Thiol-based angiotensin-converting enzyme 2 inhibitors: P1 modifications for the exploration of the S1 subsite

Article abstract of DOI:10.1016/j.bmcl.2007.11.048

Screening of a metalloprotease library led to the identification of a thiol-based dual ACE/NEP inhibitor as a potent ACE2 inhibitor. Modifications of the P¹ benzyl moiety led to improvements in ACE2 potency as well as to increased selectivity versus ACE and NEP.

Full text of DOI:10.1016/j.bmcl.2007.11.048

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 732–737
Thiol-based angiotensin-converting enzyme 2 inhibitors:
P1 modifications for the exploration of the S1 subsite
David N. Deaton,a,* Enoch N. Gao,b Kevin P. Graham,b Jeffrey W. Gross,b
Aaron B. Millerc and John M. Strelowb
aDepartment of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
bMolecular Discovery Research, Screening & Compound Profiling, GlaxoSmithKline, Upper Providence, PA 19426, USA
cMolecular Discovery Research, Computational and Structural Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 13 September 2007; revised 12 November 2007; accepted 14 November 2007
Available online 19 November 2007
Abstract—Screening of a metalloprotease library led to the identification of a thiol-based dual ACE/NEP inhibitor as a potent
ACE2 inhibitor. Modifications of the P1 benzyl moiety led to improvements in ACE2 potency as well as to increased selectivity ver-
sus ACE and NEP.
Ó 2007 Elsevier Ltd. All rights reserved.
Angiotensin-converting enzyme 2 (ACE2) is a recently
identified clan MA, family M2 monocarboxypeptidase
with highest homology to the dicarboxypeptidase angio-
tensin-converting enzyme (ACE, EC 3.4.15.1).1,2 This
membrane-associated and secreted metalloprotease is
expressed in heart, kidney, testes, intestine, and lung,
and has been implicated in cardiovascular disease, kid-
ney disease, obesity, and lung disease.3–5
littermates.8 Supporting a role for ACE2 in cardiac
function, transgenic mice overexpressing ACE2 in the
heart have lower mean arterial pressure with rare focal
myocyte vacuolization, myofibril splaying, and nuclear
enlargement. Many of these mice develop terminal ven-
tricular fibrillation with lethal arrhythmias.9
Furthermore, male ACE2 (ꢀ/Y) mice, but not female
ACE2 (ꢀ/ꢀ) mice, also accumulate fibrillar collagen in
the renal glomerular mesangium, leading to develop-
ment of glomerulosclerosis of the kidneys.10 In addition,
ACE2 (ꢀ/ꢀ) mice exhibit lower body weights than wild
type mice with reduced fat mass.11 Moreover, ACE2 is
also utilized by the severe acute respiratory syndrome
(SARS) coronavirus as the receptor for infection.12
ACE2 (ꢀ/ꢀ) mice are resistant to SARS corona virus
infection.13 Finally, ACE2 (ꢀ/ꢀ) mice have enhanced
vascular permeability, increased lung edema, and wors-
ened lung function in several murine acute respiratory
distress syndrome (ARDS) models.14 With the many po-
tential functions of ACE2, small molecule inhibitors of
this enzyme could be utilized to help further define the
physiological roles of this protease.
ACE2 processes angiotensin I and the AT1 and AT2
receptor agonist angiotensin II to produce angiotensin
(1–9) and the mas receptor agonist angiotensin (1–7),
respectively, but its exact role in the renin-angiotensin
system (RAS) needs to be clarified. One group has re-
ported that C57BL/6 ACE2 (ꢀ/ꢀ) mice exhibit a severe
reduction in cardiac contractility, which is rescued in the
ACE (ꢀ/ꢀ)/ACE2 (ꢀ/ꢀ) double knockout.6 In contrast,
another group has disclosed that ACE2 (ꢀ/ꢀ) mice of
129/SvEv, C57BL/6, and mixed background do not ex-
hibit cardiac contractility defects, but have increased
susceptibility to angiotensin II-induced hypertension.7
While, a third group has revealed that male ACE (ꢀ/Y)
mice are more susceptible to heart failure and death
after transverse aortic constriction than their normal
ACE2 belongs to the zinc metalloprotease family and it
has been reported that classical ACE inhibitors such as
captopril and lisinopril do not attenuate ACE2 enzyme
activity. As part of a strategy to discover lead molecules
for an ACE2 inhibitor program, a directed screen of
ACE2 versus a set of metalloprotease inhibitors from
Keywords: Angiotensin-converting enzyme 2; Metalloproteases; Pro-
tease inhibitors; Thiols.
