Noncompetitive inhibition of indolethylamine-N-methyltransferase by N, N-dimethyltryptamine and N, N-dimethylaminopropyltryptamine

Article abstract of DOI:10.1021/bi500175p

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N′,N′-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a K_i of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N- methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were ～6.34 and ～7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

Full text of DOI:10.1021/bi500175p

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Article
Noncompetitive Inhibition of Indolethylamine N-Methyltransferase by
N,N Dimethyltryptamine (DMT) and N,N-Dimethylaminopropyltryptamine
Uyen B Chu, Sevahn Kayaneh Vorperian, Kenneth A Satyshur, Kelsey Eickstaedt,
Nicholas V Cozzi, Timur Mavlyutov, Abdol R. Hajipour, and Arnold E. Ruoho
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi500175p • Publication Date (Web): 14 Apr 2014
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Noncompetitive Inhibition of Indolethylamine N-Methyltransferase by N,N
Dimethyltryptamine (DMT) and N,N-Dimethylaminopropyltryptamine
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Uyen B. Chu1,†, Sevahn K. Vorperian1,2,†, Kenneth Satyshur3, Kelsey Eickstaedt1, Nicholas V.
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5,
1,§
§
Cozzi , Timur Mavlyutov , Abdol R. Hajipour , and Arnold E. Ruoho
1Department of Neuroscience, University of Wisconsin School of Medicine and Public Health,
1300 University Avenue, Madison, WI 53706
2West High School, 30 Ash St., Madison, WI 53726
3University of Wisconsin Carbone Cancer Center, Small Molecule Screening and Synthesis
Facility, Wisconsin Institutes for Medical Research, 1111 Highland Avenue, Madison, WI 53705
4Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and
Public Health, 1300 University Avenue, Madison, WI 53706
5Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology,
Isfahan 84156, Iran
†These authors contributed equally to the work in this manuscript
§Address correspondence to Arnold. E. Ruoho, PhD, Department of Neuroscience, University of
Wisconsin School of Medicine and Public Health, 1300 University Avenue, Madison, Wisconsin
53706; Tel: (608) 263ꢀ5382; Fax: (608) 265ꢀ5512; Email: aeruoho@wisc.edu
§For the PAT and PDAT chemistry, address correspondence to Abdol R. Hajipour, PhD,
Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology,
Isfahan 84156, Iran. Email: arhajipour@wisc.edu
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Running title: Noncompetitive Inhibition of INMT by DMT and PDAT
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Research Funding: This research was supported by National Institute of Health Grant #
NS075820 (to A.E.R.), the PhRMA Foundation (to U.B.C) and the Science Research Intern
Program, Madison Metropolitan School District, 545 West Dayton Street, Madison, WI 53703
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Abbreviations:
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DMT, N,NꢀDimethyltryptamine
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hINMT, human IndolethylamineꢀNꢀmethyltransferase
hNNMT, human NicotinamideꢀNꢀmethyltransferase
hPNMT, human PhenethanolamineꢀNꢀmethyltransferase
PAT, Propyl Amino Tryptamine or Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)propaneꢀ1,3ꢀdiamine
PDAT, Propyl Dimethyl Amino Tryptamine or Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀN',N'ꢀ
dimethylpropaneꢀ1,3ꢀdiamine
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rabINMT, rabbit IndolethylamineꢀNꢀmethyltransferase
SAH, SꢀadenosylꢀLꢀhomocysteine
SAM, SꢀadenosylꢀLꢀmethionine
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Abstract
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IndolethylamineꢀNꢀmethyltransferase (INMT) is a Class 1 transmethylation enzyme
known for its production of N,Nꢀdimethyltryptamine (DMT), a hallucinogen with affinity for
various serotonergic, adrenergic, histaminergic, dopaminergic, and sigmaꢀ1 receptors. DMT is
produced via the action of INMT on the endogenous substrates tryptamine and SꢀadenosylꢀLꢀ
methionine (SAM). The biological, biochemical, and selective small molecule regulation of
INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit
lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were
determined. After applying MichaelisꢀMenten and LineweaverꢀBurk analyses for inhibition,
DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against
tryptamine. The novel tryptamine derivative, Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀN',N'ꢀdimethylpropaneꢀ
1,3ꢀdiamine (Propyl Dimethyl Amino Tryptamine or PDAT), was shown to inhibit rabINMT by
a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 ꢁM. No
inhibition was observed by PDAT at 2 mM when tested against structurally similar Class 1
methyltransferases, such as human phenylethanolamine Nꢀmethyltransferase (hPNMT) and
human nicotinamide Nꢀmethyltransferase (hNNMT), indicating selectivity for INMT. The
demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an
inhibitory allosteric site. In silico analyses using the computer modeling software, Autodock and
the rabINMT sequence threaded onto the human INMT (hINMT) structure (PDB code 2A14),
identified an Nꢀterminal HelixꢀLoopꢀHelix nonactive site binding region of the enzyme. The
binding energies for DMT and PDAT to this region of rabINMT, as determined by Autodock,
were ꢀ6.34 kcal/mol and ꢀ7.58 kcal/mol, respectively. Assessment of the allosteric control of
INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.
