Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for target engagement challenges in situ

Article abstract of DOI:10.1016/j.bmcl.2018.04.017

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide using S-adenosyl-L-methionine (SAM) as a methyl donor and, through doing so, can modulate cellular methylation potential to impact diverse epigenetic processes. NNMT has been implicated in a range of diseases, including cancer and metabolic disorders. Potent, selective, and cell-active inhibitors would constitute valuable probes to study the biological functions and therapeutic potential of NNMT. We previously reported the discovery of electrophilic small molecules that inhibit NNMT by reacting with an active-site cysteine residue in the SAM-binding pocket. Here, we have used activity-based protein profiling (ABPP)-guided medicinal chemistry to optimize the potency and selectivity of NNMT inhibitors, culminating in the discovery of multiple alpha-chloroacetamide (αCA) compounds with sub-μM IC₅₀ values in vitro and excellent proteomic selectivity in cell lysates. However, these compounds showed much weaker inhibition of NNMT in cells, a feature that was not shared by off-targets of the αCAs. Our results show the potential for developing potent and selective covalent inhibitors of NNMT, but also highlight challenges that may be faced in targeting this enzyme in cellular systems.

Full text of DOI:10.1016/j.bmcl.2018.04.017

Accepted Manuscript
Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evi-
dence for target engagement challenges in situ
Hsin-Yu Lee, Radu M. Suciu, Benjamin D. Horning, Ekaterina V. Vinogradova,
Olesya A. Ulanovskaya, Benjamin F. Cravatt
PII:
S0960-894X(18)30324-X
https://doi.org/10.1016/j.bmcl.2018.04.017
BMCL 25764
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2018
6 April 2018
7 April 2018
Please cite this article as: Lee, H-Y., Suciu, R.M., Horning, B.D., Vinogradova, E.V., Ulanovskaya, O.A., Cravatt,
B.F., Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for target engagement
challenges in situ, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.04.017
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Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for
target engagement challenges in situ
Hsin-Yu Lee, Radu M. Suciu, Benjamin D. Horning, Ekaterina V. Vinogradova, Olesya A.
Ulanovskaya, Benjamin F. Cravatt*
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92307,
United States
* Corresponding author. Email: Benjamin F. Cravatt (cravatt@scripps.edu)
Abstract
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide using S-
adenosyl-L-methionine (SAM) as a methyl donor and, through doing so, can modulate cellular
methylation potential to impact diverse epigenetic processes. NNMT has been implicated in a
range of diseases, including cancer and metabolic disorders. Potent, selective, and cell-active
inhibitors would constitute valuable probes to study the biological functions and therapeutic
potential of NNMT. We previously reported the discovery of electrophilic small molecules that
inhibit NNMT by reacting with an active-site cysteine residue in the SAM-binding pocket. Here,
we have used activity-based protein profiling (ABPP)-guided medicinal chemistry to optimize the
potency and selectivity of NNMT inhibitors, culminating in the discovery of multiple alpha-
chloroacetamide (αCA) compounds with sub-µM IC50 values in vitro and excellent proteomic
selectivity in cell lysates. However, these compounds showed much weaker inhibition of NNMT
in cells, a feature that was not shared by off-targets of the αCAs. Our results show the potential
for developing potent and selective covalent inhibitors of NNMT, but also highlight challenges
that may be faced in targeting this enzyme in cellular systems.
1

