The kinetic analysis of the N-methylation of 4-phenylpyridine by nicotinamide N-methyltransferase: Evidence for a novel mechanism of substrate inhibition

Article abstract of DOI:10.1016/j.biocel.2018.03.010

The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-phenylpyridine toxicity i

Full text of DOI:10.1016/j.biocel.2018.03.010

Accepted Manuscript
Title: The kinetic analysis of the N-methylation of
4
-phenylpyridine by nicotinamide N-Methyltransferase:
evidence for a novel mechanism of substrate inhibition
Authors: Matthijs J. van Haren, Martin G. Thomas, Davide
Sartini, David J. Barlow, David B. Ramsden, Monica
Emanuelli, F a´ bio Klamt, Nathaniel I. Martin, Richard B.
Parsons
PII:
DOI:
Reference:
S1357-2725(18)30064-5
https://doi.org/10.1016/j.biocel.2018.03.010
BC 5325
To appear in:
The International Journal of Biochemistry & Cell Biology
Received date:
Revised date:
Accepted date:
27-9-2017
23-2-2018
12-3-2018
Please cite this article as: van Haren MJ, Thomas MG, Sartini D, Barlow DJ, Ramsden
DB, Emanuelli M, Klamt F, Martin NI, Parsons RB, The kinetic analysis of the N-
methylation of 4-phenylpyridine by nicotinamide N-Methyltransferase: evidence for a
novel mechanism of substrate inhibition, International Journal of Biochemistry and
Cell Biology (2010), https://doi.org/10.1016/j.biocel.2018.03.010
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Kinetics of 4-PP N-methylation by NNMT
THE KINETIC ANALYSIS OF THE N-METHYLATION OF 4-PHENYLPYRIDINE
BY NICOTINAMIDE N-METHYLTRANSFERASE: EVIDENCE FOR A NOVEL
MECHANISM OF SUBSTRATE INHIBITION
a†
b†
c
b
Matthijs J. van Haren , Martin G. Thomas , Davide Sartini , David J. Barlow , David B.
d
c
e
a*
b*
Ramsden , Monica Emanuelli , Fábio Klamt , Nathaniel I. Martin , Richard B. Parsons
aUtrecht University, Utrecht Institute for Pharmaceutical Science, Universiteitsweg 99, 3584
CG Utrecht, The Netherlands
bKing’s College London, Institute of Pharmaceutical Science, 150 Stamford Street, London
SE1 9NH, UK
cUniversitá Politecnica delle Marche, Department of Clinical Sciences, School of Medicine,
Ancona, Italy
dUniversity of Birmingham, Institute of Metabolism and Systems Research, Edgbaston,
Birmingham B15 2TH, UK
eUniversidade Federal do Rio Grande do Sul, Departmento de Bioquímica, Instituto de
Ciêncas Básicas de Saúde, Rua Ramiro Barcelos, Porto Alegre, RS 90035 003, Brazil
†Both of these authors contributed equally to this work
CO-CORRESPONDING AUTHORS:
Dr Nathaniel I. Martin
Tel: +31 (0)6 1878 5274
Email: n.i.martin@uu.nl
1

Kinetics of 4-PP N-methylation by NNMT
Dr Richard B. Parsons (to whom all correspondence should be addressed)
Tel: +44 (0)20 7848 4048
Email: richard.parsons@kcl.ac.uk
RUNNING TITLE: Kinetics of 4-PP N-methylation by NNMT
ABBREVIATIONS:
4
-PP: 4-phenylpyridine; DMSO: dimethylsulphoxide; DV: dependent variable; hNNMT:
recombinant human NNMT; IV: independent variable; kcat/K : catalytic efficiency; MeNH:
N-methylnorharman; MMP: mitochondrial membrane potential; MPP+: 1-methyl-4-
m
2
phenylpyridinium ion; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NNMT: nicotinamide N-methyltransferase; PD: Parkinson’s disease
KEYWORDS: enzyme kinetics; neurotoxicity; N-methylation; substrate inhibition; substrate
specificity
2

Kinetics of 4-PP N-methylation by NNMT
GRAPHICAL ABSTRACT
HIGHLIGHTS



4-Phenylpyridine is a poor substrate for nicotinamide N-methyltransferase
4-Phenylpyridine metabolism demonstrates substrate inhibition kinetics
This arises due to docking in the active site in both an inhibitory and catalytic
orientation


4-phenylpyridine metabolism is subject to competitive inhibition by
dimethylsulphoxide
Nicotinamide N-methyltransferase does not increase 4-phenylpyridine toxicity in vitro
Abstract
The N-methylation of 4-phenylpyridine produces the neurotoxin 1-methyl-4-
phenylpyridinium ion (MPP+). We investigated the kinetics of 4-phenylpyridine N-
methylation by nicotinamide N-methyltransferase (NNMT) and its effect upon 4-
phenylpyridine toxicity in vitro. Human recombinant NNMT possessed 4-phenylpyridine N-
methyltransferase activity, with a specific activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg
NNMT. Although the K for 4-phenylpyridine was similar to that reported for nicotinamide,
m
-5
-5 -1
-1
-1
its kcat of 9.3 x 10 ± 2 x 10 s and specificity constant, kcat/K , of 0.8 ± 0.8 s M were
m
less than 0.15% of the respective values for nicotinamide, demonstrating that 4-
phenylpyridine is a poor substrate for NNMT. At low (<2.5 mM) substrate concentration, 4-
phenylpyridine N-methylation was competitively inhibited by dimethylsulphoxide, with a K
i
of 34 ± 8 mM. At high (>2.5 mM) substrate concentration, enzyme activity followed
substrate inhibition kinetics, with a K
i
of 4 ± 1 mM. In silico molecular docking suggested
3

