Yongzhi Gao1, Matthijs J van Haren1, Ed E Moret, Johannes J M Rood, Davide Sartini2, Alessia Salvucci2, Monica Emanuelli2, Pierrick Craveur3, Nicolas Babault3,4, Jian Jin4, Nathaniel I Martin1. 1. Biological Chemistry Group, Institute of Biology Leiden , Leiden University , Sylviusweg 72 , 2333 BE Leiden , The Netherlands. 2. Department of Clinical Sciences , Universitá Politecnica delle Marche , Via Ranieri 65 , 60131 Ancona , Italy. 3. Synsight , Genopole Entreprises , 4 Rue Pierre Fontaine , 91000 Évry , France. 4. Center for Chemical Biology and Drug Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , New York 10029 , United States.
Abstract
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.
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-2humanoral cancer cell line.
Nicotinamide N-methyltransferase (NNMT) is an
important metabolic enzyme that catalyzes the transfer of a methyl
group from the cofactor, S-adenosyl-l-methionine
(SAM), onto its various substrates, most notably nicotinamide (NA)
and other pyridines, to form 1-methyl-nicotinamide (MNA) or the corresponding
pyridinium ions.[1−3] The past decade has seen a renewed interest in the
biological function of NNMT in a range of human diseases. While it
was previously assumed that NNMT’s primary roles were limited
to nicotinamide metabolism and xenobiotic detoxification of endogenous
metabolites, broader roles of NNMT in human health and disease are
becoming clearer.[4] NNMT has been found
to be overexpressed in a variety of diseases, including metabolic
disorders,[5−7] cardiovascular disease,[8,9] cancer,[10−14] and Parkinson’s disease.[15,16] In general,
overexpression of NNMT has been linked to disease progression in the
aforementioned afflictions, with the exception of its role in Parkinson’s
disease where NNMT seems to be neuroprotective.[17,18] Collectively, NNMT appears to play a unique role in the regulation
of post-translational modifications and signal transduction, making
it an attractive and viable therapeutic target.Despite the
growing interest, few small-molecule NNMT inhibitors
have been described to date. Among these structures, the product of
the enzymatic reaction, MNA, is a known inhibitor of NNMT and has
generally been used in biochemical activity assays.[19] Recently, Cravatt and co-workers reported chloroacetamide-based
covalent NNMT inhibitors that react with cysteine C165 in the SAM-binding
pocket of the enzyme.[20] Notably, Sanofi
researchers have also recently reported a series of nicotinamide analogues
that inhibit NNMT activity, leading to decreased MNA production, stabilization
of insulin levels, glucose regulation, and weight loss in mouse models
of metabolic disorders.[21,22] In another approach,
the group of Watowich focused on the development of inhibitors based
on NNMT’s alternative substrate, quinoline. Their compounds
showed improvement of symptoms in diet-induced obesemice.[23] Previous work in our group has focused on bisubstrate
inhibitors designed to mimic the transition state of the methylation
reaction catalyzed by NNMT with compound 1 (Figure ) showing activity
on par with the known general methyltransferase inhibitor, sinefungin.[24]
Figure 1
Schematic overview of the design strategy of the second
generation
of inhibitors based on trivalent bisubstrate compounds 1[24] and 2.[31]
Schematic overview of the design strategy of the second
generation
of inhibitors based on trivalent bisubstrate compounds 1[24] and 2.[31]Designing bisubstrate analogues as inhibitors is
an established
and effective strategy that has been applied to a range of methyltransferase
enzymes, including catechol O-methyltransferase,[25,26] histone lysinemethyltransferases,[27] argininemethyltransferases,[28−30] and more recently nicotinamide N-methyltransferase.[24,31] A recently published co-crystal
structure of a bisubstrate inhibitor bound to NNMT [Protein Data Bank
(PDB) ID: 6CHH] clearly delineates key interactions with residues in the enzyme
active site, providing valuable information for further optimization
of improved bisubstrate-like inhibitors.[31] The work here described builds on our previous findings for “trivalent”
inhibitor 1, which is assumed to simultaneously bind
in the adenosine, amino acid, and nicotinamide binding pockets of
the NNMT active site. Based on insights provided by recent NNMT crystal
structures, we have designed new inhibitors, wherein the nicotinamide
moiety is replaced by other aromatic substituents accompanied by variation
in the length of the linker connecting the amino acid moiety. Based
on the high conservation of the residues in the adenosine binding
pocket, no changes were made to the adenosine group. A schematic overview
of the design strategy is presented in Figure .
Results and Discussion
Design
The ternary
crystal structure of NNMT (PDB ID: 3ROD) reveals the interactions
of nicotinamide and S-adenosyl-l-homocysteine
(SAH) with the active site residues.[32] The
active site can be roughly divided into three binding regions for
the adenosine group, the amino acid moiety, and the nicotinamide unit.
The starting point was a trivalent bisubstrate compound, 1, which was designed to bind all three binding regions. To find the
optimal substitutions, a systematic approach was applied, where variations
were made to the nicotinamide mimic on the one hand and the amino
acid moiety on the other. The benzamide group, representing nicotinamide,
was also replaced by methyl benzoate or benzoic acid moieties. Notably,
the crystal structure of the NNMT–nicotinamide–SAH ternary
complex reveals π–π stacking between tyrosine (Tyr)
residue Y204 and the nicotinamide substrate.[32] We, therefore, also prepared an analogue bearing a naphthalene unit
in the presumed nicotinamide position with the aim of introducing
stronger π–π stacking with the tyrosine residues
of the NNMT active site. We also explored variation of the amino acid
moiety as part of our design strategy: in some analogues the amine
of the amino acid unit was omitted to reduce charge and in others
the carboxylic acid was replaced by the corresponding primary amide.
In addition, variation in the length of the carbon chain linking the
amino acid moiety was examined. Furthermore, inspired by the structure
of histone methyltransferase DOTL1 inhibitor pinometostat,[33] we also investigated the incorporation of an
isopropyl group to replace the amino acid moiety entirely.
