in urothelial bladder cancer
A 2026 review of the whole NNMT-inhibitor field explaining why, despite encouraging animal data, poor drug properties and unproven safety have kept every compound in this class - including 5-Amino-1MQ - out of human trials so far.
Emerging opportunities for nicotinamide N-methyltransferase (NNMT) inhibitor clinical translation
Bottom line
5-Amino-1MQ has a genuinely interesting and repeatable story in animal research for fat loss, blood sugar, and muscle recovery, but it remains an unproven research chemical with no human trials, no established dose, and no long-term safety data - anyone using it today is experimenting on themselves.
Research papers
Studies we have on file for 5-Amino-1MQ. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
35 papers
NNMT inhibition in cancer-associated fibroblasts restores antitumour immunity.
Cancer-associated fibroblasts (CAFs) have a pivotal cancer-supportive role, yet CAF-targeted therapies are lacking1,2. Here, using spatial transcriptomics and single-cell RNA sequencing, we investigate the role of nicotinamide N-methyltransferase (NNMT) in high-grade serous ovarian cancer. Mechanistically, NNMT-induced H3K27me3 hypomethylation drives complement secretion from CAFs, attracting immunosuppressive myeloid-derived suppressor cells (MDSCs) to the tumour. Nnmt knockout in immunocompetent mice impairs tumour growth in syngeneic ovarian, breast and colon tumour models through enhanced CD8+ T cell activation. Using high-throughput screening, we develop a potent and specific NNMT inhibitor that reduces the tumour burden and metastasis in multiple mouse cancer models and restores immune checkpoint blockade efficacy by decreasing CAF-mediated recruitment of MDSCs and reinvigorating CD8+ T cell activation. Our findings establish NNMT as a central CAF regulator and a promising therapeutic target to mitigate immunosuppression in the tumour microenvironment.
ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver development.
N-nicotinamide methyltransferase (NNMT) is emerging as an important enzyme in the regulation of metabolism. NNMT is highly expressed in the liver. However, the exact regulatory mechanism(s) underlying NNMT expression remains unclear and its potential involvement in alcohol-related liver disease (ALD) is completely unknown. Both traditional Lieber-De Carli and the NIAAA mouse models of ALD were employed. A small-scale chemical screening assay and a chromatin immunoprecipitation assay were performed. NNMT inhibition was achieved via both genetic (adenoviral short hairpin RNA delivery) and pharmacological approaches. Chronic alcohol consumption induces endoplasmic reticulum (ER) stress and upregulates NNMT expression in the liver. ER stress inducers upregulated NNMT expression in both AML12 hepatocytes and mice. PERK-ATF4 pathway activation is the main contributor to ER stress-mediated NNMT upregulation in the liver. Alcohol consumption fails to upregulate NNMT in liver-specific Atf4 knockout mice. Both adenoviral NNMT knockdown and NNMT inhibitor administration prevented fatty liver development in response to chronic alcohol feeding; this was also associated with the downregulation of an array of genes involved in de novo lipogenesis, including Srebf1, Acaca, Acacb and Fasn. Further investigations revealed that activation of the lipogenic pathway by NNMT was independent of its NAD+-enhancing action; however, increased cellular NAD+, resulting from NNMT inhibition, was associated with marked liver AMPK activation. ER stress, specifically PERK-ATF4 pathway activation, is mechanistically involved in hepatic NNMT upregulation in response to chronic alcohol exposure. Overexpression of NNMT in the liver plays an important role in the pathogenesis of ALD. In this study, we show that nicotinamide methyltransferase (NNMT) - the enzyme that catalyzes nicotinamide degradation - is a pathological regulator of alcohol-related fatty liver development. NNMT inhibition protects against alcohol-induced fatty liver development and is associated with suppressed de novo lipogenic activity and enhanced AMPK activation. Thus, our data suggest that NNMT may be a potential therapeutic target for the treatment of alcohol-related liver disease.
NNMT inhibition counteracts tubular senescence and fibrosis in early stages of chronic kidney disease.
Chronic kidney disease (CKD) is projected to become the fifth leading cause of mortality by 2040. Tubular senescence drives kidney fibrosis, but current treatments do not target senescent cells. Here, we identify nicotinamide-N-methyltransferase (NNMT) as a critical mediator of tubular senescence and kidney fibrosis. Human CKD microarrays link NNMT to senescence and fibrosis transcriptomic signatures, and diabetic kidney disease (DKD) biopsies show NNMT protein associating with p21, fibrosis, and kidney function decline. Spatial transcriptomics in human biopsies demonstrates that NNMT-positive tubules are senescent, fibrotic, and surrounded by a pro-inflammatory microenvironment. Importantly, this pattern is conserved in aged and DKD mice, mimicking early-stage CKD features. Mechanistically, NNMT overexpression in tubular epithelial cells exacerbates senescence and partial epithelial-to-mesenchymal transition, while selective NNMT inhibition in senescent kidney cells, organoids, and in vivo is protective. Altogether, these findings position NNMT as a promising therapeutic target to reduce tubular senescence and fibrosis in early CKD.
NAD+ metabolism enzyme NNMT in cancer-associated fibroblasts drives tumor progression and resistance to immunotherapy by modulating macrophages in urothelial bladder cancer.