Kinetics of 4-PP N-methylation by NNMT
that 4-phenylpyridine binds to the active site of NNMT in two non-redundant poses, one a
substrate binding mode and the other an inhibitory mode. Finally, the expression of NNMT in
the SH-SY5Y cell-line had no effect cell death, viability, ATP content or mitochondrial
membrane potential. These data demonstrate that 4-phenylpyridine N-methylation by NNMT
is unlikely to serve as a source of MPP+. The possibility for competitive inhibition by
dimethylsulphoxide should be considered in NNMT-based drug discovery studies. The
potential for 4-phenylpyridine to bind to the active site in two binding orientations using the
same active site residues is a novel mechanism of substrate inhibition.
4

Kinetics of 4-PP N-methylation by NNMT
1
. INTRODUCTION
Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to
produce 1-methylnicotinamide using S-adenosylmethionine as cofactor (Aksoy et al., 1994).
NNMT is a cytosolic enzyme of approximately 29 kDa which is expressed in a variety of
tissues, with highest level of expression in the liver (Parsons et al., 2002). NNMT expression
in the healthy population follows a bimodal distribution, with 25% of subjects within each of
low and high expressor categories (Smith et al., 1998; Parsons et al., 2002, 2003). NNMT has
central roles in cellular energy homeostasis, signalling, survival and morphology (Parsons et
al., 2011; Williams et al., 2013; Thomas et al., 2013), and in the human naïve embryonic
stem cell it has been proposed as one of the main controllers of the epigenetic landscape
(Sperber et al., 2015). The enzyme also has been proposed as a therapeutic target for the
treatment of many diseases such as cancers (Kim et al., 2009; Emanuelli et al., 2010; Jung et
al., 2017).
Early studies upon the substrate specificity of porcine NNMT revealed a number of non-
physiological substrates, comprising close structural analogues of nicotinamide, for example
thionicotinamide and 3-acetylpyridine (Alston and Abeles, 1988). These studies have been
extensively cited in the literature to justify the identification of NNMT as responsible for the
N-methylation of a range of pyridine-containing compounds such as quinolines and β-
carbolines (Parsons et al., 2002, 2003; Williams and Ramsden, 2005). Our recent studies have
shown that the majority of these are poor NNMT substrates, with only isoquinoline
demonstrating a catalytic efficiency (kcat/K
m
) value greater than 30% of that observed for
nicotinamide (van Haren et al., 2016; Thomas et al., 2016).
5

Kinetics of 4-PP N-methylation by NNMT
The potential N-methylation of these endogenously-available compounds by NNMT is of
interest as their N-methylated derivatives share close structural similarity to the potent
Complex I inhibitor and dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+)
(Parsons et al., 2002, 2003; van Haren et al., 2016; Thomas et al., 2016). We have shown that
NNMT is expressed in the dopaminergic neurones of the substantia nigra, which are
destroyed in Parkinson’s disease (PD) (Parsons et al., 2002). Significantly, the enzyme is
overexpressed in the cerebellum and caudate nucleus of brains of patients who have died of
PD (Parsons et al., 2002, 2003). Thus it has been suggested that N-methylation may
contribute to PD pathogenesis (Gearhart et al., 2000; Matsubara et al., 2002; Parsons et al.,
2
002, 2003; Williams and Ramsden, 2005).
4
-Phenylpyridine (4-PP) is of interest because its N-methylated derivative is MPP+. 4-PP is
an exogenously-derived compound found in, amongst others, oranges (Thomas and Bassols,
992) and mint (Snyder and D’Amato, 1985). 4-PP N-methylation by NNMT provides a
1
direct metabolic pathway for the endogenous production of MPP+ (Figure 1). Due to its small
size (155 Da) and hydrophobic nature (predicted logP = 2.6), 4-PP is likely to cross the blood
brain barrier. Once inside the brain, its N-methylation by NNMT may lead to the
accumulation of MPP+ and thus contribute to toxicity.
We have previously reported that 4-PP is a substrate of NNMT, although its kinetics and its
effect upon toxicity in vitro were not investigated (van Haren et al., 2016). Hence, in this
study, we have (1) investigated the kinetics of 4-PP N-methylation using recombinant human
NNMT (hNNMT), and (2) investigated the effect of NNMT expression upon 4-PP toxicity in
vitro.
6