*
Corresponding author. Tel.: +1 919 483 6270; fax: +1 919 315
0430; e-mail: david.n.deaton@gsk.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.048

D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 732–737
733
the GlaxoSmithKline compound collection was per-
formed. Surprisingly, although confirming that classical
ACE inhibitors like captopril were inactive in the screen,
the thiol acid 1a was identified as a potent ACE2 inhib-
itor (Ki App = 86 nM).15 This biphenyl analog 1a is a
known dual ACE and M13 metalloprotease neutral
endopeptidase (neprilysin, NEP, EC 3.4.24.11) inhibitor
(ACE Ki App = 30 nM, NEP Ki App = 1.1 nM).16 It also
inhibits the M14 metalloprotease carboxypeptidase A1
(CpA, EC 3.4.17.1, Ki App = 1,200 nM). Speculating that
the thiol functioned as a binding group for the active site
zinc and that the carboxylic acid served as a recognition
element for the enzyme’s monocarboxypeptidase activ-
amino acids 2a–2b, 2d, 2f–2j, 2l, and 2o–2s (X@NH), by
diazotization of the amines to afford the bromides,
then subsequent displacement of the bromides, by
potassium thioacetate, with stereochemical inversion.
Some stereochemical erosion, presumably resulting
from formation of the a-lactone before nucleophilic
trapping, resulted during the Mitsunobu and diazotiza-
tion reaction steps, but after coupling with the amine 4,
the minor diastereomer could be removed via chro-
matography.
The structure/activity relationships of P1 analogs are de-
picted in Table 1. The (R) isomer 1b derived from L-
phenylalanine displays the P1 benzyl substituent in the
unnatural configuration. This orientation of the P1 sub-
stituent was detrimental to the ACE2 inhibitory activity
of diastereomer 1b (Ki App = 1400 nM) as well as that of
ACE (Ki App = 520 nM). This loss in inhibition is not
surprising, since nature has evolved proteases to recog-
nize the natural configuration of physiological peptide
substrates. In contrast, NEP (Ki App = 1.3 nM) accom-
modated the alternate configuration with no loss in
potency.
ity, the benzyl and methylene p-biphenyl moieties were
0
surmised to be the P1 and P1 substituents. With this pre-
mise, the structure activity relationships of the P1 posi-
tion of the lead inhibitor were explored with the goal
of improving potency and reducing ACE and NEP
inhibitory activity.
O
H
N
Complete removal of the P1 moiety caused a modest loss
HS
OH
O
in inhibitory potency versus ACE2 (H, Ki
=
App
1a
320 nM). This decrease in potency could arise from re-
duced van der Waals interactions due to the lack of a
P1 substituent, the increased entropic cost for the inhib-
itor to bind to the protease given the inhibitor’s in-
creased rotational freedom, or a combination of these
factors. In contrast to the D-isomer 1b, the ACE activity
(Ki App = 16 nM) of the glycine-like analog 1c was not
affected by elimination of the P1 element, while NEP po-
tency (Ki App = 13 nM) of 1c decreased by over an order
of magnitude.