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IndolethylamineꢀNꢀmethyltransferase (INMT), as a transmethylation enzyme, transfers
one or more methyl groups from SꢀadenosylꢀLꢀmethionine (SAM) to appropriate amino group
acceptors on indoleꢀcontaining compounds. The activity of this enzyme was first reported in
rabbit lung homogenate by Julius Axelrod (1) and rabbit and human INMT were eventually
cloned and sequenced by Thompson and collegues in the late 1990s (2, 3). INMT is a member
of a large family of Nꢀmethyltransferases that can methylate a variety of small molecule
acceptors such as tryptamine (2), serotonin, and other endogenous indoleꢀcontaining compounds
(4-7). This enzyme is widely distributed in mammalian tissues including the lungs, adrenal
gland, thyroid, placenta, heart, pancreas, lymph nodes (2-6), retina, pineal gland, and spinal cord
ventral horn motoneurons (8, 9). Tryptamine, the substrate normally associated with INMT, is
derived from the in vivo decarboxylation of tryptophan. Transmethylation produces Nꢀ
methyltryptamine (MMT) and N,Nꢀdimethyltryptamine (DMT). DMT is found in trace amounts
in humans and other animals (6, 10-17). For a thorough review on the synthesis of DMT, its
regulation and metabolism, refer to Barker et al. (7). Recently, Barker and colleagues, using
microdialysis techniques, detected DMT in realꢀtime in the pineal glands of living rats (18).
The precise role of INMT in biological systems is currently unresolved. However, several
observations indicate the pleiotropic importance of the INMT gene. For example, expression of
the INMT gene has been shown to be downꢀregulated in prostate cancer (19) and lung cancer
(20) implying a role for INMT in inhibiting tumor progression. Further, successful implantation
of the embryo in the mouse appears to be supported by the presence of the INMT/TEMT gene
(together with several other genes) (21). INMT enzyme activity is elevated in the rabbit fetal
lung (22). Refer further to a recent review of the possible peripheral (in addition to CNS) role(s)
of INMT/DMT in mammalian systems involving the sigmaꢀ1 receptor by Frecska and colleagues
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(23).
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Increased INMT activity had been suggested to be involved in schizophrenia and stressꢀ
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related psychoses in humans and this served as the basis for early work on the proposed utility of
inhibiting in vivo INMT enzyme activity to treat schizophrenia (24-26). Further, the INMT
product DMT, when ingested, produces a psychological state characterized by colorful visual
illusions, altered time and space perceptions, and changes in body image (27). Because of these
known psychoactive effects of DMT, the proposition that INMT activity and DMT production
may be involved in producing exceptional mental states may be worth investigating.
The psychoactive effects of DMT are mediated through various mechanisms including
binding to and activating serotonin receptors (28-30), exhibiting substrateꢀlike behavior at
serotonin and vesicular uptake transporters (31, 32), and inhibiting monoamine oxidase enzymes
(31-34). The sigmaꢀ1 receptor is the latest identified receptor target for DMT, where it binds at
low micromolar concentrations, inhibits voltageꢀactivated sodium ion channels via sigmaꢀ1
receptor interactions at higher concentrations, and induces a hypermobility response in wildꢀtype
mice that is abolished in sigmaꢀ1 receptor knockout mice (35). INMT has been shown to coꢀ
localize with the sigmaꢀ1 receptors in primate spinal cord motoneurons containing unique
synapses called Cꢀterminals (9) and may be involved in future therapeutic strategies for the
treatment of amyotrophic lateral sclerosis (ALS) (36, 37). Whether INMT coꢀlocalizes with
sigmaꢀ1 receptors in other neural tissue remains unknown.
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Product inhibition of INMT by SꢀadenosylꢀLꢀhomocysteine (SAH) has been
demonstrated. SAH’s inhibitory mechanism has been reported to be competitive against SAM
and noncompetitive against Nꢀmethylserotonin (22). Little is known regarding the relationship
between INMT structure/activity with synthetic or endogenous small molecule regulators or their
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kinetic mechanisms. Thompson et al. (3) identified several compounds, including the beta
carboline, norharmane, that when compared to tryptamine, served as relatively effective
substrates for rabbit INMT. Several inhibitors of rabINMT were identified by Thompson et al.
(3) but the kinetic mechanism(s) were not reported. The in vivo activity of INMT appears to be
inhibited by uncharacterized dialyzable endogenous compounds (38, 39). A competitive
inhibition mechanism by 1,8ꢀdiaminooctane and 1,7ꢀdiaminoheptane of rabbit lung INMT when
measured against tryptamine has been reported (40). Here, we report the mechanism of
inhibition of rabINMT by DMT and a novel derivative of tryptamine, Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀ
yl)ethyl)ꢀN',N'ꢀdimethylpropaneꢀ1,3ꢀdiamine (PDAT). Inhibition by PDAT was compared to Nꢀ
(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)propaneꢀ1,3ꢀdiamine (PAT) which is lacking the two methyl groups on
the Nꢀpropyl amino moiety.