Kinetics of 4-PP N-methylation by NNMT
2
. MATERIALS AND METHODS
2
.1. Materials
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Poole, UK) and
were of the highest grade available. Cell culture reagents were purchased from Life
Technologies (Paisley, UK). 4-PP was purified of contaminating MPP+ using preparative
HPLC.
2
.2. 4-Phenylpyridine N-methyltransferase enzyme assay
The ability of NNMT to N-methylate 4-PP was assessed using our previously-described
NNMT assay (van Haren et al., 2016). hNNMT prepared as previously described (Thomas et
al., 2016) was incubated with 1 mM 4-PP prepared in dimethylsulphoxide (DMSO) (5% v/v
final concentration, approximately 700 mM) for 32 min. Samples were taken immediately
after the addition of 0.5 mM S-adenosylmethionine (100 µM final concentration) and
subsequently at time points shown in Figure 2A. MPP+ content was calculated using a MPP+
standard curve and expressed as pmol MPP+ produced ± SD. Linearity of pmol MPP+
produced vs. time was determined by linear regression analysis using Prism v5.0 (GraphPad,
San Diego, CA, USA). Specific activity was calculated and expressed as pmol MPP+
produced/h/mg hNNMT ± SD (n = 3).
2
.3. Kinetic analysis
hNNMT was incubated as described in section 2.2 with 0 – 10 mM 4-PP. Samples were taken
at time points as described above, and initial velocity was calculated and expressed as nmol
MPP+ produced/h. Data were modelled initially using Michaelis-Menten non-linear
regression analysis. Due to the presence of substrate inhibition, two further analyses were
7

Kinetics of 4-PP N-methylation by NNMT
performed as previously described (Patel et al., 2013): (1) non-linear regression analysis
using the following substrate inhibition kinetics equations (Lin et al., 2001; Wu, 2011):
푉푚푎푥.[푠]
푣 = 퐾푚+[푆](1+ (equation 1)
[
ꢁ
ꢀ]
)
ꢂ
2
훽[ꢀ]
훼ꢁꢈꢁꢂ
[
ꢁ
ꢀ]
ꢇ
ꢈꢉ+ꢊ
ꢋ
v = ꢃꢄꢅꢆ. 1
(equation 2)
2
[ꢀ]
[
ꢁ
ꢀ] [ꢀ]
+
+ +
ꢈ ꢁꢂ 훼ꢁꢈꢁꢂ
and (2) Michaelis-Menten non-linear regression analysis of data from 0 – 1 mM 4-PP, i.e.
prior to the onset of substrate inhibition at high substrate concentrations and thus
representative of kinetics if NNMT were not to undergo substrate inhibition; such an
approach has been used by ourselves and others (Lin et al., 2001; Patel et al., 2013; Bomati
and Noel, 2005; Han et al., 2017). The kinetic constants K
m
, kcat and K
i
were calculated for
-1
each modelling paradigm and expressed as µM ± SD and s ± SD respectively (n = 3 for
-1
-1
both). K
m
and kcat were used to calculate kcat/K , which was expressed as s M ± SD (n = 3).
m
2
.4. Effect of DMSO concentration upon 4-phenylpyridine N-methylation
Kinetic analysis of 4-PP N-methylation was performed as described in section 2.2, with 4-PP
prepared in 1%, 3% and 5% DMSO (v/v final concentrations in reaction samples,
approximately 140, 420 and 700 mM respectively). Data were calculated and expressed as
nmol 4-PP produced/h ± SD (n = 3) and subjected to various analyses as described in section
3
.2.
2
.5. Molecular docking of 4-phenylpyridine to the active site of NNMT
8

Kinetics of 4-PP N-methylation by NNMT
Docking of 4-PP into the active site of human NNMT was performed using the Molegro
Virtual Docker software (Thomsen and Christensen, 2006), the 2.72 Å resolution X-ray
crystal structure of the NNMT enzyme complexed with S-adenosyl homocysteine and
nicotinamide (Protein Databank entry, 3ROD (Peng et al., 2011), downloaded from the PDBe
website http://www.ebi.ac.uk/pdbe (Berman et al., 2016; Young et al., 2017), and a 3D model
of 4-PP downloaded from the NCBI Pubchem website (https://pubchem.ncbi.nlm.nih.gov/
(Kim et al., 2016)). The docking of the 4-PP ligand was confined within an 8 Å radius sphere
centred on the known nicotinamide binding site, and with the S-adenosyl homocysteine
retained in its bound position. Following a rigid body docking of the 4-PP, the conformation
and position of the ligand were optimised to establish and/or improve hydrogen bonding to
the protein.
2
.6. Cell culture
SH-SY5Y human neuroblastoma cells (which have no endogenous NNMT expression) and
S.NNMT.LP cells (comprising SH-SY5Y stably expressing a recombinant NNMT C-
terminally fused with the V5 epitope) were cultured as previously described (Parsons et al.,
2
011; Thomas et al., 2013, 2016). NNMT protein and mRNA expression were confirmed
using Western blotting and RT-PCR respectively (Parsons et al., 2011) (data not shown).
2
.7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay
Cells were seeded at a density of 15,000 cells/well into the wells of a 96-well plate (Thermo
Scientific, Loughborough, UK) in quadruplicate and incubated with 4-PP (12.5 – 1600 µM)
for 48 and 120 h. As a positive control, cells were incubated with 500 µM 2N-
methylnorharman (MeNH). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as previously described (Thomas
9