The thiol analogs 1a–1s were prepared as depicted in
Scheme 1. The acids 3a–3s were activated in situ via
the carbodiimide, converted into the activated esters
with the aza-hydroxybenzotriazole, and then coupled
to the amine hydrochloride 4 to produce the fully pro-
tected amides. Subsequent hydrolysis of the methyl es-
ter, as well as the thioacetate, with lithium hydroxide
afforded the thiol acids 1a–1s.17
The alanine-derived analog 1d (Me, Ki App = 6.9 nM)
decreases rotational freedom and was an even more
potent ACE2 inhibitor than the starting lead 1a, while
increasing selectivity versus both ACE (Ki App = 21 nM)
and NEP (Ki App = 23 nM). In contrast, the geminal
dimethyl analog 1e (ACE2 Ki App = 2300 nM) decreased
inhibitory potency versus not only ACE2, but also ACE
(Ki App = 3,400 nM) and NEP (Ki App = 730 nM). Similar
to the methyl analog 1d, the linear P1 ethyl and n-butyl
analogs 1f (Et, Ki App = 1.4 nM) and 1g (n-Bu, Ki
App = 1.8 nM) were also potent ACE2 inhibitors, but
these extensions in P1 also increased activity versus
ACE (1f Ki App = 8.6 nM, 1g Ki App = 9.3 nM) and even
more so versus NEP (1f Ki App = 0.80 nM, 1g Ki
App = 1.2 nM).
The thiol acids 3a–3s were produced by three different
routes. Acids 3c and 3e were prepared by reaction of
the commercially available thiols 2c and 2e (X@S) with
acetyl chloride. In contrast, the acids 3k, 3m, and 3n
were produced from the alcohols 2k, 2m, and 2n
(X@O) via the Mitsunobu reaction with thioacetic
acid.18 Alternatively, the acids 3a–3b, 3d, 3f–3j, 3l, and
3o–3s were synthesized from the commercially available
O
O
O
H
N
XH
O
R1
R2
HCl∗H2N
OH
a,
R1
With the hope of improving the selectivity of these thiol-
based inhibitors versus ACE and NEP, the effect of
branching along the P1 side chain of this inhibitor class
was explored. Branching along the P1 chain was well tol-
erated in the S1 subsite of ACE2. The a-branched
iso-propyl 1h (i-Pr, Ki App = 1.5 nM), (R)-sec-butyl 1i
((R)-s-Bu, Ki App = 1.5 nM), (S)-sec-butyl 1j ((S)-s-Bu,
Ki App = 1.6 nM), cyclobutyl 1k (Cyb, Ki App = 2.4 nM),
O
+
b,
O
SH
R2
or
c-d
S
HO
2a-s
OH
e, f
Ph
R2 R1
O
3a-s
4
1a-s
Ph
Scheme 1. Reagents and conditions: (a) 2c and 2e, X@S, AcCl, NEt3,
dioxane, 0 °C to rt, 15–37%; (b) 2k, 2m, and 2n, X@O, AcSH, DIAD,
PPh3, THF, 0 °C to rt, 24–67%; (c) 2a–2b, 2d, 2f–2j, 2l, 2o–2s, X@NH,
HBr, NaNO2, H2O, 0 °C, 25–80%; (d) AcSꢀK+, DMF, 0 °C to rt, 11–
72%; (e) EDC, HOAt, i-Pr2NEt, CH2Cl2, 48–96%; (f) LiOHÆH2O,
THF, H2O, 30–94%.
and cyclopentyl 1l (Cyp, K
= 1.8 nM) analogs and
i
App
the b-branched iso-butyl 1o (i-Bu, Ki App = 1.4 nM)

734
D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 732–737
Table 1. Inhibition of human ACE2, ACE, NEP, and CpA
R2 R1
O
H
N
HS
OH
O
1a-s
Ph
a
ACE2 Ki App (nM)
b
ACE Ki App (nM)
c
NEP Ki App (nM)
d
CpA Ki App (nM)
#
R1
R2
1a
1b
1c
1d
1e
1f
CH2Ph
H
H
86
30
1.1
1.3
13
1200
310
CH2Ph
H
1400
320
6.9
2300
1.4
1.8
1.5
1.5
1.6
2.4
1.8
65
520
16
H
Me
1700
H
21
23
14,000
>50,000
9,300
Me
Et
Me
H
3400
8.6
730
0.80
1.2
13
1g
1h
1i
n-Bu
i-Pr
H
9.3
8900
H
200
490
200
620
700
13,000
>10,000
3.2
17,000
11,000
15,000
12,000
12,000
12,000
>50,000
11,000
22,000
17,000
32,000
5,000
(R)-s-Bu
(S)-s-Bu
Cyb
H
27
1j
H
2.4
5.4
38
1k
1l
H
Cyp
Cyh
H
1m
1n
1o
1p
1q
1r
1s
H
1300
410
0.28
2.6
130
140
2.6
Ph
i-Bu
H
84
H
1.4
7.1
420
550
860
CH2t-Bu
CH2Cyh
CH2b-Np
(CH2)2Ph
H
2,700
840
210
93
H
H
H
a Inhibition of recombinant human ACE2 activity in a fluorescence assay using 0.4 nM ACE2, 30 lM MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-
OH as substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The average percent
coefficient of variance for the Ki App values was 45%.
