Structure-activity relationships of potent and selective factor Xa inhibitors: benzimidazole derivatives with the side chain oriented to the prime site of factor Xa.

Article abstract of DOI:10.1016/j.bmcl.2004.06.009

A series of benzimidazole derivatives with the side chain on the nitrogen atom oriented to the prime site of factor Xa (FXa) were designed and synthesized. Compounds with substituted aminocarbonylmethyl groups as the side chain showed potent FXa inhibitor

Full text of DOI:10.1016/j.bmcl.2004.06.009

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4217–4220
Phenylethanolamine N-methyltransferase inhibition:
re-evaluation of kinetic data
Qian Wu,a Kevin R. Criscione,b Gary L. Grunewaldb and Michael J. McLeisha,*
aCollege of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA
bDepartment of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, USA
Received 29 April 2004; revised 3 June 2004; accepted 7 June 2004
Available online 26 June 2004
Abstract—Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosyn-
thesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we demonstrate that a
routinely used assay for screening PNMT inhibitors is not appropriate for those inhibitors having Ki values less than 1 lM. A revised
assay has been developed that shows some inhibitors bind two orders of magnitude more tightly than previously reported.
Ó 2004 Elsevier Ltd. All rights reserved.
Phenylethanolamine N-methyltransferase (PNMT; E.C.
2.1.1.28) catalyzes the terminal step in catecholamine
biosynthesis, that is, the conversion of norepinephrine to
epinephrine (adrenaline).1;2 Although epinephrine (Epi)
makes up 5–10% of the total catecholamine content of
the brain, its function within the central nervous system
(CNS) is not well understood.3 CNS epinephrine has
been implicated in a variety of activities including cen-
tral control of blood pressure4 and respiration,5;6
secretion of hormones from the pituitary7 and may even
be responsible for some of the neurodegeneration found
in Alzheimer’s disease.8 Delineation of the role of cen-
tral Epi will require an agent that can be used to regulate
epinephrine levels within the CNS. An inhibitor of
PNMT may provide such an agent that may be of
potential pharmaceutical benefit.
series of structurally diverse substrates and inhibitors
with hPNMT and the bovine adrenal enzyme.10 These
compounds were evaluated using a standard radio-
chemical assay11 that has been used for substrates12 and
inhibitors.13 Although there were some differences in
substrate/inhibitor specificity, the results showed that
the conclusions from early structure–activity studies
with bovine adrenal PNMT broadly held true for
hPNMT.10 Recently we obtained X-ray structures of
hPNMT with several different inhibitors bound in the
active site.14;15 These structures guided a mutagenic
study of the PNMT active site, the results of which will
be described elsewhere. However, during this study, we
observed several anomalous kinetic results with the
inhibition of hPNMT mutants. These results suggested
that the standard assay used for PNMT inhibitors may
not be appropriate in all cases, particularly for those
with sub-micromolar affinity. In light of this we have
carried out a detailed re-evaluation of the standard
PNMT assay, as described below. The study prompted
the development of a revised assay procedure, in which
several inhibitors are shown to have Ki values for
hPNMT 2–100-fold lower than those previously re-
ported.10
Over the years, many inhibitors of PNMT have been
developed. Unfortunately, most interact with other
biologically important sites, such as the a2-adrenocep-
tors, which limits their utility as pharmacological tools.
The majority of those inhibitors were tested against
partially purified preparations of mammalian, primarily
bovine, PNMT. Recently, we described the purification
of human PNMT (hPNMT) expressed in Escherichia
coli.9 This permitted us to compare the kinetics of a
PNMT assays generally follow the transfer of either a
14C- or 3H-labeled methyl group from S-adenosyl-
L-
methionine (AdoMet) to norepinephrine or phenyl-
ethanolamine.2;16 The majority of inhibitors described
to date are competitive with substrate. Consequently,
the inhibition assay is generally carried out at a fixed
Keywords: PNMT; Epinephrine; Inhibition; Kinetics.