Kinetics of 4-PP N-methylation by NNMT
et al., 2015). Results were calculated and expressed as percentage cell viability compared
with that of untreated cells ± SD (n = 3 for 48 h, n = 5 for 120 h). Concentration was log-
transformed and subsequently data were modelled using non-linear regression analysis using
the following equation:
푚푎푥ꢍ푚ꢎ푚−푚ꢍꢏꢍ푚ꢎ푚
+10(퐿표ꢐ퐸퐶5ꢑꢒꢓ)∗ℎꢂ푙푙ꢔ푙표푝푒
y = ꢄ푖푛푖ꢄ푢ꢄ ꢌ 1
(equation 3)
with a constraint of y = 0 for the minimum. From this, logEC50 was calculated for each
replicate from which an average logEC50 ± SD (n = 3 for 48 h, n = 5 for 120 h) was
calculated. To aid comprehension, this logEC50 value was antilogged to obtain EC50, which
was expressed as µM; SD values were not antilogged due to the asymmetry obtained when
the antilog of a logSD is obtained (Hancock et al., 1988).
2
.8. Lactate dehydrogenase cell death assay
Cells were seeded and incubated with 4-PP as outlined in section 2.7 for 120 h, after which
cell death was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher
Scientific, Paisley, UK) as previously described (Thomas et al., 2015). Results were
calculated, expressed and modelled using non-linear regression as described in section 2.7,
with a constraint of y = 0 for the maximum (n = 4).
2
.9. Cellular ATP content assay
Cells were seeded as described in section 2.7 into the wells of a poly-L-lysine-coated white
6-well plate (Greiner Bio-One Ltd., Stonehouse, UK) and incubated with 4-PP as described
in section 2.7 for 120 h. ATP content was measured as previously described (Thomas et al.,
9
1
0

Kinetics of 4-PP N-methylation by NNMT
2
016) and expressed as percentage cellular ATP content compared with that of untreated cells
±
SD (n = 5). Non-linear regression analysis was performed as described in section 2.7.
2
.10. Mitochondrial membrane potential
Cells were seeded as described in section 2.7 into the wells of a poly-L-lysine-coated black
6-well plate (Thermo Scientific, Loughborough, UK) and incubated with MPP+ as
9
described in section 2.7 for 120 h. Membrane mitochondrial potential (MMP) was measured
using the JC-1 mitochondrial membrane potential assay kit (Cayman Chemical Company,
Michigan, USA) as previously described (Parsons et al., 2011) and expressed as percentage
observed in untreated cells ± SD (n = 4). Non-linear regression analysis was performed as
described in section 2.7.
2
.11. Statistical Analysis.
All analyses were undertaken using Prism v5.0. In all instances, P < 0.05 was taken to
indicate statistical significance. Statistical analysis of cell viability, cell death, cellular ATP
content and MMP was performed as previously described (Thomas et al., 2016). Briefly,
comparison of all four endpoint measures at each concentration with values observed in
untreated cells was performed using one-way ANOVA with Tukey post-hoc comparisons,
with concentration as independent variable (IV) and endpoint measure as dependent variable
(
DV). Comparison of toxicity between cell lines and time points for each concentration was
performed using two-way ANOVA with Dunnett’s post-hoc comparisons, with cell line and
-PP or MeNH concentration as IV and endpoint measure as DV. Statistical comparison of
4
EC50s obtained from the MTT reduction assay was performed using two-way ANOVA, with
cell line and length of incubation as IVs and logEC50 as DV. Statistical comparison of cell
death, cellular ATP content and MMP EC50 values were performed using Student’s t-test with
1
1

Kinetics of 4-PP N-methylation by NNMT
Welch correction, with cell line as IV and logEC50 as DV. Statistical comparisons were
performed upon logEC50 values as they follow a normal distribution (Hancock et al., 1988).
3
. RESULTS
3
.1. NNMT demonstrated 4-phenylpyridine N-methyltransferase activity and substrate
inhibition kinetics
To determine whether NNMT was capable of N-methylating 4-PP, we incubated hNNMT
with 1 mM 4-PP prepared in DMSO (5% v/v final concentration, approximately 700 mM)
(Figure 2). MPP+ production increased linearly with time (Figure 2A), with a specific
activity of 1.7 ± 0.03 nmol MPP+ produced/h/mg hNNMT.
To estimate kinetic constants for 4-PP N-methylation, we incubated NNMT with 4-PP (0 – 10
mM), and performed Michaelis-Menten non-linear regression analysis upon resulting initial
velocities (Figure 2B). The results are summarised in Table 1. Comparison with values
published for nicotinamide (van Haren et al., 2016) revealed that 4-PP is a poor substrate for
NNMT. Michaelis-Menten analysis produced a K
reported for nicotinamide, whereas kcat and kcat/K
m
similar to that we have previously
m
were only 0.1% and 0.15% of those for
2
nicotinamide. Due to the poor goodness of fit obtained (R = 0.54), the data was subsequently
modelled using non-linear substrate inhibition kinetics which, although provided significantly
2
better goodness of fit (R = 0.96), returned ambiguous values for Vmax, K
m
and K (data not
i
shown). Data was then modelled with a variety of substrate inhibition equations in order to
obtain a best kinetic fit (Lin et al., 2001; Wu, 2011). K was 12-fold higher than that reported
for nicotinamide, with kcat and kcat/K being 0.6% and 0.04% of the respective values. The k
m
m
i
for 4-PP substrate inhibition was similar to those reported for other NNMT substrates which
demonstrated substrate inhibition, which were in the range 4.7 – 20.2 mM (van Haren et al.,
1
2