b Inhibition of recombinant human ACE activity in a fluorescence assay using 0.5 nM ACE, 10 lM MCA-Ala-Ser-Asp-Lys-Dap(DNP)-OH as
substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The average percent coefficient of
variance for the Ki App values was 58%.
c Inhibition of recombinant human NEP activity in a fluorescence assay using 0.15 nM NEP, 2 lM FAM-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-
Lys(TAMRA)-NH2 as substrate in 1 lM Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The average
percent coefficient of variance for the Ki App values was 43%.
d Inhibition of recombinant human CpA activity in a fluorescence assay using 37 nM CpA, 30 lM Abz-Gly-Gly-Nph-OH as substrate in 1 lM
Zn(OAc)2, 100 lM TCEP, 50 mM Hepes, 300 lM CHAPS, and 300 mM NaCl at pH = 7.5. The average percent coefficient of variance for the
Ki App values was 38%.
and neo-pentyl 1p (CH2t-Bu, Ki App = 7.1 nM) analogs
maintained their ACE2 inhibition. Only the bulkier
branched analogs, like the a-branched cyclohexyl 1m
(Cyh, Ki App = 65 nM) and phenyl 1n (Ph, Ki
App = 84 nM), the b-branched methylene cyclohexyl 1q
(CH2Cyh, Ki App = 420 nM) and methylene b-naphthyl
1r (CH2b-Np, Ki App = 550 nM), and the c-branched
ethylene phenyl 1s ((CH2)2Ph, Ki App = 860 nM), were
too large to be accommodated by the ACE2 S1 subsite.
(Ki App = 38 nM), cyclohexyl 1m (Ki App =1300 nM), and
phenyl 1n (Ki App = 410 nM) analogs lost some inhibi-
tory activity versus NEP relative to the ethyl analog 1f
(Ki App = 0.80 nM). Thus, a-branching at the P1 group
enhanced selectivity for ACE2 protease inhibition
relative to ACE and NEP. The (R)-sec-butyl analog 1i
(Ki App = 1.5 nM) has 330-fold selectivity versus ACE
and 18-fold selectivity versus NEP, while the cyclopentyl
1l (Ki App = 1.8 nM) has 390-fold selectivity versus ACE
and 21-fold selectivity versus NEP.
In contrast, a-branching of the P1 moiety was poorly
tolerated by the ACE S1 subsite, resulting in substantial
decreases in ACE inhibitory activity for the a-branched
iso-propyl 1h (Ki App = 200 nM), (R)-sec-butyl 1i
(Ki App = 490 nM), (S)-sec-butyl 1j (Ki App = 200 nM),
In comparison to a-branching, both ACE and NEP S1
subsites were more permissive to b-branching in P1
groups. Thus, the iso-butyl 1o analog is not only a po-
tent ACE2 inhibitor (Ki App = 1.4 nM), but also a low
nanomolar ACE (Ki App = 3.2 nM) and subnanomolar
NEP inhibitor (Ki App = 0.28 nM). ACE is less tolerant
of bulky b-branching, with the neo-pentyl 1p analog
exhibiting potent dual ACE2 (Ki App = 7.1 nM) and
NEP (Ki App = 2.6 nM) inhibition with good selec-
tivity over ACE (Ki App = 2700 nM). Larger b-branched
substituents like the methylene cyclohexyl 1q
(ACE2 Ki App = 420 nM, ACE Ki App = 840 nM, NEP
cyclobutyl 1k (Ki App = 620 nM), cyclopentyl 1l (Ki App
=
700 nM), cyclohexyl 1m (Ki App = 13,000 nM), and
phenyl 1n (Ki App = >10,000 nM) analogs. The S1 subsite
of NEP is more accepting of a-branching in P1 sub-
stituents than the ACE enzyme, but less liberal than
ACE2. The a-branched iso-propyl 1h (Ki App = 13 nM),
(R)-sec-butyl 1i (K
= 27 nM), (S)-sec-butyl 1j (Ki App =
i
App
2.4 nM), cyclobutyl 1k (Ki App = 5.4 nM), cyclopentyl 1l

D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 732–737
735
Ki App = 130 nM) and methylene b-naphthyl 1r (ACE2
Ki App = 550 nM, ACE Ki App = 210 nM, NEP Ki App
=
140 nM) attenuate inhibitory activity at all three enzymes.