This study comprehensively investigates the association between the expression of nicotinamide N-methyltransferase (NNMT) and clinical outcomes of urothelial bladder cancer (UBC), as well as the molecular mechanisms by which NNMT in cancer-associated fibroblast (CAF) modulates tumor progression and immunotherapy resistance in UBC. Single-cell transcriptomic analyses, immunohistochemical and immunofluorescence assays were performed on bladder cancer samples to validate the relationship between NNMT expression and clinical outcomes. A series of experiments, including chromatin immunoprecipitation assay, liquid chromatography tandem mass spectrometry assay, and CRISPR‒Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) knockout, together with in vivo models, have been established to determine the molecular functions of NNMT in CAFs in UBC. We demonstrated that elevated expression of the nicotinamide adenine dinucleotide (NAD+) metabolism enzyme NNMT in CAFs (NNMT+ CAFs) was significantly associated with non-response to programmed death-ligand 1 (PD-L1) blockade immunotherapy in patients with UBC and predicted the unfavorable prognosis of UBC in two independent large cohorts. Targeting NNMT using the inhibitor 5-Amino-1-methylquinolinium iodide significantly reduced tumor growth and enhanced the apoptotic effects of the anti-PD-L1 antibody in UBC mouse models. Mechanistically, NNMT+ CAFs recruit tumor-associated macrophages via epigenetic reprogramming of serum amyloid A (SAA) to drive tumor cell proliferation and confer resistance to programmed death-1/PD-L1 blockade immunotherapy. NNMT+ CAFs were significantly associated with non-response to PD-L1 blockade immunotherapy in patients with UBC. Elevated NNMT, specifically in CAFs, upregulates SAA expression and enhances the recruitment and differentiation of macrophages in the tumor microenvironment, thereby directly or indirectly promoting tumor progression and conferring resistance to immunotherapies in bladder cancer.
Nicotinamide-N-methyltransferase inhibition improves cardiac function and structure in a heart failure with preserved ejection fraction mouse model.
HFpEF is a major and global disease with limited treatment options and novel therapeutics are eagerly awaited. A potential treatment option may be Nicotinamide N-methyltransferase (NNMT) inhibition. This study aimed to investigate the cardiac protective effects of the NNMT enzyme inhibitor AMO-NAM in a HFpEF mouse model. Aged (18-22 months old) female mice developed a cardiometabolic HFpEF phenotype using a multiple hit strategy with high-fat diet (HFD) and angiotensin II (AngII) infusion. NNMT inhibitor 4-amino-6-methoxynicotinamide (AMO-NAM) was added to HFD and mice were treated for four weeks. Cardiac function was assessed by echocardiography, molecular (RT-qPCR; O-link, LC-MS assay) and histological analyses (Masson staining; macrophage staining) were performed to evaluate AMO-NAMs drug-specific effects. We observed significant increases in 1-MNA (+121.6 %), the product of the reaction catalysed by NNMT, and its downstream metabolites levels, 2PY (+274.7 %), and 4PY (+296.4 %) in the left ventricle (LV) of the HFpEF model. Treatment with the AMO-NAM did not affect NAD levels in the HFpEF model but markedly decreased 1-MNA (-77.1 %), 2PY (-66.2 %), and 4PY (-71.1 %) levels. NNMT inhibition led to notable improvements in cardiac function, evidenced by enhanced global longitudinal strain and reversed peak longitudinal strain rate alongside significant reductions in LV hypertrophy and fibrosis. This was accompanied by decreased pro-inflammatory and pro-fibrotic gene expression in plasma and LV tissue and reduced macrophage infiltration in LV and visceral adipose tissue, highlighting the anti-inflammatory and anti-fibrotic effects of NNMT inhibition. Targeting the NNMT is cardioprotective and holds promise for treating HFpEF patients with an unfavorable cardiometabolic phenotype.
Nicotinamide N-methyl transferase (NNMT): An emerging therapeutic target.
Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (NA) to generate 1-methyl nicotinamide. Since its discovery 70 years ago, the appreciation of the role of NNMT in human health has evolved from serving only metabolic functions to also being a driving force in diseases, including a variety of cancers. Despite the increasing evidence indicating NNMT as a viable therapeutic target, the development of cell-active inhibitors against this enzyme is lacking. In this review, we provide an overview of the current status of NNMT inhibitor development, relevant in vitro and in vivo studies, and a discussion of the challenges faced in the development of NNMT inhibitors.
High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT).
Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nicotinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression has been linked to diabetes, obesity, and various cancers. In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM → NAM methyl transfer reaction. NS1 was synthesized in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray cocrystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes.
Nicotinamide N -methyltransferase promotes M2 macrophage polarization by IL6 and MDSC conversion by GM-CSF in gallbladder carcinoma.
Nicotinamide N -methyltransferase (NNMT), an enzyme responsible for the methylation of nicotinamide, is involved in many metabolic pathways in adipose tissue and the liver. However, the role of NNMT in editing the tumor immune microenvironment is not well understood. Here, we identified that NNMT can promote IL6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by decreasing the tri-methyl-histone H3 levels on the promoters of IL6 and CSF2 (encoding GM-CSF) and CCAAT/Enhancer Binding Protein, an essential transcription factor for IL6 expression, thus promoting differentiation of macrophages into M2 type tumor-associated macrophages and generation of myeloid-derived suppressor cells from peripheral blood mononuclear cells. Treatment of xenografted tumor models overexpressing NNMT gallbladder carcinoma (GBC) cells with the NNMT inhibitor JBSNF-000088 resulted in compromised tumor development and decreased expression levels of IL6, GM-CSF, tumor-associated macrophage marker CD206, and myeloid-derived suppressor cell marker CD33 but increased expression levels of CD8. In addition, elevated expression of NNMT in tumors of patients with GBC was correlated with increased expression levels of CD206 and CD33 but with decreased levels of CD8 and survival of patients. These data highlight the critical role of NNMT in GBC progression. Inhibition of NNMT by JBSNF-000088 is a potential molecular target for GBC immunotherapy.
Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) with Enhanced Activity.
Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 μM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.
Discovery of Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT).
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of pyridine-containing compounds using the cofactor S-5'-adenosyl-l-methionine (SAM) as the methyl group donor. Through the regulation of the levels of its substrates, cofactor, and products, NNMT plays an important role in physiology and pathophysiology. Overexpression of NNMT has been implicated in various human diseases. Potent and selective small-molecule NNMT inhibitors are valuable chemical tools for testing biological and therapeutic hypotheses. However, very few NNMT inhibitors have been reported. Here, we describe the discovery of a bisubstrate NNMT inhibitor MS2734 (6) and characterization of this inhibitor in biochemical, biophysical, kinetic, and structural studies. Importantly, we obtained the first crystal structure of human NNMT in complex with a small-molecule inhibitor. The structure of the NNMT-6 complex has unambiguously demonstrated that 6 occupied both substrate and cofactor binding sites. The findings paved the way for developing more potent and selective NNMT inhibitors in the future.
Emerging opportunities for nicotinamide N-methyltransferase (NNMT) inhibitor clinical translation.
Nicotinamide N-methyltransferase (NNMT) is an enzyme that helps regulate how cells balance energy and methylation potential; however, its abnormal activation can disrupt this balance, fueling diseases such as diabetes, obesity, fibrosis, and cancer. As a result, small-molecule inhibitors targeting NNMT have been developed to reduce this elevated activity, but insufficient target engagement, reduced bioavailability, and unknown safety profiles have led to limited preclinical success across diseases. Advances in NNMT small-molecule inhibitor development have yielded a number of compounds that address these concerns, resulting in drug-like compounds with high affinities for cellular NNMT and promising safety profiles. In this review, we assess the growing therapeutic potential of NNMT inhibitors by highlighting recent developments in NNMT pathophysiology and inhibitor medicinal chemistry.
Nicotinamide N-Methyl Transferase as a Predictive Marker of Tubular Fibrosis in CKD.
Chronic kidney disease (CKD) progression is complex. There are not standardized methods for predicting the prognosis of CKD. Nicotinamide N-methyltransferase (NNMT) has been shown to be associated with renal fibrosis. This study aimed to validate NNMT as a prognostic biomarker of progressive CKD. We explored the relationship between NNMT expression and CKD-related outcome variables using the NephroseqV5 and GEO databases. Additionally, a validation set of 37 CKD patients was enrolled to measure the correlation between NNMT expression levels and CKD outcomes. Furthermore, single-cell RNA sequencing data and the Human Protein Atlas were reanalyzed to investigate the expression specificity of NNMT in the kidney. Finally, to detect the status of NNMT expression with tubular fibrosis in vivo, we constructed a unilateral ureteral obstruction (UUO) mouse treated with an NNMT inhibitor. Analyzing the datasets showed that NNMT was expressed mainly in proximal tubule compartments. And patients with high NNMT expression levels had a significantly lower overall survival rate compared to those with low NNMT expression levels (P = 0.013). NNMT was independent of prognosis factors in the multivariate Cox regression model, and the AUCs for CKD progression at 1, 3, and 5 years were 0.849, 0.775, and 0.877, respectively. Pathway enrichment analysis indicated that NNMT regulates the biological processes of tubulointerstitial fibrosis (TIF). In the validation group, NNMT levels were significantly higher in the CKD group combined with interstitial fibrosis. In vivo, NNMT was a high expression in the UUO group, peaking at postoperative day 21. Treatment with an NNMT inhibitor improved renal tubular interstitial fibrosis, and expression levels of FN, α-SMA, VIM, and TGF-β1 were decreased compared with UUO (P < 0.05). NNMT was expressed mainly in tubular renal compartments, and associated with CKD prognosis. It holds potential as a diagnostic biomarker for tubular fibrosis in CKD.
Nicotinamide N-methyltransferase promotes drug resistance in lung cancer, as revealed by nascent proteomic profiling.
Kinase inhibitors have achieved great success in targeted cancer therapy, yet the evident limitations in their effectiveness persist due to therapeutic resistance. To gain insight into the molecular mechanisms and thwart resistance, we profiled the time-resolved nascent protein perturbations in response to drug therapy using metabolic labeling and facilitated the identification of 2238 proteins via liquid chromatography tandem mass spectrometry (LC-MS/MS). Among these, 51 proteins exhibited upregulation, whereas 105 proteins showed downregulation following a 24-h drug treatment. Clustering analysis revealed that the differential proteins were mainly enriched in metabolic-related pathways. Combined with changes in whole-protein levels, we noticed significant fluctuations in the metabolism-related protein nicotinamide N-methyltransferase (NNMT). Additionally, NNMT overexpression diminished drug effectiveness, whereas its inhibition enhanced therapeutic efficacy. An increase in NNMT was also found in drug-resistant cells, and the NNMT inhibitor JBSNF-000088 inhibited the proliferation of resistant cells. Subsequent phosphoproteomic analysis indicated that the effects of NNMT overexpression on transcription factors, proteins involved in the Rho GTPases cycle, and cell-cycle-related proteins may be related to tumor resistance. In summary, our study provides unique insights into nascent protein perturbations during the initial stages of drug therapy and identified NNMT as a promising target for delaying and overcoming therapeutic resistance.
Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics.
Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.
Nicotinamide N-Methyltransferase: A Promising Biomarker and Target for Human Cancer Therapy.