Kinetics of 4-PP N-methylation by NNMT
2
016). To estimate kinetic parameters for 4-PP N-methylation in the absence of substrate
inhibition, data between 0 – 1 mM 4-PP, i.e. prior to the onset of substrate inhibition, were
2
subjected to Michaelis-Menten analysis (R = 0.96). The resulting K
m
was 8-fold higher, and
k
cat and kcat/K values were 0.4 and 0.04%, of values for nicotinamide.
m
3
.2. DMSO is a competitive inhibitor of 4-phenylpyridine N-methylation by NNMT
As DMSO is reported to be a competitive inhibitor of a number of enzymes (Plummer et al.,
983; Banasik et al., 2004; Kwak et al., 2009), we investigated whether DMSO was a
1
competitive inhibitor of 4-PP N-methylation by NNMT (Figure 3).
Specific activity decreased with increasing DMSO concentration (Figure 3A), resulting in
specific activities of 2.6 ± 0.06, 1.8 ± 0.05 and 1.5 ± 0.1 nmol MPP+ produced/h/mg hNNMT
at 1, 3 and 5% DMSO (v/v final concentration) respectively (P < 0.001). Non-linear substrate
inhibition regression analysis of the data (Figure 3B) revealed that increasing DMSO
concentration decreased velocity, v, at 4-PP concentrations below 2.5 mM, i.e. prior to the
onset of substrate inhibition. As it is likely that physiological concentrations of 4-PP are
below 2.5 mM, Michaelis-Menten kinetic modelling was performed using data truncated at
2
.5 mM (Figure 3C), from which apparent K
m
(K
m
app) and Vmax (Vmaxapp) values for each
app
concentration of DMSO were calculated, which are summarised in Table 2. Vmax values did
app
not alter significantly whereas K
m
values increased with increasing DMSO concentration,
hence DMSO is a competitive inhibitor of 4-PP N-methylation. As a confirmatory exercise,
the same data were modelled using an Eadie-Hofstee plot, v vs. v/[S] (Figure 3D), which
confirmed the presence of competitive inhibition of 4-PP N-methylation by DMSO. The kcat
and kcat/K
m
values for all concentrations of DMSO were calculated and are summarised in
decreased
Table 2. As expected for a competitive inhibitor, kcat did not alter whereas kcat/K
m
1
3

Kinetics of 4-PP N-methylation by NNMT
with increasing DMSO concentration. Catalytic efficiency was significantly reduced by all
three concentrations of DMSO, with an 82% reduction at 1% v/v DMSO.
app
To estimate the K
m
of 4-PP in the absence of DMSO, K
m
was plotted against DMSO
was calculated via
for DMSO substrate inhibition was calculated by first
concentration (Plummer et al., 1983) (Figure 3E), from which K
extrapolation as 155 µM. The K
m
i
calculating the inhibition factor, α, for each concentration of DMSO using the following
equation:
ꢕꢖꢄ = ꢖꢄ푎ꢗꢗ
i
K was then calculated using the following equation:
[
DMSO]
ꢕ = ꢘ ꢌ
ꢖꢍ
All three K values were then averaged to obtain a mean of 34 ± 8 mM.
i
K
m
and Vmax values of 155 µM and 48 pmol MPP+ produced/h (Vmax being derived from the
app
average of Vmax for all three concentrations of DMSO) were then used to calculate the
activity of 4-PP N-methylation in the absence of DMSO using Michaelis-Menten regression
analysis (Figure 3C). Specific activity in the absence of DMSO was extrapolated from a plot
of specific activity vs. DMSO concentration (Figure 3F), from which the specific activity was
estimated to be 2.8 ± 0.2 nmol MPP+ produced/h/mg hNNMT.
3
.3. 4-Phenylpyridine docks to the hNNMT active site in two non-redundant orientations
1
4

Kinetics of 4-PP N-methylation by NNMT
Molecular docking was used to investigate the interaction of 4-PP with the NNMT active site.
To confirm the accuracy of the docking protocol, initially hNNMT was docked with
nicotinamide (Figure 4A). As expected (Peng et al., 2011), the pyridine ring of nicotinamide
formed an electrostatic interaction with Y20 and was also held within a hydrophobic clamp
comprising Y204 and L164. The amide group of nicotinamide formed hydrogen bonds with
S201, S213 and D197.
A total of 50 docking trials for 4-PP were made, yielding just two non-redundant 4-PP poses
(taking an RMSD ≤ 1 Å on atomic co-ordinates as the criterion of pose similarity), each of
which was returned 25 times. The first pose presented 4-PP in a presumed substrate binding
mode (Figure 4B), in which the 4-PP pyridine ring interacted with Y20, Y204 and L164 as
described for nicotinamide. However, due to the lack of an amide group, there were no
hydrogen bonding interactions between 4-PP and S201, S213 and D197. There were no
interactions between the aromatic phenyl ring of 4-PP and the active site of hNNMT.
o
In the second pose, 4-PP was orientated almost 180 with respect to the first pose (Figure
4
C). This second pose most likely represents an inhibitory binding mode due to the lack of
proximity between the pyridine nitrogen and the catalytically essential Y20 (Peng et al.,
011). The phenyl ring formed a hydrophobic interaction with L164 and pi-pi stacking with
2
Y204. The pyridine nitrogen of 4-PP is orientated in a similar position to the amide nitrogen
of nicotinamide, hence forming hydrogen bond interactions with S201 and S213, but not
D197.
Docking studies were also performed using isoquinoline (Figure 5), a substrate of NNMT
which also demonstrates substrate inhibition (van Haren et al., 2016). As observed with 4-PP,
1
5