Finally, asthe ethylenephenylanalog1 s shows, like ACE2
(Ki App = 860 nM), ACE (Ki App = 93 nM) was less tolerant
of c-branching in P1 groups, while NEP (Ki App = 2.6 nM)
accepted it. None of these thiol-based inhibitors was a po-
tent inhibitor of CpA.
A model of the thiol 1i19,20 docked into the active site of
ACE2 based on the recent X-ray crystal structure21 is
shown in Figure 1. It provides insight into the SAR of
the thiol series. In addition to binding to the imidazole
nitrogens of 374His and 378His and the carboxylate of
402Glu, the active site zinc coordinates the thiol of the
inhibitor. Also, the terminal carboxylate of the inhibitor
forms electrostatic interactions with the guanidine of
273Arg and accepts hydrogen bonds from the imidazoles
of 345His and 505His. Moreover, the carbonyl of 346Pro
accepts a hydrogen bond from the amide nitrogen of
Figure 2. A close up of the P1 pocket of the model of the thiol
compound 1i bound to the active site of ACE2 based on a X-ray co-
crystal structure (PDB code 1R4L). The ACE2 carbons are colored
cyan with inhibitor 1i carbons colored green. 510Tyr is colored in
orange to highlight its putative role in the selectivity of 1i. The semi-
transparent gray surface represents the molecular surface. This figure
was generated using PYMOL version 1.00 (Delano Scientific,
www.pymol.org).
the inhibitor, while the amide carbonyl is stabilized by
0
a hydrogen bond to 515Tyr. Furthermore, the P1 meth-
ylene biphenyl substituent of 1i forms significant lipo-
0
philic interactions with the quite large S1 channel
composed of the lengthwise canal between the two sub-
domains, including residues 274Phe, 276Thr, 346Pro,
367Asp, 370Leu, 371Thr, and 374His. This biphenyl substi-
tuent is hypothesized to occupy a different part of this
large pocket than the carboxyl inhibitor co-crystallized
in 1R4L. The P1 (R)-sec-butyl group of the inhibitor
forms van der Waals interactions with the S1 pocket
composed of 347Thr, 504Phe, 510Tyr, and 514Arg. As
shown in the close-up of the S1 subsite depicted in Fig-
ure 2, 510Tyr forms part of a pocket which the a-
branched methyl of the P1 (R)-sec-butyl group occupies.
Larger substituents are disfavored as they would require
movement of the phenol side chain of 510Tyr. Thus, this
Figure 3. A model of the thiol compound 1i bound to the active site of
ACE using the X-ray co-crystal structure of ACE and lisinopril (PDB
code 1O86). The ACE carbons are colored magenta with inhibitor 1i
carbons colored green. 518Val is colored orange to highlight its
hypothesized role in the selectivity of 1i. The semi-transparent gray
surface represents the molecular surface. This figure was generated
using PYMOL version 1.00 (Delano Scientific, www.pymol.org).
model agrees with the SAR, since the S1 subsite can tol-
erate cyclobutyl and cyclopentyl substituents, but is less
accepting of a cyclohexyl group.