* Corresponding author. Tel.: +1-734-6151787; fax: +1-734-6153079;
e-mail: mcleish@umich.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.009

4218
Q. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4217–4220
concentration of AdoMet with the concentrations of
amine substrate and inhibitor being varied as appro-
priate. The standard assay described in Grunewald
et al.10 maintains AdoMet at 1 mM, while phenyletha-
nolamine (PEA) is varied between 30 and 100, and 60
and 200 lM for bovine adrenal and human PNMT,
respectively. A wide range of inhibitor concentrations
was used, depending on the value of Ki but the lowest
concentration used was 100 nM. The bovine adrenal
enzyme was only partially purified and required
200–300 lg protein/assay (in a total assay volume of
250 lL). The exact concentration was determined
experimentally for each new enzyme preparation by
assessing the amount of protein required to transfer
5–10% of the 3H label to the amine substrate, ostensibly
ensuring that the kinetics were measured under initial
rate conditions. The human enzyme had been purified to
homogeneity9 and required only ꢀ4 lg/assay to meet the
same requirements.
for those inhibitors with Ki values below 1 lM (data not
shown). The previous study on the inhibition of
hPNMT10 employed Michaelis–Menten kinetics (and
the inherent steady-state approximations) to determine
Ki values. It has been suggested that this approach is not
valid when the Ki value of the inhibitor is less than 1000-
fold greater than the total enzyme concentration.17;18
Given that the earlier study10 was carried out at a
hPNMT concentration of ꢀ4 lg/assay or ca. 500 nM,
this would apply to many of the inhibitors. For those
inhibitors, the population of free inhibitor molecules will
be significantly depleted by the formation of the en-
zyme–inhibitor complex, that is, the inhibitors will be
effectively tight-binding, and the data should be treated
according to Eq. 1.18
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ð½Eꢂ þ ½Iꢂ þ KiappÞ ꢁ ð½Eꢂ þ ½Iꢂ þ KiappÞ ꢁ 4½Eꢂ½Iꢂ
vi
v0
¼ 1 ꢁ
2½Eꢂ
ð1Þ
Examination of these assay conditions reveals three
potential problems: (i) the percentage conversion to
product is based on AdoMet, which is not the limiting
substrate, (ii) relatively high concentrations of PNMT
are required for timely conversion to product, and (iii)
not only is hPNMT potentially susceptible to substrate
inhibition, but also AdoMet often contains some
Here, v0 and vi are the reaction rates in the absence and
presence of inhibitor, respectively, [E] is the total enzyme
concentration, [I] is the inhibitor concentration, and
Kiapp is the apparent enzyme–inhibitor dissociation
constant. As demonstrated in Table 2, this is not a major
problem for the weaker inhibitors as errors brought
about by the steady-state treatment of data do not
become really significant until Ki=½Eꢂ is less than five.
S-adenosyl-L-homocysteine (AdoHcy) as a contami-
nant. We have looked at each of these, in turn, with
particular emphasis on the human enzyme as it has yet
to be studied in any great detail.
Conversely, for better inhibitors, not only will the Ki
data obtained using a steady-state treatment of data
have large errors, it may also be very misleading. For
example, if an inhibitor is tight binding, it will interact
with the enzyme in a nearly stoichiometric manner.
Therefore, as the enzyme concentration increases, more
In Table 1 we show the effect of using sufficient hPNMT
(1.25 lM) to ensure that, at 200 nM PEA, ꢀ5% of the 3H
label is transferred to the substrate. The data clearly
demonstrate that, for any concentration of PEA, less
than 5% of the AdoMet (initially 1 mM) is consumed in
the reaction. However, at an initial PEA concentration
of 30 lM, almost 50% of the PEA is converted to
product. Indeed, at all concentrations of PEA, the
reaction has gone to at least 25% completion. This
clearly shows that the earlier assays were not being
carried out under the initial rate conditions necessary to
use Michaelis–Menten kinetics for the determination of
Ki values.