Kinetics of 4-PP N-methylation by NNMT
a total of 50 docking trials yielded just two non-redundant poses, each of which was returned
2
5
5 times. The first pose presented isoquinoline in a presumed substrate binding mode (Figure
A) and the second a presumed inhibitory binding mode (Figure 5B), as determined by the
relative proximity of the pyridine nitrogen to Y20. Each pose used the same active site
residue interactions with the phenyl and pyridine rings as observed for 4-PP.
3
.4. Neither NNMT expression nor length of incubation influenced 4-phenylpyridine
toxicity in vitro
The MTT reduction cell viability assay, an early-stage measure of toxicity (Thomas et al.,
2
1
015), was used to determine whether NNMT increased 4-PP toxicity in vitro after 48 and
20 h incubation (Figure 6 and Tables 3 & 4). 4-PP was toxic towards both SH-SY5Y and
S.NNMT.LP cells at concentrations of 400, 800 and 1600 µM at both time points (Table 3).
The length of incubation had no effect upon 4-PP toxicity at any of the concentrations
investigated for either cell-line. There was no significant difference in logEC50 for each cell-
line at either time point (Table 4). As a positive control for the effect of NNMT expression
upon toxicity, the effect of 0.5 mM MeNH, a compound whose in vitro toxicity we have
shown to increase with length of incubation, was investigated. MeNH was significantly more
toxic towards both SH-SY5Y and S.NNMT.LP cells after 120 h compared with 48 hr
incubation (SH-SY5Y: 78.5 ± 3% vs. 10.7 ± 6.1%, P < 0.001, n = 3; S.NNMT.LP: 52.2 ±
3
.8% vs. 22.1 ± 18.4%, P < 0.01, n = 4). MeNH was significantly more toxic towards
S.NNMT.LP compared to SH-SY5Y cells after 48 hr incubation (P < 0.05), although after
20 h incubation there was no difference in toxicity.
1
The LDH cytotoxicity assay, a late-stage measure of toxicity (Thomas et al., 2015), was used
as an additional confirmatory toxicity assay (Figure 7A and Table 3). 4-PP was toxic towards
1
6

Kinetics of 4-PP N-methylation by NNMT
SH-SY5Y cells at 1600 and 800 µM, and towards S.NNMT.LP cells at 1600, 800 and 400
µM. There was no significant difference in logEC50 values for each cell-line (-2.51 ± 0.3 vs. -
2
.17 ± 0.1, equivalent to 3090 µM and 6761 µM respectively, P = 0.2).
The low catalytic efficiency of 4-PP N-methylation by NNMT (Table 2) in tandem with the
lack of effect of NNMT expression upon 4-PP toxicity in vitro suggested that the degree of
intracellular 4-PP N-methylation by NNMT is insignificant. As a confirmatory exercise, we
investigated the effect of NNMT expression after 120 hr incubation upon two parameters of
mitochondrial function - cellular ATP content and MMP (Parsons et al., 2011). Our
hypothesis was that increased 4-PP N-methylation by NNMT would result in increased
MPP+ production and accumulation within the cell, the consequence of which would be
Complex I inhibition, a decrease in the MMP (Figure 7B and Table 3) and a subsequent
reduction in intracellular ATP content (Figure 7C and Table 3). The MMP of SH-SY5Y cells
was significantly decreased at 200, 400, 800 and 1600 µM 4-PP, and in S.NNMT.LP cells at
4
00, 800 and 1600 µM 4-PP. There was no significant difference in MMP between each cell-
line at any concentration of 4-PP investigated. There was no significant difference in logEC50
for each cell-line (-2.69 ± 0.32 vs. -2.17 ± 0.14, equivalent to 2023 vs. 2280 µM, P = 0.5).
Intracellular ATP content of SH-SY5Y cells was significantly decreased at 800 and 1600 µM
4
-PP, yet was increased at 100, 200 and 400 µM. In S.NNMT.LP cells, 4-PP decreased
intracellular ATP content at concentrations of 800 and 1600 µM, with no concentration
increasing intracellular ATP concentration. Intracellular ATP content was significantly higher
in SH-SY5Y cells compared with that of S.NNMT.LP cells at 200 (157.6 ± 19.7% vs. 109.8 ±
3
4.4%, P < 0.01) and 400 µM (135.2 ± 23% vs. 95.2 ± 25.9%, P < 0.05) 4-PP. There was no
1
7

