BSA, 2mM Dithiothreitol and 1% DMSO. The fluorescent product
was measured using a Tecan reader with excitation at 375nm and
emission at 430nm. The IC50 values were determined by fitting the
inhibition curves (percent inhibition versus inhibitor
concentration) using a four parametric sigmoidal dose response in
GraphPad Prism. Two independent experiments (n1, n2) were
performed in duplicates. The average of the two experiments was
taken and the Standard errors were calculated for compounds
where we were able to obtain defined IC50 values. Cell based
assays were conducted using Human bone osteosarcoma (U2OS)
cell line or differentiated 3T3L1 cells. The cells were cultured
using ATCC recommended media. Cells were counted and seeded
into a 96 well cell culture plate for testing the compound. Cell
culture media was replaced with 100µL of medium/ inhibitor
mixture at different concentrations and incubated for 24 hours at
37°C, 5% CO2, and 95% humidity. Medium/Compound mixture
was removed and washed twice followed by addition of 100µL
Acetonitrile containing internal standard d4-MNA( 20ng/mL) to
the wells. The plate was centrifuged at 5000×g for 10 minutes.
150µL of supernatant was transferred into 96 well plate (Costar
3364) and analyzed by LC-MS/MS or RapidFire. The IC50 values
were determined by fitting the inhibition curves (percent inhibition
versus inhibitor concentration) using a four parametric sigmoidal
dose response in GraphPad Prism.
15) Κannt, Aimo, Pfenninger, Anja, Bluher, Matthias, A Novel
Soluble Biomarker for Insulin Resistance. WO 2015/189325Α1
16) Harshini Neelakantan, Hua-Yu Wang,Virginia Vance,Jonathan D.
Hommel,Stanton F. McHardy,and Stanley J. Watowich,
Structure−Activity Relationship for Small Molecule Inhibitors of
Nicotinamide N‑Methyltransferase. J. Med. Chem., 2017, 60 (12),
pp 5015–5028
17) Swaminathan S, Birudukota S, Thakur MK, Parveen R, Kandan
S, Juluri S, Shaik S, Anand NN, Burri RR, Kristam R, Hallur
MS, Rajagopal S, Schreuder H, Langer T, Rudolph C, Ruf
S, Dhakshinamoorthy S, Gosu R, Kannt A. Crystal structures of
monkey and mouse nicotinamide N-methyltransferase (NNMT)
bound with end product, 1-methyl nicotinamide. Biochem Biophys
Res Commun. 2017, 491(2):416-422
18) a) The HTS for NNMT inhibitors was performed using a
fluorescence assay as described by Sano et al, 1992 and van Haren
et al, 2016. In brief, 3 µl of human NNMT (Emelca Bioscience)
were added to 3 µl of test compound (30 µM) in a black 384-well
small volume microtiter plate. After incubation for 30 minutes at
room temperature, the enzymatic reaction was started by addition
of 3 µl substrate solution containing 60 µM nicotinamide and 60
µM SAM. The composition of the reaction mixture was 100 mM
Tris-HCl, pH 7.5, 0.04 % BSA, 2 mM DTT, 1 % (v/v) DMSO, 0.4
µg/ml NNMT, 10 µM test compound, 20 µM nicotinamide and 20
µM SAM. After incubation for 60 min at 37 °C, the reaction was
stopped and derivatization of MNA to 2,7-naphthyridine started
by addition of 6 µl acetophenone/ethanol (25/75 % v/v) and 3 µl
of KOH (5 M). After 10 min at room temperature, 6 µl of formic
acid were added and, after an additional incubation for 60 min at
room temperature, the formed 2,7-naphthyridine was quantified by
measuring its fluorescence at 375/430 nm (ex/em). The overall
number of compounds screened was 1057156.
20) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;
Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shaw, D.
E.; Shelley, M.; Perry, J. K.; Francis, P.; Shenkin, P. S., "Glide: A
New Approach for Rapid, Accurate Docking and Scoring. 1.
Method and Assessment of Docking Accuracy," J. Med.
Chem., 2004, 47, 1739–1749
21) Male C57Bl6/N mice (Vivo Bio Tech Ltd, India) were used for
the target engagement study. Animals were dosed with compound
12, 50 mg kg-1, p.o. and at every time point 3 animals were bled
and sacrificed. Animals were bled and sacrificed after 0.25, 0.5, 1,
2, 4, 6, 8 and 24h of dosing. 3 animals were bled and sacrificed at
0 min (no treatment). Plasma was harvested by centrifuging the
blood and analyzed for MNA using LC-MS/MS. Quantitation was
achieved by MS/MS detection in positive ion mode for MNA and
internal standard (d4–MNA). Detection of the ions was performed
in the multiple reaction monitoring (MRM) mode, monitoring the
transition of the m/z 137 precursor ion to the m/z 92 product ion
for MNA and m/z 141 precursor ion to the m/z 84 product ion for
d4-MNA (internal standard). The retention times of MNA and d4-
MNA were 2.92 min and 2.91 min, respectively.
b) Sano, A., Endo, N. Takitani, S. Fluorometric assay of rat
tissue N-methyltransferases with nicotinamide and four
isomeric methylnicotinamides. Chem Pharm Bull (Tokyo).
1992, 40, 153-156
c) Van Haren, M.J. et al. A Rapid and Efficient Assay for the
Characterization of Substrates and Inhibitors of
Nicotinamide N-Methyltransferase. Biochemistry. 2016, 55,
5307-5315
19) The screening of compounds from the focused library for NNMT
activity was carried out using fluorescence method. MNA formed
in the NNMT reaction reacts with acetophenone in the presence of
KOH and formic acid and forms a fluorescent product, 2, 7-
naphthyridine which was measured. The final assay reaction
mixture contained a buffer of 100mM Tris Hcl pH 7.5, 0.04%
22) All synthetic procedures and IC50 curves of selected compounds
are given in the supplementary information