Cancer cells typically exhibit a tightly regulated program of metabolic plasticity and epigenetic remodeling to meet the demand of uncontrolled cell proliferation. The metabolic-epigenetic axis has recently become an increasingly hot topic in carcinogenesis and offers new avenues for innovative and personalized cancer treatment strategies. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme involved in controlling methylation potential, impacting DNA and histone epigenetic modification. NNMT overexpression has been described in various solid cancer tissues and even body fluids, including serum, urine, and saliva. Furthermore, accumulating evidence has shown that NNMT knockdown significantly decreases tumorigenesis and chemoresistance capacity. Most importantly, the natural NNMT inhibitor yuanhuadine can reverse epidermal growth factor receptor tyrosine kinase inhibitor resistance in lung cancer cells. In this review, we evaluate the possibility of NNMT as a diagnostic biomarker and molecular target for effective anticancer treatment. We also reveal the exact mechanisms of how NNMT affects epigenetics and the development of more potent and selective inhibitors.
Nicotinamide-N-methyltransferase is a promising metabolic drug target for primary and metastatic clear cell renal cell carcinoma.
The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target. NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single-cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT-depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2-deoxy-D-glucose for glycolysis and BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl-sulfide) for glutamine metabolism was investigated in RCC cell lines (786-O, A498) and in two 2D ccRCC-derived primary cultures and three 3D ccRCC air-liquid interface models. NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10-16 ) and ccRCC-derived metastases (p = 3.92 × 10-20 ), irrespective of metastatic location, versus non-tumour tissue. Single-cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC-hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5-12.4; KIRC-HR = 3.3, 95% CI: 2.0-5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT-depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2-deoxy-D-glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis-derived models. In two out of three patient-derived ccRCC air-liquid interface models, NNMTi treatment induced cytotoxicity. Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small-molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.
Nicotinamide N-methyltransferase inhibition mitigates obesity-related metabolic dysfunction.
To assess the effects of a small-molecule nicotinamide N-methyltransferase (NNMT) inhibitor, 5A1MQ, on body composition, metabolic variables, fatty liver pathologies, and circulating biomarkers in diet-induced obese (DIO) mice, and characterize its plasma pharmacokinetics (PK) and tissue distribution in vivo. DIO mice were administered vehicle or 5A1MQ once daily for 28 days. Longitudinal measures of body composition, blood glucose and plasma insulin levels, and terminal measures of liver histopathology and serum markers, were evaluated. Plasma and tissue PK were established in age- and strain-matched mice after intravenous, oral, and subcutaneous dosing of 5A1MQ. 5A1MQ treatment dose-dependently limited body weight and fat mass gains, improved oral glucose tolerance and insulin sensitivity, and suppressed hyperinsulinaemia in DIO mice. Liver histology from 5A1MQ-treated DIO mice showed attenuated hepatic steatosis and macrophage infiltration, and correspondingly reduced liver weight, size, and triglyceride levels. 5A1MQ treatment normalized circulating levels of alanine transaminase, aspartate transaminase, and ketone bodies, supporting an overall improvement in liver and metabolic functions. The pharmacodynamic effects of 5A1MQ were further corroborated by its high systemic exposure and effective distribution to metabolically active tissues, including adipose, muscle and liver, following subcutaneous dosing of mice. This work validates NNMT inhibition as a viable pharmacological approach to ameliorate metabolic imbalances and improve liver pathologies that develop with obesity.
Nicotinamide N-methyltransferase: At the crossroads between cellular metabolism and epigenetic regulation.
The abundance of energy metabolites is intimately interconnected with the activity of chromatin-modifying enzymes in order to guarantee the finely tuned modulation of gene expression in response to cellular energetic status. Metabolism-induced epigenetic gene regulation is a key molecular axis for the maintenance of cellular homeostasis, and its deregulation is associated with several pathological conditions. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) using the universal methyl donor S-adenosyl methionine (SAM), directly linking one-carbon metabolism with a cell's methylation balance and nicotinamide adenine dinucleotide (NAD+) levels. NNMT expression and activity are regulated in a tissue-specific-manner, and the protein can act either physiologically or pathologically depending on its distribution. While NNMT exerts a beneficial effect by regulating lipid parameters in the liver, its expression in adipose tissue correlates with obesity and insulin resistance. NNMT upregulation has been observed in a variety of cancers, and increased NNMT expression has been associated with tumor progression, metastasis and worse clinical outcomes. Accordingly, NNMT represents an appealing druggable target for metabolic disorders as well as oncological and other diseases in which the protein is improperly activated. This review examines emerging findings concerning the complex NNMT regulatory network and the role of NNMT in both NAD metabolism and cell methylation balance. We extensively describe recent findings concerning the physiological and pathological regulation of NNMT with a specific focus on the function of NNMT in obesity, insulin resistance and other associated metabolic disorders along with its well-accepted role as a cancer-associated metabolic enzyme. Advances in strategies targeting NNMT pathways are also reported, together with current limitations of NNMT inhibitor drugs in clinical use. NNMT is emerging as a key point of intersection between cellular metabolism and epigenetic gene regulation, and growing evidence supports its central role in several pathologies. The use of molecules that target NNMT represents a current pharmaceutical challenge for the treatment of several metabolic-related disease as well as in cancer.
Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.
There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.
Potent Uncompetitive Inhibitors of Nicotinamide N-Methyltransferase (NNMT) as In Vivo Chemical Probes.