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Experimental Procedures
Materials. All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI)
and utilized without further purification unless stated otherwise. [14C]ꢀSꢀadenosylꢀLꢀmethionine
([14C]ꢀSAM) was purchased from PerkinElmer Life Sciences (Wellesley, MA). Frozen rabbit
lungs were purchased from PelꢀFreez (Rogers, AR), histidineꢀtagged human indolethylamineꢀNꢀ
methyltransferase was obtained from the Structural Genomics Consortium, University of
Toronto, Ontario, Canada, recombinant human phenylethanolamineꢀNꢀmethyltransferase
(hPNMT) and nicotinamideꢀNꢀmethyltransferase (hNNMT) were purchased from Creative
BioMart (Shirley, New York). All reagents for the hPNMT assay were prepared in 50 mM Trisꢀ
HCl buffer (pH = 8.5) unless stated otherwise, excluding PDAT which was dissolved in dimethyl
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sulfoxide (DMSO). All reagents for the hNNMT assay were prepared in 50 mM TrisꢀHCl buffer
(pH = 7.2).
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Synthesis of N,N-dimethyltryptamine (DMT). DMT was synthesized by the method of
Speeter and Anthony (41) with minor modifications. Briefly, indole, dissolved in iceꢀcold
diethyl ether, was reacted with a solution containing two equivalents of oxalylchloride in diethyl
ether followed by a 20% wt/vol solution of dimethylamine in diethyl ether until the pH reached
9ꢀ10, while maintaining the reaction at 0 °C with an ice bath. The resulting 3ꢀindoleglyoxylꢀN,Nꢀ
dimethylamide was reduced to DMT with lithium aluminum hydride (LAH) in refluxing dioxane
essentially as described (42). After workꢀup and solvent removal, DMT freeꢀbase was obtained.
The DMT was purified by several recrystallizations from hot heptane followed by air drying.
The product migrated as a single spot on silica gel thin layer plates (mobile phase 4:1:1
nꢀBuOH:AcOH:H2O) and coꢀmigrated with authentic DMT. The melting point for DMT was
determined to be 64 – 65oC and is consistent with previously reported data (43). [1H] NMR was
consistent with the assigned structure.
Syntheses of N-(2-(1H-indol-3-yl)ethyl)propane-1,3-diamine (PAT). This compound
was prepared as the nonmethylated derivative of PDAT to assess the possible importance of the
N,Nꢀdimethylation of the aminopropyl side chain. The synthesis occurred in two steps according
to the schemes outlined in Figure 1, parts A & B. In reaction A, Nꢀ(3ꢀbromopropyl)ꢀphthalimide
(1 mmol, 0.268 g) and 5 equivalents of tryptamine (5 mmol, 0.8 g) were refluxed together in
ethanol and 2 mmol of potassium carbonate (0.27 g) for 5 h. The solid was filtered off and the
solvent was evaporated under reduced pressure using a rotary evaporator. Then the reaction
mixture was quenched with water and extracted three times with 5 mL ethyl acetate. The
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combined extracts were dried in MgSO4 and evaporated under reduced pressure. The product
was purified by column chromatography using silica gel and nꢀhexane/EtOAc 4:6 giving a 90%
yield. The product was subsequently taken to the next step without further characterization.
The phthalimide tryptamine product from reaction A was subsequently reduced with
hydrazine in a Gabriel synthesis as shown in Figure 1B. Briefly, 1 mmol (0.349 g) of
phthalimide tryptamine and 2 mmol (0.064 g) hydrazine were refluxed together in ethanol for 1.5
h. The reaction was subsequently cooled to room temperature and a 5 molar ratio of concentrated
HCl was added drop wise. The reaction was refluxed for an additional 4 h and quenched with
H2O. Workꢀup procedures for this compound were performed similarly to those described for
reaction A. The product was purified by column chromatography using silica gel and
toluene/diethylamine 9:1 solvent system giving 82% yield.
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Synthesis of N-(2-(1H-indol-3-yl)ethyl)-N',N'-dimethylpropane-1,3-diamine
(PDAT). This compound was prepared according to the synthetic scheme shown in Figure 1C.
The reaction was carried out using a straightforward alkylation using 1 mmol (0.224 g) of 3ꢀ(2ꢀ
bromoethyl) indole and 5 mmol (0.52 g) of 3ꢀ(dimethylamino)ꢀ1ꢀpropylamine. The reaction
mixture was refluxed in ethanol for 5 h and quenched with H2O. Workꢀup procedures for this
compound were performed similarly to those described for reaction A. The product was purified
by column chromatography using silica gel and toluene/diethylamine 9:1 solvent system giving
80% yield.
Characterizations of PAT and PDAT. Yields refer to isolated products after column
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at 300 MHz in CDCl3 relative to TMS (0.00 ppm).