Synthesis
Key aldehyde intermediates (compounds 6, 8, 9, 16, 17, 22, 23, 27, and 28) required
for the synthesis of the various bisubstrate
analogues pursued were prepared from commercially available materials,
in good overall yields, as summarized in scheme –. The
trivalent inhibitors were then prepared via a convenient double-reductive
amination strategy starting from the commercially available 2′-3′-O-isopropylidene-6-aminomethyl-adenosine starting material
and the corresponding aldehydes (Schemes and ).
Scheme 1
Synthetic Route for Aldehydes 6, 8, and 9
Reagents and conditions:
(a)
NaOH, MeOH, room temperature (rt), 16 h (95%); (b) (i) SOCl2, reflux, 2 h, (ii) tritylamine, CH2Cl2, 0
°C to rt, 2 h (72%); (c) diisobutylaluminum hydride (DIBAL-H),
−78 °C to rt, 2 h (85%); (d) pyridinium dichromate (PDC),
CH2Cl2, rt, 2 h (53–64%); (e) NaBH4, BF3.Et2O, tetrahydrofuran (THF), 0
°C to rt, 2 h (89%); (f) LiOH, THF/H2O (2:1); (g)
2-tert-butyl-1,3-diisopropylisourea, CH2Cl2, tert-butanol (39% over two steps).
Scheme 3
Synthetic Route for Aldehydes 27 and 28
Reagents
and conditions: (a)
CH3NHOCH3·HCl, BOP, Et3N, CH2Cl2, rt, 2 h (85–88%); (b) (Boc)2O, Et3N, DMAP, CH2Cl2 (94%); (c)
DIBAL-H in hexanes (1 M), THF, −78 °C, assumed quant.
Representative Scheme for the Synthesis
of the Final Compounds; Shown
for Compounds 1 and 57–61
The same procedure was used starting
from aldehydes 30–32 to form intermediate
compounds 39–56 and 80 and final compounds 62–79 and 81 as detailed in the Experimental Section. Reagents and conditions: (a) aldehyde, NaBH(OAc)3, AcOH,
DCE, rt, overnight (49–77%); (b) (i) trifluoroacetyl (TFA),
CH2Cl2, rt, 2 h, (ii) H2O, rt, 30
min (47–73%).
Synthetic Route for Aldehydes 6, 8, and 9
Reagents and conditions:
(a)
NaOH, MeOH, room temperature (rt), 16 h (95%); (b) (i) SOCl2, reflux, 2 h, (ii) tritylamine, CH2Cl2, 0
°C to rt, 2 h (72%); (c) diisobutylaluminum hydride (DIBAL-H),
−78 °C to rt, 2 h (85%); (d) pyridinium dichromate (PDC),
CH2Cl2, rt, 2 h (53–64%); (e) NaBH4, BF3.Et2O, tetrahydrofuran (THF), 0
°C to rt, 2 h (89%); (f) LiOH, THF/H2O (2:1); (g)
2-tert-butyl-1,3-diisopropylisourea, CH2Cl2, tert-butanol (39% over two steps).
Synthetic Route for Aldehydes 16, 17, 22, and 23
Reagents and conditions: (a)
TrtCl, CH3CN, K2CO3, rt, 48 h (20–28%);
(b) KOH, EtOH, reflux, overnight (37–93%) (c) NaBH4, BF3·Et2O, THF, 0 °C to rt, 2 h
(64–81%); (d) PDC, CH2Cl2, rt, 2 h (65–78%);
(e) tert-butanol, 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide, Et3N, toluene, overnight
(25–93%).
Synthetic Route for Aldehydes 27 and 28
Reagents
and conditions: (a)
CH3NHOCH3·HCl, BOP, Et3N, CH2Cl2, rt, 2 h (85–88%); (b) (Boc)2O, Et3N, DMAP, CH2Cl2 (94%); (c)
DIBAL-H in hexanes (1 M), THF, −78 °C, assumed quant.
Representative Scheme for the Synthesis
of the Final Compounds; Shown
for Compounds 1 and 57–61
The same procedure was used starting
from aldehydes 30–32 to form intermediate
compounds 39–56 and 80 and final compounds 62–79 and 81 as detailed in the Experimental Section. Reagents and conditions: (a) aldehyde, NaBH(OAc)3, AcOH,
DCE, rt, overnight (49–77%); (b) (i) trifluoroacetyl (TFA),
CH2Cl2, rt, 2 h, (ii) H2O, rt, 30
min (47–73%).The preparation of aromatic
aldehydes 6, 8, and 9 began
with the selective mono-deprotection of
dimethyl isophthalate using sodium hydroxide (Scheme ).[34] Monomethyl
isophthalate (3) was subsequently transformed into trityl-protected
amide 4 using tritylamine via its acid chloride intermediate
and reduced by diisobutylaluminum hydride (DIBAL-H) to give alcohol 5. The alcohol was oxidized to aldehyde 6 using
pyridinium dichromate (PDC). For aldehydes 8 and 9, the carboxylic acid of 3 was selectively reduced
using a mixture of sodium borohydride and boron trifluoride diethyl
etherate.[35] The resulting alcohol (7) was oxidized using PDC to yield the corresponding aldehyde
(8). Following hydrolysis of the methyl ester in 8 and subsequent conversion to the tert-butyl
ester, aldehyde 9 was obtained.[36]Aliphatic aldehydes 16 and 17 containing
trityl-protected amide functionalities were prepared from succinimide
and glutarimide, respectively (Scheme ). The cyclic amides were first trityl-protected and
subsequently ring-opened using potassium hydroxide. Reduction to the
corresponding alcohols and oxidization using PDC gave aldehydes 16 and 17.[37,38] In an analogous fashion,
aldehydes 22 and 23, both containing tert-butyl ester moieties, were prepared by ring opening
of succinic or glutaric anhydride with tert-butyl
alcohol to obtain mono-esters 18 and 19.[39,40] The carboxylic acid functionalities were reduced to alcohols 20 and 21 and then oxidized using PDC to yield
aldehydes 22 and 23.