NNMT uses SAM as a cofactor to catalyze the methylation of nicotinamide, producing 1-methylnicotinamide. Recent studies have shown that NNMT upregulation in cancer-associated fibroblasts (CAFs) is required to maintain the CAF phenotype in high-grade serous carcinoma. These observations suggest that NNMT should be evaluated as a therapeutic target, especially in cancer. Although several small-molecule inhibitors of NNMT have been identified, there remains a need for highly potent and selective inhibitors with excellent in vivo activity and ADME properties that can be used as reliable chemical probes. We have identified azaindoline carboxamide 38 as a selective and potent NNMT inhibitor with favorable PK/PD and safety profiles as well as excellent oral bioavailability and pharmaceutical properties. Our mechanistic studies indicate that 38 binds uncompetitively with SAM but competitively with nicotinamide consistent with its binding in the nicotinamide binding site and likely forming a positive interaction with SAM.
Nicotinamide N-methyltransferase inhibition improves limb function in experimental peripheral artery disease.
Peripheral artery disease (PAD) impairs limb perfusion, walking ability, and increases the risk of amputation. Although current therapies reduce cardiovascular events, few interventions improve skeletal muscle function in PAD. Nicotinamide adenine dinucleotide (NAD+) metabolism is disrupted in PAD. Thus, it was hypothesized that inhibition of nicotinamide N-methyltransferase (NNMT), an enzyme that diverts precursors from the NAD+ salvage pathway, would improve ischemic limb function. We analyzed NAD+ pathway expression in gastrocnemius muscle from patients with and without PAD using RNA sequencing and proteomics datasets. Single-cell RNA sequencing data were used to assess NNMT expression in muscle stem cells (MuSCs) from BALB/cJ and C57BL/6J mice following hindlimb ischemia (HLI). Male BALB/cJ mice (n = 24) were randomized to either placebo or a NNMT inhibitor (NNMTi) delivered 3 h prior to HLI and daily thereafter. Functional assessments included laser Doppler perfusion imaging, muscle contractility, and a 6-min limb function test. Histological analyses were used to assess myofiber area and capillary density. NNMT mRNA and protein levels were significantly elevated in skeletal muscle from patients with PAD and were persistently elevated in MuSCs from BALB/cJ mice after HLI. NNMTi treatment did not affect limb perfusion recovery or capillary density but trended toward reduced necrosis severity (p = 0.08). Muscle mass and myofiber size were unchanged by treatment; however, NNMTi significantly improved muscle strength (p < 0.0001), power (p = 0.0305), and total work (p = 0.0367) in ischemic limbs compared to placebo. Inhibition of NNMT enhanced ischemic muscle strength and performance in a preclinical model of PAD independent of changes in perfusion.
Structure-Activity Relationship Studies on Cell-Potent Nicotinamide N-Methyltransferase Bisubstrate Inhibitors.
Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme implicated in multiple diseases, making it a promising therapeutic target. Building upon our recently reported NNMT inhibitor II399, we systematically investigate the structure-activity relationship by designing and synthesizing a series of analogues. Among them, two top inhibitors II559 (Ki = 1.2 nM) and II802 (Ki = 1.6 nM) displayed over 5000-fold selectivity for NNMT over closely related methyltransferases. Moreover, II559 and II802 showed enhanced cellular inhibition, with a cellular IC50 value of approximately 150 nM, making them the most cell-potent bisubstrate inhibitors reported to date. Furthermore, both inhibitors reduced the cell viability with a GI50 value of ∼10 μM and suppressed the migration of aggressive clear cell renal cancer cell carcinoma cell lines. Overall, II559 and II802 would serve as valuable probes to investigate the enzymatic function of NNMT in health and diseases.
Reduced calorie diet combined with NNMT inhibition establishes a distinct microbiome in DIO mice.
Treatment with a nicotinamide N-methyltransferase inhibitor (NNMTi; 5-amino-1-methylquinolinium) combined with low-fat diet (LD) promoted dramatic whole-body adiposity and weight loss in diet-induced obese (DIO) mice, rapidly normalizing these measures to age-matched lean animals, while LD switch alone was unable to restore these measures to age-matched controls in the same time frame. Since mouse microbiome profiles often highly correlate with body weight and fat composition, this study was designed to test whether the cecal microbiomes of DIO mice treated with NNMTi and LD were comparable to the microbiomes of age-matched lean counterparts and distinct from microbiomes of DIO mice maintained on a high-fat Western diet (WD) or subjected to LD switch alone. There were minimal microbiome differences between lean and obese controls, suggesting that diet composition and adiposity had limited effects. However, DIO mice switched from an obesity-promoting WD to an LD (regardless of treatment status) displayed several genera and phyla differences compared to obese and lean controls. While alpha diversity measures did not significantly differ between groups, beta diversity principal coordinates analyses suggested that mice from the same treatment group were the most similar. K-means clustering analysis of amplicon sequence variants by animal demonstrated that NNMTi-treated DIO mice switched to LD had a distinct microbiome pattern that was highlighted by decreased Erysipelatoclostridium and increased Lactobacillus relative abundances compared to vehicle counterparts; these genera are tied to body weight and metabolic regulation. Additionally, Parasutterella relative abundance, which was increased in both the vehicle- and NNMTi-treated LD-switched groups relative to the controls, significantly correlated with several adipose tissue metabolites' abundances. Collectively, these results provide a novel foundation for future investigations.
Small molecule nicotinamide N-methyltransferase inhibitor activates senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle.
Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics. We recently developed small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle. 24-month old mice were treated with saline (control), and low and high dose NNMTi (5 and 10 mg/kg) for 1-week post-injury, or control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and received 5-ethynyl-2'-deoxyuridine systemically to analyze muSC activity. In vivo contractile function measurements were conducted on the injured TA muscle and tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA) measurements to assess muscle recovery. Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by ∼70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.
Small molecule inhibitor of nicotinamide N-methyltransferase shows anti-proliferative activity in HeLa cells.
The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1-500 μM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation.IMPACT STATEMENTWhat is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously.What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased.What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy.
N6-Methyladenosine RNA Modifications Regulate the Response to Platinum Through Nicotinamide N-methyltransferase.
Development of resistance to platinum (Pt) in ovarian cancer remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. Fat mass and obesity-associated protein (FTO) is a N6-methyladenosine (m6A) RNA demethylase that we recently described as a tumor suppressor in ovarian cancer. We hypothesized that FTO-induced removal of m6A marks regulates the cellular response of ovarian cancer cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used ovarian cancer cells in which FTO was knocked down via short hairpin RNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R versus sensitive ovarian cancer cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (P < 0.05). Increased numbers of γ-H2AX foci, measuring DNA double-strand breaks, and increased apoptosis were observed after exposure to Pt in FTO-overexpressing versus control cells. Through integrated RNA sequencing and MeRIP sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO-overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO-overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target.
Adipose tissue as a source of nicotinamide N-methyltransferase and homocysteine.
Nicotinamide N-methyltransferase (NNMT) catalyses the conversion of nicotinamide to 1-methylnicotinamide and plays an important role in hepatic detoxification reactions. Here we show that, in addition to the liver, 3T3-L1 adipocytes as well as human and murine adipose tissue explants express high amounts of enzymatically active NNMT. NNMT mRNA levels and enzyme activity increased in 3T3-L1 cells in a differentiation-dependent manner. Homocysteine, the atherogenic product of the NNMT-catalyzed reaction, was secreted from 3T3-L1 cells or adipose tissue cultures. Homocysteine release increased during 3T3-L1 differentiation and was reduced when adipose tissue was treated with the NNMT inhibitor 1-methylnicotinamide. Nicotinic acid (NA), a widely used drug to lower elevated plasma lipid levels, induced NNMT enzyme activity in white adipose tissue of mice. In tissue culture nicotinamide treatment led to an increase in adipose tissue homocysteine secretion. These data support the concept that adipose tissue NNMT contributes to the increased plasma homocysteine levels in patients treated with NA.
Ligand-based in silico identification and biological evaluation of potential inhibitors of nicotinamide N-methyltransferase.
Nicotinamide N-methyltransferase (NNMT) is a protein coding gene, which methylates the nicotinamide (NA) (vitamin B3) to produce 1-methylnicotinamide (MNA). Several studies have suggested that the overexpression of NNMT is associated with different metabolic disorders like obesity and type-2 diabetes thereby making it an important therapeutic target for development of anti-diabetic agents. Here we describe a workflow for identification of new inhibitors of NNMT from a library of small molecules. In this study, we have hypothesized a four-point pharmacophore model based on the pharmacophoric features of reported NNMT inhibitors in the literature. The statistically significant pharmacophore hypothesis was used to explore the Maybridge compound library that resulted in mapping of 1330 hit compounds on the proposed hypothesis. Subsequently, a total of eight high scoring compounds, showing good protein-ligand interactions in the molecular docking study, were selected for biological evaluation of NNMT activity. Eventually, four compounds were found to show significant inhibitory activity for NNMT and can be further explored to design new derivatives around the identified scaffolds with improved activities as NNMT inhibitors.
Development & validation of LC-MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies.
5-Amino-1-methyl quinolinium (5-AMQ) is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. NNMT is an enzyme that catalyzes the N-methylation of the endogenous substrate nicotinamide, as well as exogenous xenobiotics. NNMT is fundamental to cellular metabolism; NNMT is overexpressed in select tissues (e.g., adipose tissue, skeletal muscle, etc.) in pathophysiological conditions, making it a clinically relevant target for drug development in several chronic diseases including obesity and diabetes. The objective of this study was to develop and validate a simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 5-AMQ in rat plasma and urine samples. 5-AMQ was extracted from plasma and urine by protein precipitation. Chromatographic separation was achieved using an ACE® Excel™ C18 column (2 μm, 50 × 2.1 mm) with a binary gradient solvent system comprising of water (A) and acetonitrile (B) containing 0.1 % formic acid as the mobile phase. Analysis was performed using an API 4000 QTRAP hybrid triple quadruple mass spectrometer and multiple reaction monitoring (MRM) in positive mode at m/z transitions of 159.100 → 90.00 and 162.200 → 117.200 for 5-AMQ and the internal standard, respectively. The standard curves of 5-AMQ in rat urine and plasma samples were linear in the concentration range of 10-2500 ng/mL. The intra-day and inter-day precisions and accuracies for 5-AMQ at four concentration levels in rat plasma and urine samples were found to be within the 15 % FDA acceptance range. Similarly, the accuracy and precision of 5-AMQ quantification in samples diluted up to 20-fold using blank plasma were within the 15 % acceptable range. Furthermore, the extraction recoveries and matrix effects at three concentration levels of rat plasma samples ranged from 99.5 %-110.6 % and -6.1 %-14.1 %, respectively. 5-AMQ was stable in rat plasma samples subjected to standard storage, preparation, and handling conditions, with less than 15 % variation noted at two concentration levels. The validated, sensitive, and reproducible LC-MS/MS method for 5-AMQ in rat plasma and urine samples was effectively applied to a pharmacokinetic study in rats with IV and oral administration of 5-AMQ. 5-AMQ displayed substantial plasma exposures via IV and oral route, with a mean maximum plasma concentration of 2252 ng/mL after oral administration, mean area under the curve (AUC0-∞) of 3708 h.ng/mL and 14431 h.ng/mL for the IV and oral groups, respectively, mean terminal elimination half-life of 3.80 ± 1.10 h and 6.90 ± 1.20 h respectively after intravenous and oral dose, and a good oral bioavailability (F % = 38.4).