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PAT: 1H NMR 10.1 (NH, 1H), 7.60ꢀ7.47 (m, 5 H), 5.11 (d, 2 H), 2.63ꢀ2.57 (m, 6 H), 2.3 (d,1 H),
1.84 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ 136.2, 127.41, 123.0, 119.7, 118.6, 113.4, 111.6,
49.8, 46.9, 39.3, 31.725.3. EMS [MH+] for C13H19N3, Calcd. 218.1579, Found, 218.2892.
PDAT: 1H NMR 10.2 (d, 1 H), 7.60ꢀ7.47 (m, 2 H), 2.78 (m, 2 H), 2.37ꢀ2.46 (m, 3 H), 2.26 (d, 7
H), 1.62 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ 136.8, 127.0, 123.6, 119.7, 118.6, 111.2, 54.9,
49.6, 46.5, 45.7, 26.7, 25.1. EMS [MH+] for C15H23N3, Calcd. 246.1892, Found, 246.2958.
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Preparation of rabbit lung homogenates. Rabbit lung homogenates were prepared
according to Thompson and Weinshilboum (3). Briefly, one pair of frozen rabbit lungs were
thawed in 50 mM Tris HCl pH = 7.3, minced with a scissor and homogenized using 4 bursts of
10 s each with a brinkman polytron (American Laboratory Trading Inc., East Lyme, CT) on
setting 6 on ice. Homogenates were centrifuged at 15,000 x g for 15 min at 4 oC and the
supernatant from this first low speed centrifugation was taken for a second centrifugation step at
100,000 x g for 60 min at 4 oC. The supernatant from the second high speed spin was aliquoted
and frozen at ꢀ80 oC until use in the INMT enzymatic assay.
Rabbit Lung INMT Assays. Rabbit lung INMT assays were modified from Thompson
et al. (3). A final incubation volume of 100 ꢁL in 15 mL capped tubes contained ice cold
tryptamine solutions in Tris HCl pH = 8.5 at final concentrations of 0.1, 0.3, 0.6, 0.8 and 1.0
mM, 250 ꢁg/mL bovine serum albumin (BSA), 35.5 ꢁM [14C]ꢀSꢀadenosylꢀLꢀmethionine ([14C]
SAM) [specific radioactivity varied between 28.15 and 56.3 ꢁCi/ꢁmole]. DMSO solutions of
PDAT and DMT (when tested as inhibitors) were diluted 100ꢀfold to a final concentration of 0.1
mM and the same concentration of DMSO alone was added to the nonꢀinhibitor controls. The
assay was initiated by the addition of 10 ꢁL of rabbit lung supernatant (rabINMT) in Tris HCl,
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pH = 8.5 (stock protein concentrations between 10 mg/mL and 25 mg/mL). The final volume
was adjusted to 100 ꢁL with Tris HCl pH = 8.5. The reaction was allowed to proceed at 32 °C in
a gently rotating water bath for 45 min. The assay was terminated by the addition of 0.6 mL of
ice cold 0.5 M potassium borate solution, pH = 10. Tubes without tryptamine were used as
background controls. The [14C]ꢀNꢀmonomethyltryptamine (MMT) and DMT reaction products
were extracted by addition of 5 ml of 3% isoamyl alcohol/toluene. Tubes were vortexed three
times for 5 s each and then centrifuged at 1620 x g for 2ꢀ3 min. Following centrifugation, 3.5
mL of the top organic layer was assessed for the [14C]methylated tryptamine levels using a
Beckman LS 6500 scintillation counter. The presence of authentic [14C]MMT and [14C]DMT,
which comprised greater than 95% of the reaction products, was confirmed by silica gel thin
layer chromatography using nꢀbutanol/water/acetic acid (12:5:3) followed by autoradiography
(Figure S1). Each radioactive spot was confirmed by coꢀmigration with nonradioactive MMT
(Rf = 0.48) and DMT (Rf = 0.39). Assays were performed in duplicate and each experiment was
repeated. The results reported are averages of quadruplicate counts from each duplicate sample.
hPNMT Assay. The activity of hPNMT was measured following the method of Gee et
al. (44). Briefly, phenylethanolamine (PEA) at 2.48 mM and [14C]SAM (20.95 ꢁM) were
prepared in the absence (2% DMSO) or presence of PDAT (2 ꢁM) to a final volume of 100 ꢁL
in 50 mM TrisꢀHCl pH = 8.5. The reaction was initiated with the addition of 5 ꢁL hPNMT
(0.025 ꢁg/ꢁL) and incubated for 90 minutes at 32 °C. The reaction was quenched with 0.6 mL
of 0.5 M potassium borate (pH = 10.0) and the product, [14C] NꢀmethylꢀPEA, was extracted with
5 mL 3% isoamyl alcohol in toluene, and the organic layers were separated by centrifugation for
2 min at 1620 x g in a swinging bucket centrifuge. The organic layer (approxiamtely 3.5 mL)
was combined with 6 mL scintillation solution (Ultima Gold, PerkinElmer Life Sciences,
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Wellesley, MA) and counted. The assay for hINMT was performed similarly except tryptamine
(8 mM) and hINMT (0.14 ꢁg/ꢁL) were used.