A model of the inhibitor 1i19,20 docked into the active
site of ACE, focusing on the S1 subsite, based on the
X-ray structure of lisinopril with ACE,22 is shown in
Figure 3. From the model, it is apparent that the methyl
group of the (R)-sec-butyl P1 moiety of inhibitor 1i
bumps into the enzyme, helping explain the 330-fold
selectivity for ACE2 versus ACE. The branching nature
Figure 1. A model of the thiol compound 1i bound to the active site of
ACE2 based on a X-ray co-crystal structure of a carboxylate inhibitor
bound to ACE2 (PDB code 1R4L). The ACE2 carbons are colored
cyan with inhibitor 1i carbons colored green. The semi-transparent
gray surface represents the molecular surface, while hydrogen bonds
are depicted as yellow dashed lines. Several residues were removed for
visual clarity. This figure was generated using PYMOL version 1.0
(Delano Scientific, www.pymol.org).

736
D. N. Deaton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 732–737
of 518Val (atom CG1) makes this portion of the S1 pock-
et smaller than in ACE2. 518Val is the homologous res-
idue in ACE to 510Tyr in ACE2. This 510Tyr to 518Val
substitution from ACE2 to ACE and resulting alteration
of the S1 pocket volume helps explain the reduced ability
of ACE to accommodate a-branching in P1 substituents.
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Although X-ray structures of NEP co-crystallized with a
thiol-based inhibitor (PDB code 1Y8J)23 and an analog
0
with a P1 bi-phenyl moiety (PDB code 1R1H)24 are
available, as NEP belongs to a different metalloprotease
family (M13 vs M2), no reasonable model of these thiol
inhibitors bound to NEP could be determined to help
explain the increase in NEP selectivity of a-branched
P1 analogs. Likely protein movement to accommodate
potent NEP inhibitors like 1o is required. The move-
ment of multiple amino acid residues in proteins is diffi-
cult to predict accurately.
In summary, a series of a-thiol amide-based inhibitors of
ACE2 with varied substituents at the P1 position were
synthesized. Inhibitors containing linear alkyl P1 moie-
ties were some of the more potent analogs in the
ACE2 enzymatic assay. The smaller a-branched P1 sub-
stituents, exemplified by inhibitors 1i and 1l, maintained
similar potencies, and increased selectivity versus ACE
and NEP. Information gained from these studies has
proven to be useful in the design of other ACE2 inhibi-
tors. These inhibitors will be reported in due course.
Furthermore, a potent pan ACE/ACE2/NEP inhibitor
1o and a potent dual ACE2/NEP inhibitor 1p have been
identified. These tools may prove useful in further defin-
ing the roles these proteases play in the RAS cascade.
15. Deaton, D. N.; Gao, E. N.; Graham, K. P.; Gross, J. W.;
Miller, A. B.; Strelow, J. M. In 5th General Meeting of the
International Proteolysis Society; Sotiropoulou, G., Pam-
palakis, G., Arampatzidou, M., Eds.; International Pro-
teolysis Society: Patras, Greece, 2007; p 444.
16. Bhagwat, S. S.; Fink, C. A.; Gude, C.; Chan, K.; Qiao, Y.;
Sakane, Y.; Berry, C.; Ghai, R. D. Bioorg. Med. Chem.
Lett. 1995, 5, 735.
Acknowledgment
The authors would like to thank Rob I. West for helpful
discussions.
17. Hydrolyses were performed under an argon atmosphere.
When hydrolyses were carried out with reactions open to
the atmosphere, varying amounts of disulfide products
were isolated, likely from aerobic oxidation. The enzyme
assays contain a reducing agent, tris-(2-chloroethyl)-
phosphate (TCEP), to prevent oxidation of the thiols to
disulfides during the assays. Dilutions of 10 mM stock
solutions of the thiols 1a–1s to final assay concentrations
were done with 50% aqueous acetonitrile just prior to
protease inhibition studies. Under these standard condi-
tions, both the thiols and their corresponding disulfides
showed enzyme inhibitory activity. Presumably, the disul-
fides were reduced to their corresponding thiols by TCEP
during the pre-incubation period, before substrates were
added. In contrast, if the assays were performed without
TCEP, the disulfides were completely inactive, while the
potencies of the thiols were attenuated, probably because
of partial aerobic oxidation to their corresponding disul-
fides during the pre-incubation period.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.048.
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