Binding Affinity Studies of Nicotinamide N-methyltransferase and Ligands
by Saturation Transfer Difference NMR

Tingting      Fang; Jianyu      Zhang

Abstract

Introduction: Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide with S-adenosine-L-methionine (SAM) as the methyl donor. Abnormal expression of NNMT is associated with many diseases (such as multiple cancers and metabolic and liver diseases), making NNMT a potential therapeutic target. Limited studies concerning the enzymesubstrate/ inhibitor interactions could be found to fully understand the detailed reaction mechanism.

Methods: The binding affinity and ligand binding epitopes of nicotinamide or SAH for binding NNMT and its mutants were determined using saturated transfer difference (STD) nuclear magnetic resonance (NMR) techniques combined with site-directed mutagenesis.

Results: The average dissociation constant of WT NNMT with nicotinamide and S-adenosine homocysteine (SAH) was 5.5 ± 0.9 mM and 1.2 ± 0.3 mM, respectively, while the mutants Y20F and Y20G with nicotinamide were up to nearly 4 times and 20 times that of WT and with SAH nearly 2 times and 5 times that of WT. The data suggested that WT had the highest binding affinity for nicotinamide or SAH, followed by Y20F and Y20G, which was consistent with its catalytic activity.

Conclusion: The binding affinity of nicotinamide and SAH to NNMT and its mutants were obtained by STD NMR in this study. It was found that nicotinamide and SAH bind to WT in a particular orientation, and Y20 is critical for their binding orientation and affinity to NNMT.

Graphical Abstract

[1]
Hong, S.; Zhai, B.; Pissios, P. Nicotinamide N -methyltransferase interacts with enzymes of the methionine cycle and regulates methyl donor metabolism. Biochemistry,  2018, 57(40), 5775-5779.
 [http://dx.doi.org/10.1021/acs.biochem.8b00561] [PMID: 30226369]

[2]
Lim, B.H.; Cho, B.I.; Kim, Y.N.; Kim, J.W.; Park, S.T.; Lee, C.W. Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification. Exp. Mol. Med.,  2006, 38(5), 455-465.
 [http://dx.doi.org/10.1038/emm.2006.54] [PMID: 17079861]

[3]
Ulanovskaya, O.A.; Zuhl, A.M.; Cravatt, B.F. NNMT promotes epigenetic remodeling in cancer by creating a metabolic methylation sink. Nat. Chem. Biol.,  2013, 9(5), 300-306.
 [http://dx.doi.org/10.1038/nchembio.1204] [PMID: 23455543]

[4]
Parsons, R.B.; Smith, S.W.; Waring, R.H.; Williams, A.C.; Ramsden, D.B. High expression of nicotinamide N-methyl-transferase in patients with idiopathic Parkinson’s disease. Neurosci. Lett.,  2003, 342(1-2), 13-16.
 [http://dx.doi.org/10.1016/S0304-3940(03)00218-0] [PMID: 12727306]

[5]
Kraus, D.; Yang, Q.; Kong, D.; Banks, A.S.; Zhang, L.; Rodgers, J.T.; Pirinen, E.; Pulinilkunnil, T.C.; Gong, F.; Wang, Y.; Cen, Y.; Sauve, A.A.; Asara, J.M.; Peroni, O.D.; Monia, B.P.; Bhanot, S.; Alhonen, L.; Puigserver, P.; Kahn, B.B. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature,  2014, 508(7495), 258-262.
 [http://dx.doi.org/10.1038/nature13198] [PMID: 24717514]

[6]
Kannt, A.; Rajagopal, S.; Kadnur, S.V.; Suresh, J.; Bhamidipati, R.K.; Swaminathan, S.; Hallur, M.S.; Kristam, R.; Elvert, R.; Czech, J.; Pfenninger, A.; Rudolph, C.; Schreuder, H.; Chandrasekar, D.V.; Mane, V.S.; Birudukota, S.; Shaik, S.; Zope, B.R.; Burri, R.R.; Anand, N.N.; Thakur, M.K.; Singh, M.; Parveen, R.; Kandan, S.; Mullangi, R.; Yura, T.; Gosu, R.; Ruf, S.; Dhakshinamoorthy, S. A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci. Rep.,  2018, 8(1), 3660.
 [http://dx.doi.org/10.1038/s41598-018-22081-7] [PMID: 29311619]