Table 2. Effect of ratio Ki=½Eꢂ on observed inhibition
a
b
Ki=½Eꢂ
vi=v0
%Error in vi=v0
100
20
10
5
0.501
0.506
0.513
0.525
0.562
0.618
0.707
0.839
0.910
0.2
1.2
2.5
5.0
2
12.3
23.6
41.4
67.7
82.0
1
0.5
0.2
0.1
Potentially, the biggest problem is the concentration of
enzyme used in the assay. Studies with mutant proteins
suggested that the extent of inhibition was dependent on
protein concentration and, subsequently, we were able
to demonstrate a similar effect with wild-type hPNMT
a Values calculated using Eq. 1 assuming no substrate is present and
½Iꢂ ¼ Ki. Under these conditions, if Ki ꢃ ½Eꢂ, vi=v0 ¼ 0:5.
b %Error ¼ ðvi=v0 ꢁ 0:5Þ=0:5 ꢄ 100.
Table 1. Extent of reaction as a function of substrate concentration
[PEA] (lM)
PEA (pmol/assay)
dpm/assaya;b
pmol/30 minc
%Reaction of AdoMet
%Reaction of PEA
200
150
100
60
50,000
37,500
25,000
15,000
7500
6841
6223
4961
3561
2065
12,440
11,314
9021
4.9
4.5
3.6
2.6
1.5
24.7
30.0
35.9
42.9
49.7
6475
3754
30
a Assays were carried out at 1 mM AdoMet, with 277,500 dpm methyl 3H AdoMet per assay as described in Ref. 10.
b Values are mean of three experiments and reflect the number of dpm in the product per 1 mL of organic layer.
c Data not corrected for extraction efficiency.

Q. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4217–4220
4219
inhibitor will be required to attain the same level of
inhibition. This is described by Eq. 2.19;20
have results clouded by inhibition from sources other
than the inhibitor under investigation.
IC50 ¼ 0:5½Eꢂ þ Kapp
ð2Þ
Overall, the data suggest that the current assay is not
optimal for all inhibitors, and is certainly inappropriate
for the assay of sub-micromolar inhibitors. Overcoming
these problems requires a reduction in the assay con-
centrations of both AdoMet and hPNMT. Initially, the
AdoMet concentration was lowered to 5 lM (ꢀKm),
while the concentration of labeled AdoMet was main-
tained at ca. 300,000 dpm/assay. This had the dual effect
of increasing the sensitivity of the assay and guaran-
teeing that, if the amount of product formed (in dpm) is
<5–10% of the initial number of counts, the assay will
have been carried out under initial rate conditions. The
increase in sensitivity permitted the hPNMT concen-
tration to be reduced to 30 ng/assay (ꢀ4 nM), thereby
ensuring that all but the most potent inhibitors can be
routinely assayed and the data fit to Eq. 3.
i
At an enzyme concentration of 500 nM, inhibitors with
apparent Ki values of 1, 10, and 100 nM will have
broadly similar IC50 values (251, 260, and 350 nM,
respectively). Clearly, under these assay conditions, it is
almost impossible to obtain meaningful IC50 values, and
it suggests that the similar sub-micromolar Ki values
reported earlier represent, in effect, a titration of the
enzyme rather than true values of Ki.
To some extent the problems described above result
from the use of a high concentration of AdoMet in the
assay. The ratio of labeled/unlabeled AdoMet is very
low, making the assay relatively insensitive. There are
also some other undesirable consequences. It has been
demonstrated that PNMT from a variety of species is
subject to substrate inhibition by norepinephrine.21;22
Inhibition by AdoMet is less common, but it has been
reported for some isozymes of rabbit adrenal PNMT.22
However, the studies examining substrate inhibition by
AdoMet were carried out at concentrations of less than
100 lM, significantly below those in the assay under
review.21–23 Further, and more worrying, PNMT is
strongly inhibited by its reaction product, AdoHcy with
Ki values in the low micromolar range. Commercial
preparations of AdoMet are well known to contain
small amounts of AdoHcy.24;25 Even if the contamina-
tion was as little as 1%, an assay using 1 mM AdoMet
would contain at least 10 lM AdoHcy, which is above
the Ki values reported for AdoHcy inhibition of rabbit
adrenal PNMT.24;26 We therefore examined the effect of
AdoMet concentration on the reaction catalyzed by
hPNMT.