Identification of novel human nicotinamide N-methyltransferase inhibitors: a structure-based pharmacophore modeling and molecular dynamics approach.
Human nicotinamide N-methyltransferase (hNNMT) is a cytosolic enzyme associated in the phase-II metabolism, belonging to the S-adenosyl-L-methionine (SAM)-dependent methyltransferases family. Overexpression of hNNMT was observed in diseases such as metabolic disorders and different types of cancers, which suggest NNMT as a prospective therapeutic target. In this study we propose a structure-based pharmacophore model to understand the structural features responsible for the pharmacological activity. The generated model was validated using the ROC curve (AUC), goodness of hit score (GH), specificity, sensitivity and enrichment factor (EF). The pharmacophore was employed to retrieve active molecules from the ZINC database, followed by virtual-screening and molecular docking. Six molecules with the best pharmfit score, binding energy and ADMET properties were identified in this study. A 150 ns molecular dynamics simulation was performed on the selected molecules complexed with hNNMT protein to validate the results. The molecules ZINC35464499, ZINC13311192, ZINC31159282, ZINC14650833, ZINC14819515 and ZINC00303881 were identified, which could be act as the potential hNNMT inhibitors and can also be used as direct hits for developing novel hNNMT antagonists.Communicated by Ramaswamy H. Sarma.
Targeting nicotinamide N-methyltransferase overcomes resistance to EGFR-TKI in non-small cell lung cancer cells.
Activating mutations of epidermal growth factor receptor (EGFR) contributes to the progression of non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitor (TKI)-targeted therapy has become the standard treatment for NSCLC patients with EGFR-mutations. However, acquired resistance to these agents remains a major obstacle for managing NSCLC. Here, we investigated a novel strategy to overcome EGFR TKI resistance by targeting the nicotinamide N-methyltransferase (NNMT). Using iTRAQ-based quantitative proteomics analysis, we identified that NNMT was significantly increased in EGFR-TKI-resistant NSCLC cells. Moreover, we found that NNMT expression was increased in EGFR-TKI-resistant NSCLC tissue samples, and higher levels were correlated with shorter progression-free survival in EGFR-TKI-treated NSCLC patients. Knockdown of NNMT rendered EGFR-TKI-resistant cells more sensitive to EGFR-TKI, whereas overexpression of NNMT in EGFR-TKI-sensitive cells resulted in EGFR-TKI resistance. Mechanically, upregulation of NNMT increased c-myc expression via SIRT1-mediated c-myc deacetylation, which in turn promoted glycolysis and EGFR-TKI resistance. Furthermore, we demonstrated that the combination of NNMT inhibitor and EGFR-TKI strikingly suppressed the growth of EGFR-TKI-resistant NSCLC cells both in vitro and in vivo. In conclusion, our research indicated that NNMT overexpression is important for acquired resistance to EGFR-TKI and that targeting NNMT might be a potential therapeutic strategy to overcome resistance to EGFR TKI.
Nicotinamide N-Methyltransferase Inhibition Mitigates Cerulein-Induced Pancreatic Fibrosis via Galectin-3-Mediated Regulation of Stellate Cell Activation and Macrophage M2 Polarization in Mice.
Chronic pancreatitis (CP) is a chronic disease characterized by pancreatic fibrosis driven by pancreatic stellate cell activation and M2 polarization of macrophages. Nicotinamide n-methyltransferase (NNMT) is a methylase critically involved in fibrosis. Galectin-3 (LGALS3), a member of the galectin family, drives M2 macrophage polarization. This study explores the roles of NNMT and LGALS3 in CP. CP was induced in C57BL/6 mice via repeated intraperitoneal injections of 50 µg/kg cerulein. NNMT was upregulated in pancreatic tissues of CP mice, especially in mouse pancreatic stellate cells (mPSCs). Functional inhibition of NNMT in mPSCs, via lentiviral short-hairpin RNA-mediated knockdown or treatment with NNMT inhibitor, suppressed mPSC activation, proliferation, and migration. Moreover, conditioned medium from mPSCs with NNMT inhibition reduced M2 polarization of bone marrow-derived macrophages (BMDMs). In vivo, intraperitoneal administration of 20 mg/kg NNMT inhibitor for 28 days alleviated pancreatic collagen deposition and macrophage M2 polarization, resulting in reduced pancreatic fibrosis. RNA sequencing and bioinformatics analysis identified LGALS3 as a potential downstream target of NNMT. NNMT inhibition downregulated LGALS3 expression in mPSCs, and chromatin immunoprecipitation - quantitative polymerase chain reaction (ChIP-qPCR) confirmed that this suppression was associated with increased trimethylation of lysine 27 on histone H3 (H3K27me3) enrichment at the LGALS3 promoter. Lentivirus-mediated LGALS3 overexpression reversed the inhibitory effect of NNMT knockdown on BMDM M2 polarization. In conclusion, inhibition of NNMT alleviates mPSC activation and suppresses mPSC-induced macrophage M2 polarization via H3K27me3-mediated repression of LGALS3 transcription, with the ultimate effect of mitigating cerulein-induced pancreatic fibrosis.