Scheme 2
Synthetic Route for Aldehydes 16, 17, 22, and 23
Reagents and conditions: (a)
TrtCl, CH3CN, K2CO3, rt, 48 h (20–28%);
(b) KOH, EtOH, reflux, overnight (37–93%) (c) NaBH4, BF3·Et2O, THF, 0 °C to rt, 2 h
(64–81%); (d) PDC, CH2Cl2, rt, 2 h (65–78%);
(e) tert-butanol, 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide, Et3N, toluene, overnight
(25–93%).
Aldehydes 27 and 28, both containing
protected amino acid functionalities, were prepared starting from
the appropriately protected aspartic acid and glutamic acid building
blocks (Scheme ).
Conversion of the side chain carboxylates to their corresponding Weinreb
amides yielded intermediates 24 and 25.
Reduction of aspartate-derived 24 with DIBAL-H gave amino
acid aldehyde 27 in high yield. For the preparation of
aldehyde 28, a similar route was followed with the addition
of a second Boc-protection of intermediate 25 to avoid
an intramolecular cyclization side reaction.[24,41]With the necessary aldehyde building blocks in hand, assembly
of
the bisubstrate inhibitors was performed in each case starting from
commercially available 2′-3′-O-isopropylidene-6-aminomethyl-adenosine
(Scheme ). Using a
reliable reductive amination approach, aromatic aldehydes 6, 8, and 9, and commercially available
2-naphthaldehyde were each coupled to the protected adenosine species
to yield intermediates 29–32. These
intermediates were next connected with aliphatic aldehydes 16, 17, 22, 23, 27, and 28 or acetone via a second reductive amination
step to give the corresponding protected tertiary amine intermediates 33–56 (Scheme ). Global deprotection of the acid-labile
protecting groups was carried out in CH2Cl2/TFA
(1:1) with isopropylidene group cleavage facilitated by subsequent
addition of water. The crude products were purified by preparative
high-performance liquid chromatography (HPLC) to yield bisubstrate
analogues 1 and 57–60.
Inhibition Studies
The bisubstrate analogues were next
tested for their NNMT inhibitory activity using a method recently
developed in our group.[2] This assay employs
ultra-high-performance (UHP) hydrophilic liquid interaction chromatography
(HILIC) coupled to quadrupole time-of-flight mass spectrometry (Q-TOF-MS)
to rapidly and efficiently assess NNMT inhibition by analysis of the
formation of MNA. The NNMT inhibition of all compounds was initially
screened at a fixed concentration of 250 μM for all of the compounds.
In cases where at least 50% inhibition was detected at this concentration,
full inhibition curves were measured in triplicate to determine the
corresponding half-maximal inhibitory concentration (IC50) values. As reference compounds, we included the well-established
and general methyltransferase inhibitors sinefungin and SAH. In addition,
we also synthesized two recently described NNMT inhibitors, compound 2 and 6-(methylamino)-nicotinamide, following the procedures
described in the corresponding publications.[21,31] The structures of these reference compounds are provided in Figure .
Figure 2
Chemical structures of
the reference compounds used in NNMT inhibition
studies.
Chemical structures of
the reference compounds used in NNMT inhibition
studies.The results of the NNMT inhibition
studies are summarized in Table and clearly show
that only minor adjustments to the functional groups found in the
enzyme’s natural substrates are tolerated. Among the compounds
studied, the most potent inhibition was observed when the aliphatic
moiety corresponded to the same length in the amino acid side chain
as present in the methyldonorSAM. Notably, the preferred aromatic
moiety was found to be the naphthalene group, an apparent confirmation
of our hypothesis that increased π–π stacking can
lead to enhanced binding in the nicotinamide pocket. The bisubstrate
analogue containing both of these elements (compound 78), displayed the highest inhibitory activity against NNMT with an
IC50 of 1.41 μM. Interestingly, the amino acid and
naphthyl moieties were also found to independently enhance the activity
of the other inhibitors prepared. In this way, a suboptimal moiety
at one position can be compensated for—to an extent—by
including either the SAM amino acid motif or the naphthalene unit
at the other position. For example, bisubstrate analogues containing
the benzamide, benzoic acid, or methyl benzoate groups only show inhibitory
activity if they also contain the amino acid motif (compounds 1, 2, 66, and 72) with
IC50 values of 4.36–23.4 μM, respectively.
On the other hand, among the bisubstrate analogues lacking the amino
acid motif, inclusion of the naphthalene moiety (compounds 74–79) enhances NNMT inhibition, albeit with moderate
IC50 values in the range of 52.6–129.9 μM.
Table 1
Tabulated Overview of the Chemical
Structures and Inhibition Results of the Final Compounds and Reference
Compounds
Assays performed in triplicate on
at least six different inhibitor concentrations. Standard errors of
the mean reported.
Assays performed in triplicate on
at least six different inhibitor concentrations. Standard errors of
the mean reported.Other
notable findings were the results obtained with the reference
compounds. The general methyltransferase inhibitors, sinefungin and
SAH, showed inhibitory activities in line with those previously reported.[24] Interestingly, the 6-methylamino-NA compound,
recently described by Sanofi to be a submicromolar inhibitor,[21] gave an IC50 of 19.8 μM in
our assay. The recently published bisubstrate analogue 2 exhibited good activity (IC50 4.4 μM) on par with
published values.[31] Given the potent inhibition
measured for both compounds 2 and 78, we
also prepared and tested compound 81, an analogue of 78 bearing the same naphthyl moiety but with the amino acid
motif containing an additional methylene unit as in 2. Somewhat surprisingly, this linker elongation resulted in a complete
loss of inhibitory activity (IC50 > 250 μM).To gain insight into the selectivity of compound 78,
we also tested its activity against representative members of both
the arginine and lysine families of methyltransferases, PRMT1 and
NSD2, respectively. In both cases, compound 78 was tested
at a concentration of 50 μM and showed no significant inhibition
(>50% of the enzyme’s activity remained), see Table S1.
To further evaluate the binding interactions
of the most active bisubstrate
analogues with NNMT, isothermal titration calorimetry (ITC) studies
were performed. Compounds 1, 66, 72, and 78, all containing the amino acid moiety but with
varying aromatic substituents, were investigated. As illustrated in Figure , the dissociation
constants (Kd) measured for these compounds
track very well with the IC50 values measured in the in
vitro assay. Compounds 1 and 66 display
similar binding to NNMT with Kd values
of 36 and 25 μM, respectively, whereas compound 72 binds less tightly with a Kd of 124
μM. In good agreement with the results of the inhibition assay,
the most active inhibitor, compound 78, also displayed
the highest binding affinity for NNMT with a Kd of 5.6 μM. As expected, the inhibitors were each found
to bind the enzyme with a 1:1 stoichiometry.