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hNNMT Assay. The activity of hNNMT assay was performed similar to hPNMT with
modifications following Rini et al. (45). Nicotinamide (10 mM) and 5 ꢁL [14C] SAM (20.95
ꢁM) was prepared in 50 mM Tris HCl (pH = 7.2) to a final volume of 100 ꢁL in the absence (2%
DMSO) or presence of PDAT (2 ꢁM). As a positive control a similar reaction was prepared in
the presence of 1ꢀNꢀmethylnicotinamide (1 mM). The reaction was initiated by adding 5 ꢁL of
hNNMT (0.025 ꢁg/ꢁL). After 90 minutes at 32 °C, each reaction was quenched with 0.6 ml of
0.5 M potassium borate and 100 ꢁL of 1.3 M 1ꢀheptanesulfonic acid to neutralize the charge on
the product 1ꢀNꢀmethylnicotinamide (45). The product was extracted with 5 ml of 60% isoamyl
alcohol in toluene. The organic and aqueous layers were separated by centrifugation at 1620 x g
for 3.5 min after which 3.5 mL of the organic layer were extracted, combined with 6 mL
scintillation solution (Ultima Gold, PerkinElmer, Wellesley, MA) and counted.
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The Ki for PDAT was determined from the Km and Vmax values by use of the following
equation, Vmax (+ inhibitor) = Vmax (ꢀ inhibitor)/(1 + [inhibitor]/Ki) to be ~ 84 ꢁM.
In silico Modeling. RabINMT sequence was aligned with the human INMT (hINMT)
using ClustalW. The human and rabbit INMT are 90% identical in amino acid sequence (Figure
S2). This aligned rabINMT sequence was threaded onto the hINMT structure (PDB code 2A14)
by sequential mutation of residues using the 'mutate' option in the biopolymer module of the
molecular modeling program Sybyl (Tripos Corp., St. Louis). The fits were examined with
molecular graphics and the side chains torsioned to accommodate any clashes with the
surrounding residues. In order to remove residual clashes, when fully threaded, the resultant
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model was energyꢀminimized using the Tripos force field. The allosteric loop (residues 25ꢀ34)
was examined for conformational similarity to the hINMT and its suitability for docking.
All dockings were performed with Autodock4 (Scripps Inst.) with a box size adequate to
include the allosteric loop and the active site. The active site in hINMT has not ligand (other than
SAH) and is an apo conformation that excludes ligands as part of the docking process. All the
docking of ligands occurs in the allosteric loop. All minimizations and dockings included the
SAH.
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Results
The lead compound, PDAT, showed inhibition of INMT in rabbit lung homogenates
(rabINMT) with a half maximum inhibitory concentration (IC50) of 83 ꢁM (Figure 2A). PAT
was decidedly less potent than PDAT for inhibition of rabINMT, showing an IC50 of
approximately 1 mM (data not shown). These data demonstrate the importance of N,Nꢀ
dimethylation of the propylamino moiety for effective INMT inhibition. PDAT inhibition of
pure human INMT (hINMT) was also observed with an estimated IC50 of 1 mM (data not
shown).
The affinity of PDAT for two structurally related Nꢀmethyltransferases, human hPNMT
and hNNMT were assessed. PDAT did not inhibit either enzyme at a concentration of 2 mM,
supporting its selectivity for INMT (Figure 2B). Inhibitors for both PNMT and NNMT (46-48),
when measured against their respective substrates, phenylethanolamine and nicotinamide,
showed complete inhibition of these enzymes (data not shown).
DMT is a known inhibitor of INMT (3) but its kinetic mechanism of inhibition has not
been elucidated. DMT showed a mixed noncompetitive mechanism when measured against its
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substrate, tryptamine, as assessed by MichaelisꢀMenten and LineweaverꢀBurk analysis. The
rabINMT Vmax was reduced from 4.63 ± 0.14 ꢁmol/min for control to 2.75 ± 0.85 ꢁmol/min in
the presence of DMT and the Km for tryptamine was increased from 852 ± 61 to 1618 ± 275 ꢁM
(Figure 3 and S3 and Table 1).
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PDAT inhibition of rabINMT showed a pure noncompetitive inhibition mechanism when
measured against the variable substrate, tryptamine. Vmax was reduced from 2.47 ± 1.22
ꢁmol/min for control to to 1.13 ± 0.37 ꢁmol/min in the presence of PDAT but the Km for
tryptamine remained larged unaltered (from 499 ± 68 ꢁM to 523 ± 85 ꢁM) (Figure 4 and S4
and Table 1). These data are consistent with an INMT allosteric inhibitory mechanism for DMT
and DMTꢀlike molecules, such as PDAT.