Vmax½Sꢂ
½Sꢂ þ Km 1 þ K
ꢀ
ꢁ
v ¼
ð3Þ
½Iꢂ
i
For the most potent inhibitors, even an enzyme con-
centration as low as 4 nM is still too high to use steady
state approximations. In those cases the inhibitor must
be considered to be tight binding and the inhibition data
should therefore be fit to Eq. 1.
We have used the modified assay27 to re-evaluate those
inhibitors previously reported10 as having Ki values for
hPNMT of less than 1 lM. The results are shown in
Table 3.
The results show that, in all cases, a lower value of Ki
was obtained using the revised assay. The increase in
binding affinity ranged from ca. 2- to 100-fold and, not
unexpectedly, the greatest increase was shown by those
compounds with the lowest values of Ki in the original
assay. Whilst these results do not change the structure–
activity conclusions reached in the earlier paper,10 we
are now able to discern considerable differences between
inhibitors whose Ki values were essentially identical in
the previous study.
The results, shown in Figure 1, clearly demonstrates that
high concentrations of AdoMet do have an inhibitory
effect on hPNMT catalysis. It is beyond the scope of this
work to determine whether this is due to substrate or
product inhibition. While this in itself may not be a fatal
flaw in assay design, as reactions without inhibitor are
run under identical conditions, it is still not optimal to
We have now adopted a 2-fold approach to determining
Ki values for new hPNMT inhibitors. First an IC50 value
for the inhibitor is obtained at a phenylethanolamine
concentration of 100 lM (i.e., at Km), with AdoMet and
hPNMT concentrations of 5 lM and 4 nM, respectively.
For a competitive inhibitor, the value of Ki is related to
IC50 by Eq. 4.28 For those compounds with an IC50
value above 100 nM, the Ki value is then determined by
fitting to Eq. 3 using variable PEA and inhibitor data
obtained at AdoMet (5 lM) and hPNMT (4 nM).
ꢂ
ꢃ
ꢄꢅ
½Sꢂ
IC50 ¼ Kapp 1 þ
ð4Þ
i
Km
Figure 1. Effect of AdoMet concentration on the reaction catalyzed by
hPNMT. The initial phenylethanolamine concentration was 200 lM
and the hPNMT concentration was 63 nM.
For inhibitors with IC50 values below 100 nM, Kiapp=½Eꢂ
will be <12.5. The inhibitor is therefore considered to be

4220
Q. Wu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4217–4220
Table 3. Comparison of Ki values obtained using original and revised
A Generation of Progress; Lipton, M. A., DiMascio, A.,
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7. Crowley, W. R.; Terry, L. C.; Johnson, M. D. Endocri-
nology 1982, 110, 1102.
assaya
Compoundb
Ki
Ki This workd
hPNMTc
8. Burke, W. J.; Chung, H. D.; Strong, R.; Mattammal,
M. B.; Marshall, G. L.; Nakra, R.; Grossberg, G. T.;
Haring, J. H.; Joh, T. H. In Central Nervous System
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0.28
(0.03)
0.012
(0.001)
9. Caine, J. M.; Macreadie, I. G.; Grunewald, G. L.;
McLeish, M. J. Prot. Express. Purif. 1996, 8, 160.
10. Grunewald, G. L.; McLeish, M. J.; Criscione, K. R.
Bioorg. Med. Chem. Lett. 2001, 11, 1579.