Figure 3
ITC isotherms and thermograms
including thermodynamic binding parameters
measured for compounds 1, 66, 72, and 78 with human NNMT.
ITC isotherms and thermograms
including thermodynamic binding parameters
measured for compounds 1, 66, 72, and 78 with humanNNMT.
Modeling Studies
To further investigate the way in
which the inhibitors bind within the NNMT active site, modeling studies
were performed. Working from the available crystal structure of the
NNMT protein bounded to nicotinamide and SAH (PDB ID: 3ROD),[32] compounds 1, 2, 78, and 81 were modeled in the binding pocket. In an attempt
to explain the significant difference in the activity of 78 and 81, additional molecular dynamic simulations were
also performed for compounds 1, 2, 78, and 81. Although these simulations suggest
differences in the binding interaction of the compounds (Figure S1, Supporting Information), the calculated
binding energies for each are all very similar (Table S2, Supporting Information). In terms of their active
site orientations, compounds 1, 2, 78, and 81 are all predicted to position their
three branches roughly in the same regions of the active site; however,
their orientations and interactions are quite different.From
the modeling data, two distinct features are apparent. First, when
the chain linking the amino acid moiety is shorter (as in compounds 1 and 78), the formation of an intramolecular
hydrogen bond interaction was observed between the carboxylate of
the amino acid moiety and the protonated tertiary amine (see Figure ). This intramolecular
interaction is highly stable for compound 78 and less
stable for compound 1. This additional interaction reduces
the entropic energy of the ligand, thereby potentially stabilizing
its binding, and re-orients the amino acid part in the pocket, preventing
the polar interactions with neighboring residues (e.g., Y25, D61,
Y69, and T163) observed when the chain is longer (as present in compounds 2 and 81). This intramolecular hydrogen bond
may explain the difference in activity observed between compounds 78 and 81. The second distinct feature is the
tyrosine-rich environment around the naphthalene moiety of 78 compared to the nicotinamide unit of 1. The orientation
of the tyrosine residues surrounding this part of the molecule leads
to π–π stacking interactions with the naphthalene
and hints at an explanation for the strong inhibition and high affinity
of compound 78 with the NNMT protein (Figure ).
Figure 4
Modeling results for
compound 78 in the NNMT active
site (PDB ID: 3ROD). Molecular dynamics simulation indicates the presence of an intramolecular
hydrogen bond (2.7 Å, shown in cyan) specific to compound 78 (in green) that would be expected to reduce the entropic
energy of the ligand and potentially stabilize binding to NNMT (in
white). Proposed intermolecular hydrogen bond network (in yellow)
and π–π stacking interactions with Tyr residues
(in purple) stabilize compound 78 in the NNMT active
site (hydrogens omitted for clarity).
Modeling results for
compound 78 in the NNMT active
site (PDB ID: 3ROD). Molecular dynamics simulation indicates the presence of an intramolecular
hydrogen bond (2.7 Å, shown in cyan) specific to compound 78 (in green) that would be expected to reduce the entropic
energy of the ligand and potentially stabilize binding to NNMT (in
white). Proposed intermolecular hydrogen bond network (in yellow)
and π–π stacking interactions with Tyr residues
(in purple) stabilize compound 78 in the NNMT active
site (hydrogens omitted for clarity).
Cell-Based Assays
To evaluate the cellular activity
of the bisubstrate inhibitors, the compounds were tested for their
effect on cell proliferation in the humanoral cancer cell line, HSC-2.
We recently found that NNMT expression levels are high in this particular
cell line and may contribute to its proliferation and tumorigenic
capacity.[42] As shown in Figure , there were no significant
differences in the cell proliferation rate between HSC-2 cells treated
with dimethyl sulfoxide (DMSO) at 0.1% concentration and cells grown
with only the culture medium, at any time of each performed assay.
Upon treatment with the NNMT inhibitors, cell proliferation was not
significantly inhibited by compounds 1, 2, and 81 (Figure ). In contrast, relative to the DMSO control, treatment with
compound 78 led to a notable decrease in cell proliferation.
In particular, cell proliferation was significantly (p < 0.05) inhibited by compound 78 at 10 μM
(20% reduction), 50 μM (21% reduction), and 100 μM (27%
reduction) concentrations, 48 h after treatment. Interestingly, at
the longest 72 h time point taken, treatment with compound 78 leads to an even greater and significant (p <
0.01) decrease in cell proliferation (44% reduction), at the highest
concentration (100 μM) (Figure ).
Figure 5
Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide (MTT) cell viability assay on HSC-2 human oral cancer cells.
Only compound 78 showed a significant effect on cell
proliferation after 48 and 72 h.
Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide (MTT) cell viability assay on HSC-2humanoral cancer cells.
Only compound 78 showed a significant effect on cell
proliferation after 48 and 72 h.We next investigated the effect of compound 78 on
cellular NNMT activity by assessing its impact on MNA production in
the same HSC-2 cell line. Cells were treated with 100 μM of 78, and MNA levels were determined after 0, 1, 2, and 3 days.
Cells treated with compound 78 show a significant (p < 0.01) decrease in the levels of MNA (50% reduction)
compared to controls after 48 h. Interestingly, at 72 h an increase
in cellular MNA production was detected; however, the same effect
was also observed in the DMSO control (but not in the untreated control),
suggesting an effect attributable to longer term DMSO exposure. The
results of the cellular MNA analysis are presented in Figure S2, Supporting Information.