Since the crystal structure of SAH bound hINMT has been reported (49) we assessed the
potential docking site of DMT and PDAT for the hINMT using an in silico approach (Figure 5A
&B). Both molecules docked to the Nꢀterminal HelixꢀLoopꢀHelix region of INMT with binding
energies of ꢀ6.34 kcal/mole for DMT and ꢀ7.58 kcal/mol for PDAT. There are multiple
hydrogen bonds that may contribute to both DMT and PDAT binding to this loop region. As
shown in Figure 5, the indole nitrogen of DMT forms two hydrogen bonds with aspartic acidꢀ28
with the terminal nitrogen hydrogen bonding with glutamic acidꢀ34. Unlike DMT, PDAT is
predicted to form four hydrogen bonds where the middle nitrogen forms two hydrogen bonds
with aspartic acidꢀ28, and the indole nitrogen bonds with tyramineꢀ24. The terminal nitrogen of
PDAT bonds to glutamic acidꢀ34. This mechanism of binding may contribute to the lower
energy (or higher affinity) of PDAT compared to DMT.
Additionally, we threaded the sequence of rabINMT onto the sequence of hINMT (Figure
6A &B). When DMT and PDAT were assessed in silico for their interactions with rabINMT
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using the program Autodock, the highest affinity interactions of DMT and PDAT were found to
occur exclusively in the Nꢀterminal HelixꢀLoopꢀHelix region similar to the docking of DMT and
PDAT to hINMT. The proposed allosteric Nꢀterminal HelixꢀloopꢀHelix region is not within the
active site of the enzyme as assessed by the position of SAH in the co crystal structure of hINMT
(PDB code 2A14). In addition to the hydrogen bonding interactions between these compounds
and INMT, however, the rabINMT contains a glutamine at position 28 that is likely to form piꢀ
stacking interactions with the indole ring of both DMT and PDAT to contribute to the binding of
both compounds for rabINMT. The Nꢀterminal HelixꢀLoopꢀHelix sequence (amino acids 24ꢀ 34)
are aligned and identified in Figure S2.
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Discussion
INMT is a member of a large class of Nꢀmethyltransferases that utilize SAM as a methyl
donor. The enzyme INMT transfers methyl groups from SAM to the nitrogen of substrates
containing indolyl alkyl amino groups with the resultant formation of SAH (1, 3, 51). One
product of INMT Nꢀmethylation of tryptamine is DMT. In light of the recent discovery that
DMT activates the sigmaꢀ1 receptor (35), we sought to find DMTꢀlike small molecule regulators
of INMT in order to study the crosstalk between this metabolic pathway and that of the sigmaꢀ1
receptor, as well as to better understand the biology of INMT. Two Nꢀpropylamino derivatives
of tryptamine, PAT and PDAT (Scheme 1B and 1C, respectively) were synthesized and their
INMT regulatory properties were compared to DMT (Scheme 1A).
The biological role of DMT remains largely unknown. There are suggestions that DMT
may act as a neurotransmitter in humans and may be involved in psychosis, dreaming, nearꢀdeath
experiences, and spiritual exaltation (27, 51-54). INMT/DMT/Sigmaꢀ1 Receptor mechanisms
involving peripheral as well as CNS roles may also be important in mammalian biology (23).
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Recently, Nꢀmethylated derivatives of tryptamine, including Nꢀmonomethyl, N,Nꢀdimethyl and
N,N,Nꢀtrimethyltryptamines have been identified in the leaves and seeds of a wide variety of
citrus plants including Bergamot orange (Citrus bergamia) (55) and other Citrus fruits (56).
Presumably these naturallyꢀoccurring compounds are formed through INMT transmethylation of
tryptamine, but this is yet to be established. DMT and related tryptamines also occur in the
Illinois Bundleflower (Desmanthus illinoensis, common in the United States), chacruna
(Psychotria viridis, native to Central and South America), and trees of the Virola genus, native to
South American rainforests. Archeological evidence indicates that South American native
cultures have used these plants for shamanistic rituals for at least 3000 years (57).
The work reported in this paper on the inhibition of rabbit lung INMT by DMT and by a
novel synthetic tryptamine derivative, PDAT (Figure 1C), adds to our knowledge of the
mechanisms of INMT regulation. Both compounds showed noncompetitive inhibition kinetics
when measured against tryptamine at saturating concentrations of SAM. DMT showed mixed
competitive and noncompetitive kinetics (Figure 3) consistent with DMT binding to INMT alone
and to the INMT/tryptamine complex with different Ki values (see Table 1 and Figure S5A).
PDAT, on the other hand, showed pure noncompetitive kinetics (Figure 4) consistent with an
equal affinity for INMT alone and for the INMTꢀtryptamine complex (see Figure S5B). The Ki
for PDAT was determined to be 84 ꢁM (Figure 2A). The presence of the dimethyl substitution
on the propylamino side chain was found to be important for the affinity of PDAT for INMT,
since the nonmethylated propylamino tryptamine derivative, PAT (Figure 1B) was significantly
weaker in INMT inhibition (data not shown). PDAT was shown to be a selective inhibitor of
INMT since 2 mM concentrations of PDAT showed no significant inhibition of NNMT or
PNMT (Figure 2B, C, D).