Enhancing glioma immunotherapy by disrupting RBP-J-mediated NNMT signaling in tumor microenvironment.
Glioma is a highly aggressive central nervous system malignancy characterized by profound immune evasion, the underlying mechanisms of which remain incompletely defined. This study investigated how the transcription factor RBP-J drives immune suppression through activation of NNMT in cancer-associated fibroblasts (CAFs). By integrating single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST), we identified a CAF-specific NNMT-high subpopulation enriched at the tumor margin and closely associated with M2 macrophages. Bioinformatic analyses using Seurat and Monocle3 delineated a stromal-immune regulatory network and highlighted RBP-J as a potential upstream regulator of NNMT. Mechanistic experiments demonstrated that RBP-J directly binds to the NNMT promoter and activates its transcription, leading to intracellular SAM depletion, reduced H3K27me3 levels, and epigenetic upregulation of SAA3. Elevated SAA3 promoted M2 macrophage recruitment and polarization, resulting in CD8+ T cell exhaustion and immune suppression. In vivo experiments using an orthotopic glioma model confirmed that NNMT-high CAFs accelerated tumor growth, increased M2 macrophage infiltration, and diminished CD8+ T cell activity. Importantly, combined treatment with an NNMT inhibitor and αPD-1 partially reversed the immunosuppressive microenvironment and significantly enhanced therapeutic efficacy. Collectively, this study identifies the RBP-J/NNMT/SAA3 axis as a critical stromal-driven mechanism of immune evasion in glioma and provides a rationale for targeting metabolic-epigenetic pathways to improve immunotherapy outcomes. Schematic Diagram Illustrating the Molecular Mechanism by Which RBP-J Promotes Immune Evasion in Glioma via Activation of NNMT in CAFs, Leading to H3K27 Demethylation-Mediated Upregulation of SAA3 and Subsequent Reprogramming of M2 Macrophages.
Nicotinamide n-methyltransferase inhibitor synergizes with sodium-glucose cotransporter 2 inhibitor to protect renal tubular epithelium in experimental models of type 2 diabetes mellitus.
We aim to explore the potential of nicotinamide n-methyltransferase (NNMT) as a sensitive marker of renal tubular injury and the possibility of an NNMT inhibitor to combine with sodium-glucose cotransporter 2 (SGLT2) inhibitor to protect proximal tubular epithelium in vivo and in vitro model of Type 2 diabetes mellitus (T2DM), respectively. In vivo, immunohistochemical staining, Masson's trichrome staining and Sirius red staining were used to observe the changes of NNMT expression, renal tubular injury and interstitial fibrosis in renal tissue from the db/db mice. Bioinformatic analysis was also conducted to broaden the range of data validation. In vitro, Western Blot and quantitative RT-PCR were used to measure the degree of damage of HK-2 cells. Our in vivo data showed upregulation of NNMT expression paralleled renal tubular damage and interstitial fibrosis. Our in vitro data revealed both NNMT inhibitors and SGLT2 inhibitors can protect against the injury as assessed by extracellular matrix (ECM) synthesis and profibrotic phenotype transition of HK-2 cells, and the combination of these two agents can further reduce these injuries. The present study is the first to show that NNMT is a promising marker of renal tubular injury in diabetic nephropathy (DN) and NNMT inhibitors can synergize with SGLT2 inhibitors to protect HK-2 better. Our findings will provide the insight and pave the way of developing novel therapeutic strategies for chronic renal tubular injury associated with T2DM.
Nicotinamide N-methyltransferase gene silencing is associated with upregulation of X-inactive specific transcript and downregulation of stemness-related transcription factors in ovarian cancer.
BACKGROUND: X-inactive specific transcript (Xist), a long non-coding RNA and MacroH2A1, a histone variant are primarily involved in maintaining the inactive X chromosome in females. Xist downregulation was correlated with augmented cancer cell invasion, migration, cancer stemness and the reactivation of certain X-linked genes in ovarian cancer. Nicotinamide N-methyltransferase (NNMT) is increasingly associated with tumor progression and cancer stem cell enrichment as well as epithelial-mesenchymal transition (EMT). However, a potential relationship between NNMT and Xist, MacroH2A1 alongside stem cell mediating transcription factors (NANOG, Oct4, SOX2) and the EMT marker SNAI1 has not been previously explored. METHODS: OVCAR-3 and SKOV-3 ovarian cancer cell lines were treated with NNMT siRNA or the NNMT inhibitor JBSNF-000088. Expression of Xist, NANOG, Oct4, SOX2, SNAI1, MacroH2A1 and NNMT were analyzed via RT qPCR and Western Blotting. Invasion and migration assays were performed using the xCELLigence RTCA system. Database analyses assessed Xist expression, survival and methylation correlations. RESULTS: NNMT silencing significantly increased Xist and MacroH2A1 expression, while decreasing NANOG, Oct4, SOX2, and SNAI1 (p < 0.05). NNMT silencing decreased invasion and migration capabilities of cancer cells (p < 0.001). CONCLUSION: These findings suggest a potential link between NNMT expression, cancer stemness, and Xist regulation in ovarian cancer cell lines. Upregulation of Xist and decrease in the levels of stemness and EMT factors following NNMT silencing may be associated with a more favorable molecular profile. The concurrent increase in MacroH2A1 expression supports the potential role of NNMT in modulating X chromosome reactivation in ovarian cancer possibly through the repressive effects of pluripotency factors on Xist.
Quick links (PubMed)
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