[7]
Ruf, S.; Hallur, M.S.; Anchan, N.K.; Swamy, I.N.; Murugesan, K.R.; Sarkar, S.; Narasimhulu, L.K.; Putta, V.P.R.K.; Shaik, S.; Chandrasekar, D.V.; Mane, V.S.; Kadnur, S.V.; Suresh, J.; Bhamidipati, R.K.; Singh, M.; Burri, R.R.; Kristam, R.; Schreuder, H.; Czech, J.; Rudolph, C.; Marker, A.; Langer, T.; Mullangi, R.; Yura, T.; Gosu, R.; Kannt, A.; Dhakshinamoorthy, S.; Rajagopal, S. Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase. Bioorg. Med. Chem. Lett.,  2018, 28(5), 922-925.
 [http://dx.doi.org/10.1016/j.bmcl.2018.01.058] [PMID: 29433927]

[8]
Loring, H.S.; Thompson, P.R. Kinetic mechanism of nicotinamide n -methyltransferase. Biochemistry,  2018, 57(38), 5524-5532.
 [http://dx.doi.org/10.1021/acs.biochem.8b00775] [PMID: 30148963]

[9]
Babault, N.; Allali-Hassani, A.; Li, F.; Fan, J.; Yue, A.; Ju, K.; Liu, F.; Vedadi, M.; Liu, J.; Jin, J. Discovery of bisubstrate inhibitors of nicotinamide N -methyltransferase (NNMT). J. Med. Chem.,  2018, 61(4), 1541-1551.
 [http://dx.doi.org/10.1021/acs.jmedchem.7b01422] [PMID: 29320176]

[10]
Neelakantan, H.; Vance, V.; Wang, H.Y.L.; McHardy, S.F.; Watowich, S.J. Noncoupled fluorescent assay for direct real-time monitoring of nicotinamide N -methyltransferase activity. Biochemistry,  2017, 56(6), 824-832.
 [http://dx.doi.org/10.1021/acs.biochem.6b01215] [PMID: 28121423]

[11]
Iyamu, I.D.; Vilseck, J.Z.; Yadav, R.; Noinaj, N.; Huang, R. Exploring unconventional SAM analogues to build cell‐potent bisubstrate inhibitors for nicotinamide N‐methyltransferase. Angew. Chem. Int. Ed.,  2022, 61(16), e202114813.
 [http://dx.doi.org/10.1002/anie.202114813] [PMID: 35134268]

[12]
Peng, Y.; Sartini, D.; Pozzi, V.; Wilk, D.; Emanuelli, M.; Yee, V.C. Structural basis of substrate recognition in human nicotinamide N-methyltransferase. Biochemistry,  2011, 50(36), 7800-7808.
 [http://dx.doi.org/10.1021/bi2007614] [PMID: 21823666]

[13]
Jing, Y.; Cheng, Y.; Li, F.; Li, Y.; Liu, F.; Zhang, J. Linkage of nanosecond protein motion with enzymatic methyl transfer by nicotinamide N-methyltransferase. Turk. J. Biol.,  2021, 45(3), 333-341.
 [http://dx.doi.org/10.3906/biy-2101-54] [PMID: 34377057]

[14]
Walpole, S.; Monaco, S.; Nepravishta, R.; Angulo, J. STD NMR as a technique for ligand screening and structural studies. Methods Enzymol.,  2019, 615, 423-451.
 [http://dx.doi.org/10.1016/bs.mie.2018.08.018] [PMID: 30638536]

[15]
Cala, O.; Krimm, I. Ligand-orientation based fragment selection in STD NMR screening. J. Med. Chem.,  2015, 58(21), 8739-8742.
 [http://dx.doi.org/10.1021/acs.jmedchem.5b01114] [PMID: 26492576]

[16]
Benoni, R.; Pertinhez, T.A.; Spyrakis, F.; Davalli, S.; Pellegrino, S.; Paredi, G.; Pezzotti, A.; Bettati, S.; Campanini, B.; Mozzarelli, A. Structural insight into the interaction of O- acetylserine sulfhydrylase with competitive, peptidic inhibitors by saturation transfer difference-NMR. FEBS Lett.,  2016, 590(7), 943-953.
 [http://dx.doi.org/10.1002/1873-3468.12126] [PMID: 27072053]

[17]
Monaco, S.; Ramírez-Cárdenas, J.; Carmona, A.T.; Robina, I.; Angulo, J. Inter-Ligand STD NMR: An efficient 1D NMR approach to probe relative orientation of ligands in a multi-subsite protein binding pocket. Pharmaceuticals.,  2022, 15(8), 1030.
 [http://dx.doi.org/10.3390/ph15081030] [PMID: 36015178]

[18]
Angulo, J.; Enríquez-Navas, P.M.; Nieto, P.M. Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: The binding isotherm of STD initial growth rates. Chemistry,  2010, 16(26), 7803-7812.
 [http://dx.doi.org/10.1002/chem.200903528] [PMID: 20496354]

[19]
Wang, W.; Sun, Q.; Gan, N.; Zhai, Y.; Xiang, H.; Li, H. Characterizing the interaction between methyl ferulate and human serum albumin by saturation transfer difference NMR. RSC Advances,  2020, 10(54), 32999-33009.
 [http://dx.doi.org/10.1039/D0RA05844K] [PMID: 35516494]