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These data support an allosteric inhibitory mechanism for DMT and PDAT for rabbit
INMT. An in silico analysis of DMT and PDAT binding to both the hINMT and rabINMT
structure (Figure 5 & 6) identified the Nꢀterminal HelixꢀLoopꢀHelix region of INMT as a
possible location for a proposed allosteric inhibitory site. The loop regions of PNMT and NNMT
are substantially reduced in volume due to an extra partial turn of the Nꢀterminal helix as it enters
the loop (58), consistent with the observation that PDAT did not inhibit these Nꢀ
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methyltransferases because it likely cannot bind.
There were several reports on developing inhibitors of INMT in the 1970s ꢀ1980s. These
compounds include derivatives of SAM (22) that are broadꢀbased inhibitors of SAMꢀdependent
methyltransferases. These compounds are less selective for specific Nꢀmethyltransferases such as
INMT. Cyclic amidine inhibitors of INMT were also previously developed (24-26). These
compounds were tested for inhibition of both human and rabbit INMT but their kinetic
mechanisms, with the exception of a compound identified as DBN (2,3,4,6,7,8,ꢀ
hexahydropyrrolo[1,2ꢀa]pyrimidine) (24), were not thoroughly characterized. Our work with
DMT thus provides the first kinetic analysis of a mixed competitiveꢀnoncompetitive mechanism
of product inhibition of INMT. Further, characterization of the pure noncompetitive inhibition by
PDAT suggests PDAT as a lead compound for the development of effective and selective
allosteric regulators of INMT. In the broad family of methyltransferases, unique allosteric
mechanisms of regulation may be common. As examples, a small molecule allosteric inhibition
of a protein Nꢀmethyltransferase has been recently reported (59) and the Type 1 protein arginine
methyltransferase 3 (PRMT3) asymetrically dimethylates one of the guanidine nitrogens of
arginine on 40S ribosomal protein S2 resulting in stabilization of the protein (60). Siarheyeva et
al. (59) show that a unique phenethyl thiadiazolyl urea compound is an allosteric inhibitor of
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PRMT3 by binding at the nonactive site interface of a PRMT3 dimer. Other methyltransferases
appear to be allosterically activated by interaction with their protein partners (61). For example,
the DNA methyltransferase, Dnmt 1, preferentially methylates hemimethylated CpG sites on
DNA after DNA replication (62, 63) and Bashtrykov et al. (61) report that the ubiquitinꢀlike
PHD and RING finger domains protein 1 (Uhrf1) allosterically activates Dnmt 1 by unblocking
an inhibitory domain of Dnmt 1 thus allowing better access of the CpG substrate to the active
site of Dnmtꢀ1. In a similar manner, since the sigmaꢀ1 receptor colocalizes with INMT in
primate motoneuron cell bodies (9) a regulatory allosteric interaction may occur between these
two important signaling proteins as well.
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In summary, PDAT selectively inhibited hINMT in comparison to the structurally similar
Nꢀmethyltransferases, PNMT and NNMT. DMT and PDAT were found to inhibit INMT activity
and the kinetic mechanisms for inhibition were determined to be mixed competitive and
noncompetitive, and pure noncompetitive, respectively. In silico docking analyses supported the
experimentally derived kinetic data through the identification of a putative allosteric inhibitory
binding site in the Nꢀterminal HelixꢀLoopꢀHelix region of the computer threaded structure of
rabINMT using hINMT as the template. Pharmacological approaches to regulate INMT
enzymatic activity are highly desirable to assess the biological importance of INMT and its
possible role in cancer, autoimmunity, normal and aberrant mental states, and neurodegeneration.
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Acknowledgments
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We wish to thank Dr. Masoud Vedadi from the Structural Genomics Consortium at the
University of Toronto, 101 College Street, MaRS Center, South Tower, Toronto, Ontario M5G
1L7, Canada for providing the human INMT for our work.
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Supporting Information
Supporting Figure S1 (sequence comparison of hINMT and rabINMT) and Figure S2 (
schematics of the kinetic mechanisms of inhibition of INMT by DMT and PDAT) available free
of charge via the internet at http://pubs.acs.org.
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Table 1
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Km (µM) Vmax (µmol/min)
8
9
852 ± 61
1618 ± 275
499 ± 68
4.63 ± 0.14
2.75 ± 0.85
2.47 ± 1.22
1.13 ± 0.37
Control
100 µM DMT
Control
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523 ± 85
100 µM PDAT
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Figure 1: Synthetic schemes of DMT and inhibitors of INMT. A. DMT B. Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀ
yl)ethyl)propaneꢀ1,3ꢀdiamine (PAT) and C. Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀN',N'ꢀdimethylpropaneꢀ
1,3ꢀdiamine (PDAT).