Kinetics of 4-PP N-methylation by NNMT
significant difference in logEC50 values for each cell-line (-3.13 ± 0.04 vs. -3.2 ± 0.07,
equivalent to 738 vs. 625 µM, P = 0.12).
4
. DISCUSSION
4
.1. 4-PP is a substrate of NNMT
Our kinetic analysis of 4-PP N-methylation by hNNMT demonstrated that 4-PP was rather a
poor substrate, even when the inhibitory effect of DMSO was taken into account. 4-PP is an
exogenous compound whose abundance in the diet is very low. Its content in Brassica
campestris L. ssp. Pekinensis, for example, is as little as 0.9% (Hong and Kim, 2013). Hence,
even for someone eating an exclusively vegetarian diet it is unlikely that the concentration of
4
-PP within the brain will be sufficiently high to outcompete nicotinamide as a substrate for
NNMT. Even if it were to reach such a concentration, the low rate of catalytic turnover would
more likely result in the inhibition of nicotinamide N-methylation. Hence, it is unlikely that
NNMT-mediated 4-PP metabolism is a source of MPP+. This is supported by the failure of
NNMT expression to increase the toxicity of 4-PP due to increased MPP+ production in
S.NNMT.LP cells, even at concentrations which were significantly higher than what would
be expected physiologically. As MPP+ is actively sequestered into mitochondria due to its
positive charge (Trevor et al., 1987), it could be argued that, with even such a low catalytic
turnover, over a much longer period of time the slow but continuous N-methylation of 4-PP
may result in a toxic accumulation of MPP+ within the mitochondria, resulting in Complex I
inhibition and depletion of the MMP, and ultimately cell death. It is also possible that mature
neurones, which rely predominantly upon mitochondrially-derived ATP (Zheng et al., 2016),
may be more susceptible to long-term MPP+ accumulation, compared to tumour cells in
culture which rely predominantly upon glycolysis (Vander Heiden et al., 2009). In vivo
studies using acute high-dose 4-PP administration have failed to support this (Bradbury et al.,
1
8

Kinetics of 4-PP N-methylation by NNMT
1
985; Perry et al., 1987; Irwin et al., 1987; Godin and Crooks, 1989; Carr and Basham, 1991)
and to date no study using NNMT-overexpressing in vivo models have reported such
observations.
4
.2. 4-PP N-methylation by NNMT displayed substrate inhibition kinetics
Although 4-PP is a minor substrate of NNMT, its kinetics are of interest as they reveal that
NNMT-mediated 4-PP N-methylation was subject to both substrate and competitive
inhibition. Below concentrations of 2.5 mM 4-PP, competitive inhibition by DMSO was
observed, whereas at concentrations higher than 2.5 mM, substrate inhibition occurred. This
has important consequences for NNMT-based drug discovery studies, as it is a therapeutic
target for a number of diseases, in particular cancer.
Molecular docking revealed that 4-PP is able to dock with the NNMT active site in two non-
redundant poses, one of which we propose is a substrate binding mode and the other an
inhibitory binding mode as deduced by the proximity of the pyridine nitrogen to Y20. The
substrate inhibition can thus be rationalised to arise due to competition between these two
binding modes. In its inhibitory binding mode, 4-PP interacts with the active site via both its
phenyl and pyridine rings, whereas in its substrate binding mode it interacts only via its
pyridine ring, with the increased number of interactions presumably favouring the inhibitory
binding mode at high 4-PP concentration.
Mechanistically, this is of interest, as it represents a previously unreported mechanism of
substrate inhibition. To date, two mechanistic models have been proposed. The first model
involves the formation of a ternary dead-end complex comprising the substrate interacting
with the end-product of cofactor metabolism within the active site at high substrate
1
9

Kinetics of 4-PP N-methylation by NNMT
concentrations (Wu, 2011; Tyapochkin et al., 2009). The second model predicts the presence
of two substrate binding sites, the first a catalytically-active high affinity site and the second a
catalytically-inactive low affinity site, either within or separate to the active site itself (Lin et
al., 2001; Wu, 2011). At high substrate concentration, binding to the catalytically-inactive
site prevents the binding of substrate to the catalytically-active site either via direct steric
hindrance, as for example warfarin binding within the active site of cytochrome P450 2C9
(
Williams et al., 2003), or the induction of a conformational change of the protein, as for
example dehydroepiandrosterone binding to sulphotransferase 2A1 (Tracy and Hummel,
004; Allali-Hassani et al., 2007). Our molecular docking study suggests a novel mechanism
2
in which 4-PP binds to the active site of NNMT in two orientations, one a catalytic and the
other a non-catalytic orientation, both of which bind to a single binding site using the same
amino residue interactions. The ability to bind in this manner arises due to 4-PP possessing 2-
fold rotational symmetry. In support of this, the majority of NNMT substrates which
demonstrate substrate inhibition are quinoline and its analogues, compounds which also
demonstrate two-fold rotational symmetry (Figure 8) and have K
i
values similar to that of 4-
PP (van Haren et al., 2016). Molecular docking of isoquinoline returned the same binding
poses and active site amino acid residue interactions as 4-PP, and has a K of 20 mM which is
i
similar to that for 4-PP.
4
.3. 4-PP N-methylation undergoes competitive inhibition by DMSO
Our results demonstrate that DMSO is a competitive inhibitor of 4-PP N-methylation by
NNMT. Many studies of enzyme kinetic reactions utilise DMSO as a solvent for solubilising
non-polar substrates (van Haren et al., 2016; Faulds et al., 2011; Patel et al., 2013), usually at
concentrations below 5% (approximately 700 mM). We have shown that preparing
nicotinamide in 5% DMSO results in more reliable estimation of the kinetic parameters of
2
0