Conclusions
Building from our earlier findings with first reported ternary
bisubstrateNNMT inhibitor 1,[24] we designed and prepared a focused library of novel inhibitors to
provide new structure–activity insights. In doing so, various
structural motifs were investigated for their ability to enhance inhibitor
activity and binding within the NNMT active site. By probing the SAM
and NA binding pockets with different spacers and functional groups,
we found that the optimal ligands are the endogenous amino acid side
chain and the naphthalene moiety. Among the naphthalene-containing
bisubstrate analogues prepared, compound 78 showed the
most potent NNMT inhibition. In this way, the activity of our initial
NNMT inhibitor 1 (IC50 14.9 μM) was
improved 10-fold with compound 78, displaying an IC50 value of 1.41 μM. Notably, using an assay designed
to directly measure NNMT product formation, compound 78 was shown to be more potent than most other NNMT inhibitors reported
to date. ITC-based binding studies provided additional insights into
the affinity of the inhibitors for the enzyme with the measured Kd value following a trend similar to that observed
for the IC50 data obtained in the in vitro inhibition assays.
From modeling studies, the improved activity of compound 78 can be rationalized by the apparent presence of an intramolecular
hydrogen bonding interaction predisposing the compound to an active
conformation with lower entropic cost. In addition, the modeling indicates
that the naphthalene group in 78 is properly oriented
so as to benefit from additional π–π stacking interactions
with several tyrosine residues in the nicotinamide binding pocket
of the enzyme. The cellular data obtained for compound 78 show a significant inhibitory effect on cell proliferation in HSC-2oral cancer cells. These promising results provide important new insights
for the design and further optimization of potent NNMT inhibitors.
Experimental Procedures
General Procedures
All reagents employed were of American
Chemical Society grade or finer and were used without further purification
unless otherwise stated. For compound characterization, 1H NMR spectra were recorded at 400 MHz with chemical shifts reported
in parts per million downfield relative to tetramethylsilane, H2O (δ 4.79), CHCl3 (7.26), or DMSO (δ
2.50). 1H NMR data are reported in the following order:
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; and
m, multiplet), coupling constant (J) in hertz (Hz)
and the number of protons. Where appropriate, the multiplicity is
preceded by br, indicating that the signal was broad. 13C NMR spectra were recorded at 101 MHz with chemical shifts reported
relative to CDCl3 (δ 77.16), methanol (δ 49.00),
or DMSO (δ 39.52). The 13C NMR spectra of the compounds
recorded in D2O could not be referenced. High-resolution
mass spectrometry (HRMS) analysis was performed using a Q-TOF instrument.
Compounds 1,[24]2,[31]3,[34]7,[34]8,[36]9,[37]10,[43]12,[38]14,[38]16,[44]18,[40]19,[40]20,[45]21,[46]22,[47]23,[40]24,[41]25,[41]26,[48]27,[41] and 28(48) were prepared as previously described and had NMR spectra
and mass spectra consistent with the assigned structures. Purity was
confirmed to be ≥95% by analytical reversed-phase HPLC using
a Phenomenex Kinetex C18 column (5 μm, 250 × 4.6 mm2) eluted with a water–acetonitrile gradient moving
from 0 to 100% CH3CN (0.1% TFA) in 30 min. The compounds
were purified via preparative HPLC using a ReproSil-Pur C18-AQ column
(10 μm, 250 × 22 mm2) eluted with a water–acetonitrile
gradient moving from 0 to 50% CH3CN (0.1% TFA) over 60
min at a flow rate of 12.0 mL/min with UV detection at 214 and 254
nm.
Methyl 3-(Tritylcarbamoyl)benzoate (4)
Monomethyl isophthalate 3 (0.98 g, 5.4 mmol)
was refluxed in 10 mL of SOCl2 at 90 °C for about
1 h (until the reaction mixture was a clear solution). SOCl2 was removed under reduced pressure and the acid chloride intermediate
was redissolved in 15 mL of dry CH2Cl2 and transferred
to a cooled (ice bath) solution of tritylamine (1.41 g, 5.4 mmol)
and 2 mL of triethylamine in 30 mL of CH2Cl2. The reaction was stirred overnight under a N2 atmosphere,
allowing the mixture to warm to room temperature. After the reaction
was completed [monitored by thin-layer chromatography (TLC) (petroleum
ether/CH2Cl2 = 1:1)], the reaction mixture was
washed with water and brine, and the organic phase was dried over
Na2SO4 and concentrated. The crude product was
purified by column chromatography (petroleum ether/CH2Cl2 = 2:1) to give compound 4 as a white powder
(1.64 g, 72% yield). 1H NMR (400 MHz, CDCl3)
δ 8.45 (t, J = 1.6 Hz, 1H), 8.18 (m, 1H), 8.03
(m, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.41–7.26
(m, 15H), 3.94 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 166.3, 165.4, 144.5, 135.6, 132.5, 131.7, 130.6, 128.9,
128.7, 128.1, 128.1, 127.6, 127.2, 71.0, 52.4. HRMS [electrospray
ionization (ESI)]: calcd for C28H23NO3 [M + Na]+ 444.1576, found 444.1581.
3-(Hydroxymethyl)-N-tritylbenzamide (5)
Methyl 3-(tritylcarbamoyl)benzoate 4 (0.56 g, 1.33 mmol) was dissolved in dry CH2Cl2 (20 mL) under a N2 atmosphere, the reaction solution
was cooled down to −78 °C, and then diisobutylaluminum
hydride (DIBAL-H) (5.5 mL, 1.0 M hexane solution) was added slowly.
The reaction mixture was stirred at −78 °C for 2 h. Saturated
(sat.) aqueous (aq) NH4Cl (50 mL) was added slowly to quench
the reaction at −78 °C, followed by the addition of a
saturated Rochelle salt solution (100 mL). The mixture was stirred
at room temperature overnight, extracted with CH2Cl2, and the organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product
was purified by column chromatography (CH2Cl2/EtOAc = 9:1) to obtain 5 as a white powder (0.44 g,
85% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H), 7.78 (s, 1H), 7.75–7.71 (m,
1H), 7.47 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.36–7.18 (m, 15H), 5.26 (br, 1H), 4.54 (s,
2H). 13C NMR (101 MHz, DMSO-d6) δ 167.0, 145.3, 143.0, 135.5, 129.6, 128.9, 128.3, 127.9,
126.7, 126.5, 126.2, 79.6, 69.9, 69.9, 63.0. HRMS (ESI): calcd for
C27H23NO2 [2M + Na]+ 809.3355,
found 809.3359.