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Figure 2: Selectivity profile of PDAT for Nꢀmethyltransferases. A. Inhibition of rabINMT by
PDAT. Data are presented as percent of control after background subtraction and the absence of
PDAT was normalized as 100%. B. Inhibition of hINMT by PDAT. hINMT assays were
conducted with tryptamine as a substrate, [14C]SAM as the methyl donor, in the absence or
presence of 2 mM PDAT. C. Lack of inhibition of human recombinant PNMT by PDAT.
PNMT assays were conducted similar to hINMT assays but with phenylethanolamine (PEA) as a
substrate. D. Lack of PDAT inhibition of hNNMT. hNNMT assays were conducted as
described for hINMT assays but using nicotinamide as a substrate.
Figure 3: Binding mechanism of DMT for rabINMT. A. A representative MichaelisꢀMenten
plot for DMT in the absence or presence of cold DMT (100 ꢁM). Concentrations of tryptamine
varied from 100 ꢁM to 1000 ꢁM. B. LineweaverꢀBurk transformation of the data presented in
A. Velocity (V) was calculated by determining the [14C]ꢀDMT formed over the time course of
60 min) and [S] represents the tryptamine concentrations. Inset shows a close up image of part B
showing the intersections of the two lines. In the absence of inhibitor, the Km for DMT is 852.2
± 61.35 ꢁM and 1618 ± 275.5 ꢁM in the presence of cold DMT. The Km values were
determined using the MichaelisꢀMenten analyses from Graphpad Prism and standard error of the
mean was calculated from two separate experiments performed in duplicates.
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Figure 4: Binding mechanism of PDAT for rabINMT. A. Representative MichaelisꢀMenten plot
for DMT in the absence or presence of 100 ꢁM of PDAT. Concentrations of tryptamine varied
from 100 ꢁM to 1000 ꢁM. B. LineweaverꢀBurk transformations of the data presented in A.
Velocity (V) was calculated by determining the 14CꢀDMT formed over the time course of 60
min) and [S] represents the tryptamine concentrations. Inset shows a close up image of Part B
showing the intersections of the two lines. In the absence of inhibitor, the Km for DMT is 499.6
± 68.2 ꢁM and 523.4 ± 85.4 ꢁM in the presence of 100 ꢁM PDAT. The Km values were
determined using the MichaelisꢀMenten analyses from Graphpad Prism and standard error of the
mean was calculated from two separate experiments performed in duplicates.
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Figure 5: In silico docking of DMT and PDAT on the hINMT (PDB code 2A14). The crystal
structure of hINMT was solved with SAM bound. A. The structure of hINMT docked with
DMT. The optimal DMT docking to hINMT indicates a terminal nitrogen hydrogen bond to
Gluꢀ34, with the indole nitrogen hydrogen bonded to the loop Aspꢀ28 carboxylate. The free
energy of binding of the displayed fit is ꢀ7.06 Kcal/mol. B. The structure of hINMT docked
with PDAT. Stabilization of PDAT with the protein is predicted to be mediated by the hydrogen
bonding interactions between the terminal nitrogen and Gluꢀ34, between the middle nitrogen and
Aspꢀ28 of the protein, and with the indole nitrogen hydrogen bonded to the backbone of the Tyrꢀ
24 carbonyl of helix Leuꢀ16ꢀ Tyrꢀ24. The free energy of binding of the displayed fit is ꢀ8.24
Kcal/mol.
Figure 6: In silico docking of DMT and PDAT on the threaded structure of rabINMT. The
threaded structure of rabINMT was modeled after the hINMT (PDB code 2A14). A. The
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threaded structure of rabINMT docked with DMT. Optimal DMT docking in threaded the
rabINMT model uses a terminal hydrogen bond to Gluꢀ34, with the indole nitrogenhydrogen
bonded to the helix backbone. The free energy of binding is at ꢀ6.34 Kcal/mol. B. The structure
of rabINMT docked with PDAT. Optimal PDAT docking in the threaded rabINMT also uses a
hydrogen bond from the terminal nitrogen to Gluꢀ24, but the middle nitrogen is placed at the
negative polar end of the Leuꢀ16ꢀ Tyrꢀ24 helix. The docking free energy of binding is calculated
as ꢀ7.58 kcal/mol. A pi-pi stacking interaction may occur between the indole rings of PDAT and
DMT with Glnꢀ28.
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Figure 1
A.
O
8
9
H3C
CH3
O
Cl
O
H3C
HN
Cl
Cl
N
CH3
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N
CH3
CH3
O
O
N
H
O
Diethyl ether
0oC
Diethyl ether
0oC
LiAlH4
Dioxane
reflux
N
H
N
H
N
H
B.
(i)
O
N
O
O
NH
H2N
K2CO3/Ethanol
Reflux, 5 h
+
N
H
N
Br
N
H
O
(ii)
O
NH
NH
1. N2H4/Ethanol
2. HCl
N
N
H
H2N
N
H
O
PAT
C.
NH
Br
Ethanol
N
NH2
+
N
N
H
Reflux, 5 h
N
H
PDAT
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