Nicotinamide N-methyltransferase (NNMT) is a cytoplasmic enzyme that catalyzes the
N-methylation of nicotinamide and other pyridine compounds using S-adenosyl-L-methionine
(SAM) as a methyl donor. NNMT is also involved in the metabolism of drugs and xenobiotic
compounds.1-2 NNMT is highly expressed in liver and to lesser extents in other tissues.2 NNMT
is also elevated in numerous cancers, where it supports migration, invasion, proliferation, and in
vivo tumor growth.3-13 How NNMT is regulated in cancer remains incompletely understood, but
one process for induction of the enzyme may be the epithelial-to-mesenchymal transition
(EMT).14-16 NNMT has also been linked to metabolic disorders,17-20 where knockdown of the
enzyme in mice inhibits body weight gain, reduces fat mass, and prevents diet-induced
obesity.20 In both cancer21 and metabolic systems,20 as well as in stem cells,22 NNMT appears to
regulate not only nicotinamide metabolism, but also the broader methylation potential and
epigenetic state of cells, likely through altering the balance of SAM and S-adenosyl
homocysteine (SAH) concentrations21.
Virtually all the functional studies performed on NNMT to date have used molecular
biology methods to perturb expression of the enzyme. Selective and cell-active inhibitors of
NNMT would offer valuable, complementary tools to investigate the biological functions and
therapeutic potential of the enzyme. While much effort has been dedicated over the past decade
to the generation of protein methyltransferase inhibitors, including compounds that have entered
into clinical development,23-24 there are few selective and cell-active inhibitors of metabolic
methyltransferases like NNMT. Examples of first-generation NNMT inhibitors include
quinolinium25 and bi-substrate transition-state26 analogues that display low-µM inhibition against
purified NNMT, as well as more promiscuous methyltransferase inhibitors such as sinefungin.27
Assay development is another challenge facing the pursuit of NNMT inhibitors. Recently, a
higher-throughput substrate assay using ultra-high-performance hydrophilic interaction
chromatography have been introduced27, and our lab has described a complementary approach
that uses SAH-derived chemical proteomic probes to profile the activity of NNMT and other
methyltransferases in native biological systems (Supplementary Figure 1).28 Using SAH
probes, we identified an alpha-chloroacetamide (αCA) compound RS004 (Figure 1A) that
inhibits NNMT activity with an IC50 value of 10 µM.28 We found that RS004 inhibits NNMT by
covalently modifying a non-catalytic active site cysteine residue (C165; Figure 1B) and displays
good selectivity for NNMT over other metabolic and protein methyltransferases in human cancer
cell proteomes.28
2

A.
B.
O
F3C
N
N
H
Cl
HN
Cl
O
C165
RS004
SAH
Figure 1. (A) Structure of RS004, a previously identified inhibitor of NNMT.28 (B) Crystal
structure of SAH bound NNMT35 showing the location of cysteine C165 – the site of covalent
modification by RS004.
Here, we set out to explore the broader potential of αCA compounds as NNMT inhibitors
by synthesizing and testing a series of RS004 analogues. We first synthesized RS004
analogues with modifications to the N-alkyl substituent of the αCA group (Figure 2A,
Supplementary Figure 2A, and Supplementary Scheme 1) and tested these compounds for
activity against endogenous NNMT in soluble proteomic lysates of the human renal cell
carcinoma line 786O. The activity of NNMT can be conveniently evaluated in this cell line, which
expresses high levels of NNMT,28 by using an activity-based protein profiling (ABPP) protocol
that involves treating lysates with a fluorescent, photoreactive SAH-based activity probe
(Supplementary Figure 1), followed by SDS-PAGE and in-gel fluorescence scanning, where
an active inhibitor is found to compete for binding with the SAH probe to NNMT and reduce the
fluorescent labeling signal for the protein.28 This gel-based ABPP assay revealed that HS58A-
C2 bearing an N-isopentyl group showed improved potency against NNMT, with near-complete
blockade of SAH-probe labeling observed at 2 µM (Figure 2B). The corresponding N-isobutyl
analog HS193, as well as other N-alkyl analogues, were less active compared to HS58A-C2
(Figure 2B and Supplementary Figure 2B), and many additional N-substitutions resulted in
compounds with negligible inhibition of NNMT at the tested concentrations of 2 or 20 µM
(Figure 2B and Supplementary Figure 2B). Among these inactive analogues, we selected
HS183 as a representative negative control compound for future studies. We also explored
changes to the CF3 group of the aniline ring of RS004, but these modifications uniformly
resulted in a loss of NNMT inhibition (Figure 2 and Supplementary Scheme 2). Finally, as
expected, conversion of the αCA of RS004 to a 3-chloropropanamide produced a compound
HS177 that did not inhibit NNMT.
3

O
A.
F3C
N
Me
n
N
H
N
R =
H
Ph
tBu
Cl
N
Me
F
Me
N
HS 193
n = 1
Cl
R
HS178
HS77
HS81e
HS95
HS183
RS004
O
HS58A-C2
n = 2
O
O
O
HN
N
F3C
N
HN
N
R
N
H
N
H
R =
Cl
N
Cl
HN
HN
Cl
Cl
Cl
O
O
Cl
HS125
HS126
HS145-2
HS177
B.
µM
kDa
75
50
37
NNMT
25
Figure 2. Structure-activity analysis of αCA inhibitors of NNMT. (A), Structures of representative
αCA compounds tested for NNMT inhibition. (B) Evaluating αCA compounds for NNMT
inhibition in 786O cell proteomic lysates using a SAH-rhodamine (SAH-Rh) probe and gel-based
ABPP.
Having identified the N-isopentyl substitution of the αCA as a productive group for NNMT
interaction, we next synthesized a series of analogues of HS58A-C2 with modifications to the N-
aryl group of the distal amide (Figure 3A, Supplementary Figures 3A and 4A, and
Supplementary Scheme 3). These changes generally did not further improve potency of
NNMT inhibition, although a few analogues (e.g., HS312, HS300, HS257) showed comparable
potency to HS58A-C2 (Figure 3B and Supplementary Figures 3B and 4B).
4