3-Formyl-N-tritylbenzamide
(6)
3-(Hydroxymethyl)-N-tritylbenzamide 5 (0.20 g, 0.51 mmol) and pyridinium dichromate (PDC) (0.23
g, 0.61
mmol) were placed in a 50 mL round bottom flask and 10 mL of dry CH2Cl2 was added under a N2 atmosphere
at room temperature. The reaction was stirred till completion, as
monitored by TLC (petroleum ether/CH2Cl2 = 5:1).
The mixture was filtered and the organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The resulting crude product was purified by
column chromatography (petroleum ether/CH2Cl2 = 9:1) to obtain 6 as a white powder (0.13 g, yield
64%). 1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.31 (s, 1H), 8.39 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 7.7 Hz, 1H),
7.68 (t, J = 7.7 Hz, 1H), 7.41–7.17 (m, 15H). 13C NMR (101 MHz, CDCl3) δ 191.5, 165.1, 144.4,
136.5, 136.2, 133.0, 132.5, 129.5, 128.6, 128.5, 128.1, 127.7, 127.3,
77.2, 71.1. HRMS (ESI): calcd for C27H21NO2 [2M + Na]+ 805.3042, found 805.3047.
N-(Triphenylmethyl)glutarimide (11)
Glutarimide
(2.8 g, 25 mmol), triphenylchloromethane (7.4
g, 25 mmol), and potassium carbonate (3.7 g, 25 mmol) were added to
100 mL of acetonitrile, and the mixture was stirred at room temperature
overnight. Saturated aqueous NaHCO3 (50 mL) was added,
and the mixture was extracted with EtOAc. The combined organic layers
were dried with anhydrous Na2SO4, and the solvent
was removed under reduced pressure. The crude product was purified
by column chromatography (petroleum ether/EtOAc = 4:1) to obtain 11 as a white powder (1.8 g, yield 20%). 1H NMR
(400 MHz, DMSO-d6) δ 7.45–7.35
(m, 6H), 7.20 (t, J = 7.8 Hz, 6H), 7.08 (t, J = 7.3 Hz, 3H), 2.66 (t, J = 6.4 Hz, 4H),
2.01 (p, J = 6.5 Hz, 2H). 13C NMR (101
MHz, CDCl3) δ 172.4, 143.4, 128.5, 127.3, 125.9,
35.5, 16.7. HRMS (ESI): calcd for C24H21NO2 [M + Na]+ 378.1470, found 378.1493.
5-Oxo-5-(tritylamino)pentanoic
Acid (13)
To 2.80 g of KOH dissolved in 50 mL
of ethanol was added N-tritylglutarimide 11 (1.00 g, 2.8 mmol),
and the mixture was refluxed for 48 h. The mixture was then concentrated
to dryness and redissolved in H2O. Acidification of the
basic solution with conc. HCl to pH = 2 and filtration of the product
gave compound 13 as a white powder (0.96 g, yield 91%). 1H NMR (400 MHz, CD3OD) δ 7.30–7.17
(m, 15H), 2.37 (t, J = 7.4 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.79–1.87 (m 2H). 13C
NMR (101 MHz, CD3OD) δ 175.5, 173.3, 144.6, 128.6,
127.3, 127.2, 126.7, 126.3, 35.2, 32.6, 20.7. HRMS (ESI): calcd for
C24H23NO3 [M + Na]+ 396.1576,
found 396.1573.
5-Hydroxy-N-tritylpentanamide
(15)
To a solution of 13 (2.60
g, 6.96 mmol) in
dry THF (60 mL) cooled to 0 °C was added NaBH(OAc)3 (0.28 g, 7.3 mmol). The solution was stirred until evolution of
H2 stopped, and BF3.OEt2 (1.1 mL,
8.8 mmol) was added dropwise. The reaction was stirred at room temperature
for 4 h. The reaction was quenched by adding 50 mL of H2O at 0 °C. The mixture was extracted with EtOAc, and the combined
organic layers were washed with sat. aqNa2CO3 and brine and dried over Na2SO4. The crude
product was purified by column chromatography (100% EtOAc) to give
compound 15 as a white powder (1.60 g, 64% yield). 1H NMR (400 MHz, CDCl3) δ 7.22–6.74
(m, 15H), 6.36 (br, 1H), 3.29–3.19 (br, 2H), 2.01 (t, J = 7.2 Hz, 2H), 1.46–1.36 (m, 2H), 1.24 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 171.9, 144.7, 128.6,
127.9, 127.0, 62.0, 37.0, 32.0, 21.4. HRMS (ESI): calcd for C24H25NO2 [M + Na]+ 382.1783,
found 382.1783.
5-Oxo-N-tritylpentanamide
(17)
5-Hydroxy-N-tritylpentanamide 15 (1.30
g, 3.6 mmol) and PDC (2.00 g, 5.4 mmol) were dissolved in 50 mL of
dry CH2Cl2 and stirred for 2 h under a N2 atmosphere at room temperature. The mixture was filtered,
and the organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure.
The crude product was purified by column chromatography (100% CH2Cl2) to give compound 17 as an off-white
powder (0.84 g, 65% yield). 1H NMR (400 MHz, CDCl3) δ 9.71 (s, 1H), 7.36–7.10 (m, 15H), 6.59 (s, 1H),
2.44 (t, J = 7.0 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.97–1.88 (m, 2H). 13C NMR (101
MHz, CDCl3) δ 202.0, 170.8, 144.6, 128.6, 127.9,
127.0, 70.5, 42.9, 36.1, 17.9. HRMS (ESI): calcd for C24H23NO2 [M + Na]+ 380.1626, found
380.1629.
To a solution of compound 33 (100 mg, 0.098 mmol) in 5 mL of CH2Cl2 was
added 5 mL of TFA, and the mixture was stirred at room temperature.