[20]
Viegas, A.; Manso, J.; Nobrega, F.L.; Cabrita, E.J. Saturation-transfer difference (STD) NMR: A simple and fast method for ligand screening and characterization of protein binding. J. Chem. Educ.,  2011, 88(7), 990-994.
 [http://dx.doi.org/10.1021/ed101169t]

[21]
Monaco, S.; Tailford, L.E.; Juge, N.; Angulo, J. Differential epitope mapping by STD NMR spectroscopy to reveal the nature of protein-ligand contacts. Angew. Chem. Int. Ed.,  2017, 56(48), 15289-15293.
 [http://dx.doi.org/10.1002/anie.201707682] [PMID: 28977722]

[22]
Li, Y.; Zhang, Y.; Cheng, Y.; Du, T.; Zhang, J. Solvent inhibition profiles and inverse solvent isotope effects for enzymatic methyl transfer catalyzed by nicotinamide N‐methyltransferase. J. Phys. Org. Chem.,  2020, 33(10), 1-10.
 [http://dx.doi.org/10.1002/poc.4093]

[23]
van Haren, M.J.; Sastre Toraño, J.; Sartini, D.; Emanuelli, M.; Parsons, R.B.; Martin, N.I. A rapid and efficient assay for the characterization of substrates and inhibitors of nicotinamide N-methyltransferase. Biochemistry,  2016, 55(37), 5307-5315.
 [http://dx.doi.org/10.1021/acs.biochem.6b00733] [PMID: 27570878]

[24]
Sun, Q.; Zhai, Y.; Wang, W.; Gan, N.; Zhang, S.; Suo, Z.; Li, H. Molecular recognition patterns between vitamin B12 and human serum albumin explored through STD–NMR and spectroscopic methods. Spectrochim. Acta A Mol. Biomol. Spectrosc.,  2021, 258, 119828.
 [http://dx.doi.org/10.1016/j.saa.2021.119828] [PMID: 33930850]

[25]
Ghosh, R.; Thomas, D.S.; Arcot, J. Molecular recognition patterns between vitamin B12 and proteins explored through STD-NMR and in silico studies. Foods,  2023, 12(3), 575.
 [http://dx.doi.org/10.3390/foods12030575] [PMID: 36766105]

[26]
Khan, A.M. Atia-tul-Wahab; Farooq, S.; Ullah, A.; Choudhary, M.I. Repurposing of US-FDA approved drugs against SARS-CoV-2 main protease (Mpro) by using STD-NMR spectroscopy, in silico studies and antiviral assays. Int. J. Biol. Macromol.,  2023, 234, 123540.
 [http://dx.doi.org/10.1016/j.ijbiomac.2023.123540] [PMID: 36740128]

[27]
Reddy, R.R.; Reddy, J.G.; Kumar, B.V.N.P. NMR investigations on binding and dynamics of imidazolium-based ionic liquids with HEWL. Phys. Chem. Chem. Phys.,  2020, 22(41), 23824-23836.
 [http://dx.doi.org/10.1039/D0CP04584E] [PMID: 33073278]

[28]
Meyer, B.; Peters, T. NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew. Chem. Int. Ed.,  2003, 42(8), 864-890.
 [http://dx.doi.org/10.1002/anie.200390233] [PMID: 12596167]

[29]
Aksoy, S.; Szumlanski, C.L.; Weinshilboum, R.M. Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization. J. Biol. Chem.,  1994, 269(20), 14835-14840.
 [http://dx.doi.org/10.1016/S0021-9258(17)36700-5] [PMID: 8182091]

[30]
Neelakantan, H.; Wang, H.Y.; Vance, V.; Hommel, J.D.; McHardy, S.F.; Watowich, S.J. Structure–activity relationship for small molecule inhibitors of nicotinamide N -Methyltransferase. J. Med. Chem.,  2017, 60(12), 5015-5028.
 [http://dx.doi.org/10.1021/acs.jmedchem.7b00389] [PMID: 28548833]

[31]
Kannt, A.; Rajagopal, S.; Hallur, M.S.; Swamy, I.; Kristam, R.; Dhakshinamoorthy, S.; Czech, J.; Zech, G.; Schreuder, H.; Ruf, S. Novel inhibitors of nicotinamide-N-methyltransferase for the treatment of metabolic disorders. Molecules,  2021, 26(4), 991-1012.
 [http://dx.doi.org/10.3390/molecules26040991] [PMID: 33668468]

Cite As

Protein & Peptide Letters

Binding Affinity Studies of Nicotinamide N-methyltransferase and Ligands by Saturation Transfer Difference NMR

Abstract

Graphical Abstract