Macrocyclic peptides as allosteric inhibitors of nicotinamide <i>N</i>-methyltransferase (NNMT)

Haren, Matthijs J. van; Zhang, Yurui; Thijssen, Vito; Buijs, Ned; Gao, Yongzhi; Mateuszuk, Łukasz; Fedak, Filip A.; Kij, Agnieszka; Campagna, Roberto; Emanuelli, Monica; Chłopicki, Stefan; Jongkees, Seino A. K.; Martin, Nathaniel I.

doi:10.1039/d1cb00134e

Cited by 41 publications

(43 citation statements)

References 38 publications

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“…The first rounds of RaPID selections ( Figures 1 and S1 ) were carried out on the full-size H1 ectodomain of a new pandemic H1N1 virus of 2009 (A/California/04/2009). The selection used a library of peptides of general architecture ClAc-Y-X 15 -C-G-S-G-S-G-S (where ClAc is a chloroacetyl group and X is any amino acid encoded by an NNK codon) following published protocols, 25 with the initiating tyrosine in either d - or l -stereochemistry in two parallel selections. A strong enrichment of HA binders was already observed after three rounds ( Figure S2a,b ).…”

Section: Resultsmentioning

confidence: 99%

“…Cyanomethyl esters of chloroacetylated d - and l -tyrosine (separate reactions) were charged on tRNA CAU fMet as previously described. 14 Based on a previously reported method, 25 selections were carried out against either Fc- or Strep-tag-immobilized HA using Protein G or Strep-Tactin X magnetic beads, respectively, for both d - and l -tyrosine-initiated libraries in parallel. An overview of the protein construct and magnetic beads used in each round of the selection is listed in Table S2 .…”

Section: Methodsmentioning

confidence: 99%

“…Puromycin ligation by T4 RNA ligase (NEB), peptide translation using PURExpress (NEB), and reverse transcription by ProtoScript II Reverse Transcriptase (NEB) were all performed as reported previously. 25 cDNA/mRNA-tagged peptide libraries were diluted with stock solutions of PBS-T and BSA to achieve final concentrations of 1× and 0.1%, respectively, with a total volume of 25 μL in the first selection round and 14 μL in subsequent rounds; 1 μL of the reverse transcription reaction was diluted in 500 μL of milliQ to serve as an input sample for qPCR analysis. The translation mixes were then sequentially incubated three times for 20 min at 4 °C with 2.5 μL of magnetic beads without the target protein to preclear any peptides binding to Strep-Tactin XT or Protein G beads (omitted for the first round).…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region

et al. 2022

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Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-throughput screening of a diverse peptide library. The peptides are active against both H1 and H5 variants, with no detectable cytotoxicity. Despite the high sequence diversity across hits, all tested peptides were found to bind to the same region in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in this region identified in an escape variant confirmed the binding site. This stands in contrast to the immunodominance of the head region for antibody binding and suggests that macrocyclic peptides from in vitro display may be well suited for finding new druggable sites not revealed by antibodies. Functional analysis indicates that these peptides stabilize the prefusion conformation of the protein and thereby prevent virus–cell fusion. High-throughput screening of macrocyclic peptides is thus shown here to be a powerful method for the discovery of novel broadly acting viral fusion inhibitors with therapeutic potential.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region

et al. 2022

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“…There is active research on discovering the inhibitors of NNMT enzyme activity with some small molecule inhibitors showing promising results [167][168][169][170][171][172][173]. Although NNMT may exert a primary role in the first step of carcinogenesis by irreversibly methylating NAM, thus generating MNA [166], the effective role of this enzyme as well as NAD+ boosters on cancer development still need to be fully elucidated.…”

Section: Potential (In)direct Harmful Effects Of Increased Nad+mentioning

confidence: 99%

Current Uncertainties and Future Challenges Regarding NAD+ Boosting Strategies

2022

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Precursors of nicotinamide adenine dinucleotide (NAD+), modulators of enzymes of the NAD+ biosynthesis pathways and inhibitors of NAD+ consuming enzymes, are the main boosters of NAD+. Increasing public awareness and interest in anti-ageing strategies and health-promoting lifestyles have grown the interest in the use of NAD+ boosters as dietary supplements, both in scientific circles and among the general population. Here, we discuss the current trends in NAD+ precursor usage as well as the uncertainties in dosage, timing, safety, and side effects. There are many unknowns regarding pharmacokinetics and pharmacodynamics, particularly bioavailability, metabolism, and tissue specificity of NAD+ boosters. Given the lack of long-term safety studies, there is a need for more clinical trials to determine the proper dose of NAD+ boosters and treatment duration for aging prevention and as disease therapy. Further research will also need to address the long-term consequences of increased NAD+ and the best approaches and combinations to increase NAD+ levels. The answers to the above questions will contribute to the more efficient and safer use of NAD+ boosters.

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“…Additional NNMT inhibitors have recently been reported including other nicotinamide-related compounds such as 6-methylaminonicotinamide 19 , alpha-chloroacetamide-based compounds covalently binding to a cysteine residue in the SAM-binding pocket of the catalytic site 20 , macrocyclic peptides as allosteric NNMT inhibitors 21 , and bisubstrate-like inhibitors based on adenosyl scaffolds 22 – 26 , or 3-methyl-4-phenylpyrazole 27 that occupy both the Nam and the SAM-binding pockets. The lack of cellular activity of these bisubstrate-like inhibitors was recently overcome by Iyamu et al 28 who generated SAM mimics extending into the nicotinamide binding pocket with nanomolar potencies in biochemical and micromolar potencies in cell assays.…”

Section: Introductionmentioning

confidence: 99%

Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders

Ruf

Rajagopal

Kadnur

et al. 2022

Sci Rep

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Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.

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Macrocyclic peptides as allosteric inhibitors of nicotinamide N-methyltransferase (NNMT)

Abstract: Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-L-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly...

Cited by 41 publications

References 38 publications

Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region

Inhibition of H1 and H5 Influenza A Virus Entry by Diverse Macrocyclic Peptides Targeting the Hemagglutinin Stem Region

Current Uncertainties and Future Challenges Regarding NAD+ Boosting Strategies

Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders

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