After 2 h, 2 mL of H2O was added, and the mixture was stirred
for 1 h at room temperature. The mixture was concentrated, and the
crude product was purified by preparative HPLC affording compound 57 as a white powder. 1H NMR (400 MHz, D2O) δ 8.46–8.06 (m, 2H), 7.87–7.26 (m, 4H), 6.08
(br, 1H), 4.75–4.36 (m, 4H), 4.27 (br, 1H), 3.84–3.27
(m, 4H), 2.38 (br, 2H), 2.10 (br, 2H). 13C NMR (101 MHz,
D2O) δ 177.5, 162.8, 162.5, 149.6, 143.8, 134.8,
134.1, 132.7, 129.6, 129.1, 128.3, 118.9, 117.6, 114.7, 90.4, 77.7,
73.6, 71.5, 57.9, 54.8, 31.8, 19.0. HRMS (ESI): calcd for C22H28N8O5 [M + H]+ 485.2261,
found 485.2265.
Following the procedure described
for compound 57, compound 80 (120 mg, 0.15
mmol) was deprotected to obtain compound 81 as a white
powder (58 mg, 63% yield). 1H NMR (600 MHz, D2O) δ 8.14 (br, 1H), 7.69–6.93 (m, 8H), 5.93 (br, 1H),
4.59–4.43 (m, 2H), 4.27 (br, 2H), 4.15–3.73 (m, 2H),
3.47 (m, 4H), 2.16–1.90 (m, 4H). 13C NMR (151 MHz,
D2O) δ 171.6, 162.9, 162.7, 148.6, 131.8 127.3, 127.1,
126.8, 119.2, 117.3, 115.3, 90.7, 78.0, 74.3, 71.3, 58.8, 52.3, 26.9,
19.4. HRMS (ESI): calcd for C26H31N7O5 [M + H]+ 522.2465, found 522.2468.
Inhibition
Studies
Expression and purification of full-length
wild-type NNMT protein (NNMTwt) were performed as previously described.[32] The purity of the enzyme was confirmed using
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
with Coomassie blue staining, and NNMT identity was confirmed using
SDS-PAGE and Western blotting. Catalytic activity of the recombinant
protein was evaluated with 1 unit of enzyme activity representing
the formation of 1 nmol of MNA/h of incubation at 37 °C. The
specific activity of the batch used in the inhibitory activity assays
was 18 665 units/mg of protein at a protein concentration of
0.56 mg/mL. NNMT was used at a final concentration of 100 nM diluted
in assay buffer (50 mM Tris buffer (pH 8.4) and 1 mM dithiothreitol).
The compounds were dissolved in DMSO and diluted with water to concentrations
ranging from 0.1 to 500 μM (DMSO was kept constant at 1.25%
final concentration). The compounds were incubated with the enzyme
for 10 min at 37 °C before initiating the reaction with a mixture
of NA and AdoMet at their KM values of
200 and 8.5 μM, respectively. The formation of MNA was measured
after 30 min at 37 °C. The reaction was quenched by addition
of 15 μL of the sample to 70 μL of acetonitrile containing
50 nM deutero-methylated nicotinamide as internal standard. The enzymatic
activity assays were performed using UHP-HILIC-MS/MS as previously
described with minor modifications.[24] The
UHP-HILIC-MS/MS system consisted of a binary ultra-HPLC system, consisting
of two LC-30AD pumps, a SIL30-ACmp auto-sampler, a CTO-20AC column
oven, and a DGU-20A5R degasser (all from Shimadzu, ’s-Hertogenbosch,
The Netherlands). Isocratic elution was performed after 1 μL
injections on a Waters Acquity BEHAmide HILIC column (3.0 ×
100 mm, 1.7 μm particle size, Waters, Milford), using water
containing 300 μM formic acid and 550 μM NH4OH (pH 9.2) at 40% v/v and acetonitrile at 60% v/v, with a runtime
of 3 min. Calibration samples were prepared using 75 μL of internal
standard d3-MNA at 50 nM in acetonitrile
and 25 μL of an aqueous solution of reference standard MNA with
concentrations ranging from 2500 to 1.221 nM. For detection, a Sciex
QTRAP 5500 triple quadrupole mass spectrometer, with Analyst 1.6.2
and MultiQuant 3.0.1 software (Sciex, Ontario, Canada), was used.
Settings used for the ionization source were as follows: curtain gas,
40 psi; collision gas, “medium”; ionspray voltage, 5000
V; temperature, 600 °C; ion source gas 1, 60 psi; and ion source
gas 2, 80 psi. Dwell times were 10 ms, and the entrance potential
was set to 10 V; specific parameters of the compounds can be found
in Table . The whole
eluate was transferred to the electrospray probe from 1.0 till 2.8
min using the MS diverter valve. Ratios of the sums of the MNA and d3-MNA transitions were calculated and plotted
versus concentration.
Table 2
Tuned MS/MS Parameters
for All Quantified
Componentsa,b
compound
Q1 (m/z)
Q3 (m/z)
DP
CE
CXP
MNA
137.101
94.0
136
27
12
92.0
136
29
12
78.0
136
35
10
MNA-d3
140.128
97.1
121
29
12
95.1
121
31
12
78.0
121
35
10
The entrance potential was set at
10 V for all compounds, dwell time was 10 ms.
The entrance potential was set at
10 V for all compounds, dwell time was 10 ms.Q1: quadrupole 1, Q3: quadrupole
3, z: charge, DP: declustering potential, CE: collision
energy, CXP: collision cell exit potential.
Isothermal Titration Calorimetry
Expression and purification
of full-length wild-type NNMT protein (NNMTwt) were performed as previously
described.[31] Isothermal titration calorimetry
(ITC) measurements were made at 25 °C on a MicroCal ITC200 Instrument
(Malvern Instruments) with 2 μL injections. NNMTwt was diluted
at 200 μM in ITC buffer [50 mM Tris (pH 8.0), 150 mM NaCl] supplemented
with 4% DMSO. Compounds were dissolved in DMSO at 50 mM and diluted
to 2 mM in ITC buffer with a final DMSO concentration of 4%. Binding
constants were calculated by fitting the data using the ITC data analysis
module in Origin 7.0 (OriginLab Corp.).Docking computations were performed
using Autodock 4.2.[49] Compounds 1, 2, 78, and 81 were docked
into the catalytic pocket of the structure taken from PDB ID: 3ROD.[32] Four molecular dynamic simulations were performed with
GROMACS 2018.2[50] using the AMBER03 force
field.[51] Each structure was immersed in
a cubic box using TIP3P water molecules[52] and neutralized with counter ions. A production step of 250 ns was
carried out using the Parrinello–Rahman algorithm[53] for temperature and pressure control, with coupling
constants of T = 0.1 ps and P =
2.0 ps, for compounds 1, 2, and 81 and extended to 450 ns for compound 78, to reach equilibrium
of the system. Coordinates were saved every 200 ps, and the protein/ligand
binding energy was estimated using g_mmpbsa calculations[54,55] on the last 50 ns of each trajectory. The conformation of minimal
energy in these 50 ns was extracted from the simulations and minimized
to represent the interactions between the ligands and NNMT protein.