0.30
(0.03)
0.0049
(0.0006)
11. A typical assay mixture consisted of phosphate buffer
(pH 8.0, 0.5 M, 50 lL), unlabeled AdoMet (10 mM, 25 lL),
[methyl–3H]AdoMet, (ca. 3 ꢄ 105 dpm, 5 lL), substrate
solution (25 lL), inhibitor solution (25 lL), enzyme prep-
aration (25 lL), and sufficient water to achieve a final
volume of 250 lL. After incubation for 30 min at 37 °C,
the mixture was quenched with borate buffer (pH 10.0,
0.5 M, 250 lL), and extracted with toluene/isoamyl alcohol
(7:3, 2 mL). A portion of the organic layer (1 mL) was
removed, transferred to a scintillation vial and diluted
with scintillation cocktail (5 mL) for counting. Inhibition
assays were run in duplicate (at least) with four substrate
concentrations and three inhibitor concentrations, both
over a 5-fold range. Km and Ki values were determined by a
hyperbolic fit of the data.
0.30
(0.04)
0.0031
(0.0006)
0.58
(0.04)
0.28
(0.02)
0.66
(0.07)
0.073
(0.004)
0.81
(0.09)
0.090
(0.006)
12. Grunewald, G. L.; Grindel, J. M.; Vincek, W. C.;
Borchardt, R. T. Mol. Pharmacol. 1975, 11, 694.
13. Grunewald, G. L.; Borchardt, R. T.; Rafferty, M. F.;
Krass, P. Mol. Pharmacol. 1981, 20, 377.
14. Martin, J. L.; Begun, J.; McLeish, M. J.; Caine, J. M.;
Grunewald, G. L. Structure (Camb) 2001, 9, 977.
15. McMillan, F. M.; Archbold, J.; McLeish, M. J.; Caine, J.
M.; Criscione, K. R.; Grunewald, G. L.; Martin, J. L.
J. Med. Chem. 2004, 47, 37.
0.95
(0.09)
0.27
(0.03)
a Values of Ki are expressed in lM (SEM).
b Compound numbers from Ref. 10 are 12, 18, 19, 21, 24, 13, and 26, as
listed from top to bottom.
c Data from Ref. 10.
d Assayed as described in Ref. 27. Ki values were determined by a
hyperbolic fit of kinetic data to Eq. 1.
16. Saavedra, J. M.; Palkovitis, M.; Brownstein, J.; Axelrod,
J. Nature 1974, 248, 695.
17. Goldstein, A. J. Gen. Physiol. 1944, 27, 529.
18. Copeland, R. A. In Enzymes Second Edition: A Practical
Introduction to Structure, Mechanism and Data Analysis;
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tight binding, and the variable PEA and inhibitor data
are fit to Eq. 1.
19. Cha, S. Biochem. Pharmacol. 1975, 24, 2177.
20. Williams, J. W.; Morrison, J. F. In Methods Enzymol.;
Purich, D. L., Ed.; New York: Academic, 1979; pp 437–
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21. Connet, R. J.; Kirshner, N. J. Biol. Chem. 1970, 245, 329.
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Acknowledgements
This work was supported by the National Institutes of
Health (NIH HL 34193).
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27. The revised inhibition assay comprises phosphate buffer
(50 mM, pH 8.0), unlabeled AdoMet (5 lM), [methyl–
3H]AdoMet, (ca. 3 ꢄ 105 dpm), hPNMT (ꢀ4 nM), phen-
ylethanolamine, and inhibitor in a final volume of 250 lL.
Inhibition assays were run using four–five concentrations
of both phenylethanolamine and inhibitor, both over a 10-
fold range, and reaction workup was as described previ-
ously.
2. Axelrod, J. J. Biol. Chem. 1962, 237, 1657.
3. Fuller, R. W. In Epinephrine in the Central Nervous
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New York: Oxford University Press, 1988; pp 366–369.
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€
6. Goldstein, M.; Lew, J. Y.; Matsumoto, Y.; Hokfelt, T.;
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