Cl
A.
N
H
N
N
H
O
N
H
N
H
N
H
CF3
R =
F
O
NO2
F3C
R
HS238
HS270
HS300
HS312
HS257
HS308
N
Cl
N
N
N
O
N
NHBoc
N
H
N
H
N
H
N
H
N
H
N
H
Cl
HS58A-C2
HS244
HS250-2
HS302
HS245
HS249
B.
µM
kDa
75
50
37
NNMT
25
Figure 3. Further structure-activity analysis of lead NNMT inhibitor HS58A-C2. (A) Structures of
representative analogs of HS58A-C2 tested for NNMT inhibition. (B) Evaluating αCA
compounds for NNMT inhibition in 786O cell proteomic lysates using a SAH-rhodamine (SAH-
Rh) probe and gel-based ABPP.
A broader concentration-dependent inhibition profile confirmed similar potencies for
HS58A-C and HS312 (Figure 4A). We next measured IC50 values for NNMT inhibition by both
compounds using the SAH activity probe and a nicotinamide substrate assay (Supplementary
Figure 5), and both assays furnished similar IC50 values of ~200-400 nM for HS58A-C and
~150-350 nM for HS312 (Figure 4B). Importantly, HS58A-C2 and HS312 displayed
concentration-dependent inhibition of wild type-NNMT, but not the C165A mutant of NNMT as
measured by a nicotinamide substrate assay, supporting that the inhibitory mechanism occurs
by covalent reactivity with C165 (Figure 4C). We also determined kobs/[I] values for HS58A-C
5

and HS312 of 200 82 M-1s-1 and 285 59 M-1s-1, respectively, using the SAH activity probe
assay (Supplementary Figure 6), and measured IC50 values for several other analogues of
these inhibitors (Supplementary Figure 7). Finally, we confirmed that the control compound
HS183 showed very weak inhibition of NNMT measured using either the SAH activity probe or a
nicotinamide substrate assay (IC50 values > 15 µM; Figure 4B).
.
Figure 4. Potency of lead NNMT inhibitors HS58A-C2 and HS312. (A) Concentration-
dependent inhibition of NNMT by HS58A-C2 and HS312 as measured by gel-based ABPP of
786O cell proteome. (B) In vitro IC50 values for HS58A-C2 and HS312 and inactive control
HS183, as measured in 786O proteome using gel-based ABPP (black) or a nicotinamide
substrate assay (green). (C) HS58A-C2 and HS312 inhibit NNMT activity in a concentration-
dependent manner for WT-NNMT, but not C165A mutant NNMT as measured with a
nicotinamide substrate assay. Data represent average values +/- standard deviation, n = 4 per
group.
6

We next investigated the selectivity of NNMT inhibitors HS58A-C2 and HS312 using the
quantitative chemical proteomic method isoTOP-ABPP (isotopic tandem orthogonal proteolysis-
activity-based protein profiling), which can survey reactivity and drug engagement for thousands
of cysteines in parallel in native biological systems.29-31 786O cell lysates were treated with
DMSO or inhibitor (1 or 5 µM, 1 h) and then exposed to the broad-spectrum cysteine-reactive
probe iodoacetamide (IA)-alkyne. Proteins bearing IA-alkyne-modified cysteine residues from
DMSO- and inhibitor-treated samples were then conjugated by copper-catalyzed azide-alkyne
cycloaddition (CuAAC) to isotopically differentiated azide-biotin tags (heavy and light,
respectively) bearing TEV protease-cleavable linkers. The samples were combined, precipitated,
enriched by streptavidin beads, digested sequentially on-bead with trypsin and TEV protease,
and the released peptides analyzed by multidimensional liquid chromatography-mass
spectrometry (LC/LC-MS/MS). Cysteine residues targeted by HS58A-C2 and HS312 show
substantially altered MS1 chromatographic peak ratios (R values ≥ 4) for heavy/light samples.
These chemical proteomic data confirmed strong engagement of C165 of NNMT by both
inhibitors at 5 µM (Figure 5A-C and Supplementary Table 1) and good overall proteomic
selectivity across the > 1200 cysteine residues quantified per isoTOP-ABPP experiment in
786O cell lysates. HS58A-C2 displayed fewer off-targets than HS312 (Figure 5A, B), and at
least some of these off-targets (e.g., HMOX2) were also engaged by the inactive control
compound HS183, which did not interact with C165 of NNMT (Figure 5D and Supplementary
Table 1). We also noted that C165 of NNMT was more strongly engaged by HS312 than
HS58A-C2 when tested at 1 µM (Supplementary Figure 8 and Supplementary Table 1).
7