Enzyme Assay for Selectivity
Methyltransferase inhibition
assays were performed as described[56] by
using commercially available chemiluminescent assay kits for PRMT1
and NSD2 (purchased from BPS Bioscience). The enzymatic reactions
were conducted in duplicate at room temperature for 1 h (PRMT1) or
2 h (NSD2) in substrate-coated well plates at a final reaction volume
of 50 μL containing the manufacturer’s proprietary assay
buffer, AdoMet (at a concentration of 5 times the respective Km value for each enzyme), the methyltransferase
enzyme: PRMT1 (100 ng per reaction) and NSD2 (500 ng per reaction),
and inhibitor 78. Before addition of AdoMet, the enzyme
was first incubated with the inhibitor for 15 min at room temperature.
Positive controls were performed in the absence of the inhibitor using
water to keep the final volume consistent. Blanks and substrate controls
were performed in the absence of the enzyme and AdoMet, respectively.
Following the enzymatic reactions, 100 μL of primary antibody
(recognizing the respective immobilized methylated product) was added
to each well, and the plate was incubated at room temperature for
an additional 1 h. Then, 100 μL of secondary horseradish peroxidase
(HRP)-conjugated antibody was added to each well, and the plate was
incubated at room temperature for additional 30 min. Finally, 100
μL of an HRP substrate mixture was added to the wells, and the
luminescence was measured directly by using a standard microplate
reader. The luminescence data were normalized with the positive controls
defined as 100% activity and blank defined as 0%.
Cell Culture
and Treatment with Compounds
The HSC-2humanoral cancer cell line was purchased from the American Type Culture
Collection (Rockville, MD) and cultured in Dulbecco’s modified
Eagle’s medium/F12 medium, supplemented with 10% fetal bovine
serum and 50 μg/mL gentamicin, at 37 °C in a humidified
5% CO2 incubator. Compounds 1, 2, 78, and 81 were tested for their inhibitory
effect on cell proliferation of HSC-2 cells. Each compound was dissolved
in DMSO at 100 mM concentration. This stock solution was then diluted
in culture medium to final concentration values ranging between 1
and 100 μM. For each sample, DMSO was kept constant at 0.1%
final concentration.The day before starting treatment, cells
were seeded in 96-well plates, at a density of 1 × 103 cells/well. Cells were allowed to attach overnight and then incubated
with compounds at different final concentrations, or with only DMSO,
for 24, 48, and 72 h. All experiments were performed in triplicate.
MTT Assay
Cell proliferation was determined using a
colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT). The MTT assay measures the conversion of
MTT to insoluble formazan by dehydrogenase enzymes of the intact mitochondria
of living cells. HSC-2 cell proliferation was evaluated by measuring
the conversion of the tetrazolium saltMTT to formazan crystals upon
treatment with compounds or only DMSO for 24, 48, and 72 h. Briefly,
cells were incubated for 2 h at 37 °C with 100 μL of fresh
culture medium containing 5 μL of the MTT reagent (5 mg/mL in
PBS). The medium was removed, and 200 μL of isopropanol were
added. The amount of formazan crystals formed correlated directly
with the number of viable cells. The reaction product was quantified
by measuring the absorbance at 540 nm using an enzyme-linked immunosorbent
assay plate reader. Experiments were repeated three times. Results
were expressed as percentage of the control (control equals 100% and
corresponds to the absorbance value of each sample at time zero) and
presented as mean values ± standard deviation of three independent
experiments performed in triplicate. Data were analyzed using GraphPad
Prism software (GraphPad Software, San Diego, CA). Significant differences
between groups were determined using the one-way analysis of variance.
A p value <0.05 was considered as statistically
significant.
Quantitative Measurements of MNA Levels in
Cultured Cells
The analysis was performed as previously described[57] with minor modifications. Cellular MNA levels
were determined
using the same UHP-HILIC-MS/MS employed for the inhibition studies,
as described above. To determine the effect of compound 78 on NNMT activity in the HSC-2 oral cancer cell line, used cells
were treated with 78 at 100 μM (final DMSO content
0.1%) and incubated for 24, 48, or 72 h. The day prior to starting
treatment, cells were seeded in 6-well plates, at a density of 3 ×
104 cells/well. Cells were allowed to attach overnight
and were then incubated with compound 78. All experiments
were performed in duplicate. Following treatment, medium was removed,
and adherent cells were trypsinized and harvested by centrifugation
at 1000g for 3 min at 4 °C. Supernatant was
then discarded and cell pellets were stored at −80 °C
until further use. The extraction of MNA from the cell pellets was
performed as previously described.[58] Briefly,
100 μL of acetonitrile containing 50 nM d3-MNA (as internal control) was added to the cell pellets,
and the cells were lyzed for 20 min at room temperature with mild
shaking. Then, 50 μL of purified water was added, followed by
mixing, and the resulting cell debris was centrifuged for 10 min at
5000 rpm. Then, 100 μL of the resulting supernatant was transferred
to a 96-well plate and analyzed for MNA content.
Authors: Richard B Parsons; Stuart W Smith; Rosemary H Waring; Adrian C Williams; David B Ramsden Journal: Neurosci Lett Date: 2003-05-15 Impact factor: 3.046
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Figure 1
Scheme 1
Scheme 3
Scheme 4
Scheme 5
Scheme 2
Figure 2
Figure 3
Figure 4
Figure 5