Kinetics of 4-PP N-methylation by NNMT
nicotinamide N-methylation compared to nicotinamide prepared in phosphate-buffered saline,
which we ascribed to the relative insolubility of nicotinamide in phosphate-buffered saline
compared to DMSO which resulted in less nicotinamide delivered to the assay (Patel et al.,
2
013). Although DMSO has no effect upon the activity of a number of enzymes (Chauret et
al., 1998; Tsitanou et al., 1999; Easterbrook et al., 2001), DMSO is known to be a
competitive inhibitor of many enzymes (Perlman and Wolff, 1968; Plummer et al., 1983;
Chauret et al., 1998; Banasik and Ueda, 1999; Easterbrook et al., 2001; Banasik et al., 2004).
We were unable to determine, using molecular docking, how DMSO binds to the NNMT
active site. This was due to the small size of the molecule and the vast number of potential
interactions within the large active site of NNMT. Where crystal structures of DMSO
complexed with other enzymes have been obtained, in many instances DMSO interacts with
inorganic metal ions within their active sites. For example, the crystal structure of P450 BM-
3
reveals that the sulphur atom of DMSO interacts with the iron of the heme group (Kuper et
al., 2007). DMSO can also interact directly with amino acid residues. For example, the
inhibition of liver alcohol dehydrogenase involves co-ordination with amino acid residue S48
as well as the metal ion within the active site (Meijers et al., 2007). As NNMT does not
require an active site metal ion for activity, it can be assumed that DMSO interacts directly
with residues within the active site.
The limited numbers of studies which have calculated a K
inhibitor, with values ranging from 5 mM for alcohol dehydrogenase (Perlman and Wolff,
968) to 70 mM for acetylcholinesterase (Plummer et al., 1983). The K for NNMT N-
i
for DMSO reveal it to be a poor
1
i
methylation of 4-PP is in accord with these values. Although not a potent inhibitor, the
concentrations of DMSO routinely used in enzyme reactions are significantly above these K
i
values, being approximately 20-fold higher for 4-PP N-methylation by NNMT. Therefore it is
2
1

Kinetics of 4-PP N-methylation by NNMT
likely that many studies are performed using sub-optimal enzyme activity, and are thus
reporting incorrect K
m
and kcat/K values, as evidenced in our study. Hence, the use of
m
alternative solvents for kinetic studies such as acetonitrile and methanol should be
considered. Studies of cytochrome P450s, UDP glucuronosyltransferases and
phenolsulphotransferases show that concentrations of up to 1% methanol and 2% acetonitrile
had little effect upon their activities, in contrast to DMSO (Chauret et al., 1998; Easterbrook
et al., 2001). However, these solvents can also act as enzyme inhibitors (Busby et al., 1999),
so care must be taken to assess the effect of solvents upon enzyme catalytic activity in order
to choose the most appropriate.
This is of particular importance for the screening of compounds in drug discovery studies in
which the therapeutic target is NNMT. It is possible that, in addition to acting as a
competitive inhibitor of substrate metabolism, DMSO may also compete with competitive
inhibitors for the active site, thus reducing their efficacy. For example, DMSO binds to the
same binding site in the active site of lysozyme as its inhibitor N-acetylglucosamine, reducing
its inhibitory efficacy (Johannesson et al., 1997). Also, increasing concentrations of DMSO
increased the IC50 values of all lipophilic and hydrophilic inhibitors of poly(ADP-ribose)
synthetase investigated by upto 10-fold, with the IC50 of 6(5H)-phenanthridinone increasing
3
3-fold (Banasik and Ueda, 1999). Thus, the use of DMSO in enzyme assays can potentially
affect candidate inhibitor efficacy sufficiently to lead to their unwarranted removal from drug
inhibitor screens. The small size of DMSO makes its docking to protein active sites
somewhat complicated to predict, in particular for enzymes such as NNMT which have large
active sites, thus it is very difficult to predict whether DMSO may interfere with candidate
inhibitor docking and thus inhibitory activity. However, by using the kinetic approaches used
in this study, it will be possible to determine whether DMSO is an appropriate solvent for
2
2

Kinetics of 4-PP N-methylation by NNMT
inhibitor screening, thus reducing the likelihood of removing candidate inhibitors due to
incorrect determination of IC50 and K .
i
5
. CONCLUSIONS: these findings suggest that the N-methylation of 4-PP into the
dopaminergic neurotoxin MPP+ is unlikely to occur within the cell and is thus unlikely to
contribute to the pathogenic process of PD.
CONFLICTS OF INTEREST: none.
ACKNOWLEDGEMENTS: MGT was supported by a King’s College London Graduate
Schools Studentship. The funding body had no involvement in study design, collection,
analysis or interpretation of data, writing the report or the decision to submit.
2
3

Kinetics of 4-PP N-methylation by NNMT
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