A.
B.
C75 C115
C50
C165
NNMT$
GSTO1
CKAP4
HMOX2
RH/L
1.3
1.0
1.3
20
NNMT8cysteine
C.
D.
NNMT$
GSTO1
ALDH1A1
HMOX2
REEP5
NT5DC2
SCP2
PMPCA
ZYX
CKAP4
PPME1
ALDH1A1
HMOX2
TXNDC17
NNMT
Figure 5. Chemical proteomic analysis of NNMT inhibitors HS58A-C2 and HS312 in 786O
proteomic lysates. A, Representative MS1 peptide peaks for quantified cysteines in NNMT from
competitive isoTOP-ABPP experiments, showing blockade of C165 (R value = 20 for DMSO
(H)/inhibitor (L)) by HS58A-C2 (5 µM, 1 h), but not other cysteines (R values ~ 1) in NNMT. B-D,
Representative isoTOP-ABPP plots for HS58A-C2 (B), HS312 (C), and HS183 (D) (5 µM
inhibitor, 1 h) for cysteines quantified in the soluble proteome of 786O cells. Dashed lines
designate R values ≥ 4 (DMSO/compound), which was used as a cutoff to define cysteines
liganded by the indicated compounds. Data are from individual experiments representative of at
least three biological replicates.
Encouraged by the strong and selective engagement of NNMT by HS312 and HS58A-
C2 in 786O cell lysates, we evaluated the compounds for in situ activity by treating 786O cells
with HS58A-C2 or HS312 (5 µM, 4 h) followed by cell lysis and isoTOP-ABPP. Surprisingly,
however, we observed no engagement of C165 of NNMT in these in situ experiments, despite
substantial engagement of several off-targets (e.g., GSTO1, HMOX2, TXNDC17) (Figure 6 and
Supplementary Table 1). These data support that the αCA compounds are cell-permeable, but
8

for as of yet unknown reasons show limited engagement of NNMT compared to other protein
targets in situ.
HS312
Figure 6. Heat map comparing the in vitro and in situ reactivity profiles of cysteines targeted by
HS312. Cysteines are organized from top to bottom based on decreasing R values
(DMSO/HS312) in isoTOP-ABPP experiments performed in 786O cell proteome treated in vitro
with 5 µM of HS312 for 1 h. Note that, among the most HS312-sensitive cysteines (R > 10),
C165 of NNMT shows a unique loss of reactivity with HS312 in situ. Similar results were
obtained for HS58A-C2 (see Supplementary Table 1).
In summary, by screening a series of αCA compounds with an ABPP platform directed
to enzymes from the methyltransferase class, we have identified potent (sub–µM) and selective
inhibitors of NNMT. Evidence was provided to support that these compounds – HS58A-C2 and
9

HS312 – inhibit NNMT by covalently modifying an active-site cysteine (C165) that is located in
the SAM/SAH-binding pocket of the enzyme. The reduced NNMT inhibitory activity observed for
HS58A-C2 and HS312 in cancer cells is, on the surface, perplexing, given that these
compounds effectively engaged other protein targets in situ. We speculate that special features
of NNMT, such as interactions with endogenous SAM/SAH metabolites, may underlie the
observed disconnect between the in vitro and in situ activity of C165-directed covalent inhibitors.
This conundrum is reminiscent of challenges that have been described for the development of
cell-active inhibitors of kinases that display strong binding affinity to ATP.32-34 Consistent with
this hypothesis, we found that the in vitro potency of NNMT inhibition by HS312 was reduced in
the presence of increasing concentrations of SAM (10-160 µM; Supplementary Fig. 9). We are
hopeful that continued improvements in the potency and selectivity of NNMT inhibitors will
furnish compounds that can achieve sufficient in situ engagement to study the role that this
enzyme plays in regulating the methylation potential and epigenetic state of human cells.
Acknowledgements. This work was supported by the NIH (CA132630). H.-Y.L. was supported
by a Talent Development Program postdoctoral fellowship between Academia Sinica and The
Scripps Research Institute. E.V.V. was supported by the Life Sciences Research Foundation
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Highlights
•
•
•
α-Chloroacetamides (αCAs) serve as irreversible inhibitors of NNMT
αCA inhibitors show good proteomic selectivity for NNMT in vitro
The potency of αCA inhibitors of NNMT is greatly reduced in cells
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