Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: An attempt to improve the activity against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2011.07.005

Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC₅₀ values in the micromolar range, with a best value of 41 μM.

Full text of DOI:10.1016/j.bmcl.2011.07.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5403–5407
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substitution of the phosphonic acid and hydroxamic acid functionalities
of the DXR inhibitor FR900098: An attempt to improve the activity against
Mycobacterium tuberculosis
Mounir Andaloussi a, Martin Lindh a, Christofer Björkelid b, Surisetti Suresh a, Anna Wieckowska a,
Harini Iyer c, Anders Karlén a, Mats Larhed a,
⇑
a Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, SE-751 23 Uppsala, Sweden
b Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden
c AstraZeneca India Private Limited, Bellary Road, Hebbal, Bangalore 560024, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and
Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the
exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coor-
dinating functionalities. The best inhibitors provided IC50 values in the micromolar range, with a best
Received 29 May 2011
Revised 30 June 2011
Accepted 3 July 2011
Available online 20 July 2011
value of 41 lM.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Tuberculosis
DXR
Enzyme inhibitor
Fosmidomycin
FR900098
Tuberculosis (TB) is a great public health problem causing
death, illness and poverty. The World Health Organization has esti-
mated that a third of the world’s population today is infected with
the pathogenic bacteria Mycobacterium tuberculosis (Mt), and that
there were around 9.4 million new active TB cases and 1.3 million
TB deaths among HIV-negative people in 2009.1 The disease has a
huge impact not only on the patients and their families but also on
entire countries.
Two major factors have during the last decades increased the
complexity of the TB problem; the susceptibility of people infected
with the acquired immune deficiency syndrome (AIDS), which aug-
ments the risk of developing TB 100-fold and the increase in resis-
tant strains of Mt, leading to multidrug-resistant TB (MDR-TB) and
extensively drug-resistant TB (XDR-TB).1 The alarming develop-
ment of resistant strains of Mt is to a large extent caused by poor
compliance due to the lengthy, disagreeable treatment (a mini-
mum of 4 months) and associated unpleasant side-effects, in com-
bination with underfunded and underdeveloped healthcare. When
standard pharmacotherapies fail, second-line TB drugs are used,
but these drugs have a far lower efficacy and require even longer
treatment periods (18–24 months) associated with higher cost,
higher rates of adverse effects, and low cure rates.1 Since the
WHO’s declaration of TB as a public health emergency in 1993,
serious efforts have been implemented to combat the TB epidemic.
After peaking at 143 cases per 100,000 people in 2004, the TB inci-
dence level has slowly decreased.1 A new and more effective TB
drug could greatly help these efforts provided that it could bring
down costs and make TB-treatment more available. The need for
new TB drugs is perhaps greatest in the battle against MDR-TB
and XDR-TB for which the pharmacotherapy is expensive or non-
existent. New TB drugs, for which MDR-TB strains are not resistant,
can be discovered by inhibiting essential Mt-enzymes that are not
targeted by any of the currently used TB-drugs. One such potential
target is the metal-containing enzyme 1-deoxy-
D-xylulose
5-phosphate reductoisomerase (DXR).
DXR is involved in the nonmevalonate (DOXP/MEP) pathway of
isoprenoid biosynthesis, which leads to isopentenyl diphosphate,
an important isoprenoid precursor.2 Since the DOXP/MEP pathway
is absent in humans it is believed to be an excellent drug discovery
target for the development of both anti-malaria and anti-TB drugs.
Fosmidomycin, and the close analog FR900098, resemble DXR’s
natural substrate 1-deoxy-D-xylulose 5-phosphate (DXP), and are
known MtDXR inhibitors (Fig. 1). In addition, the structure of
MtDXR in complex with fosmidomycin is available.3 However, nei-
ther fosmidomycin nor FR900098 affect the growth or viability of
⇑
Corresponding author.
E-mail address: mats.larhed@orgfarm.uu.se (M. Larhed).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.005

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M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5403–5407
O
O
HO
HO
R1
P
N
A. Modification of
the phosphonic
acid part
OH
B. Modification of
the hydroxamic
acid part
R1 = H, Fosmidomycin
R
1 = CH3, FR900098
R2
O
N
N
O
O O
O
HO
O
NaO
S
HN
N
NH
O
N
N
H
S
N
O
O
O
O
H
H
N
O
H
N
O
COOH
OH
N
N
HO
O
O
O
OH
OH
O
O
P
R2= Br,
Ph, H
N
H
Ph
Figure 1. Design rationale of novel FR900098/fosmidomycin analogs.
O
O
O
iii
R
Br
i
R
N
vii
R
N
R
N
OBn
OH
OBn
3
3
3
3
R = CO2Et, 1a
1b
R = CO2Et, 2a
2b
3a
R = CO2H, 4a
R = SO3Na,
R = NHSO2CH3,
R = tetrazole, 4d
R = CO2H,
R = NHSO2CH3, 3b
3c
iv
R = Br,
4b
R = Br,
R = SO3Na,
ii
v
R = CN,
R = tetrazole,
4c
2c
vi
3d
Scheme 1. Synthetic route for the compounds 4a–4d. Reagents and conditions: (i) BnONHCOCH3, NaH, DMF, 50 °C; (ii) Na2SO3, H2O, 120 °C; (iii) NaOH (1 M), MeOH, 60 °C;
(iv) CDI, DBU, methanesulfonamide, 60 °C; (v) (a) CDI, NH3(g), THF, rt; (b) TEA, TFAA, DCM, rt; (vi). NaN3, NH4Cl, DMF, MW 140 °C, 2 h; (vii) H2, 10% Pd/C, rt.
R1
OH
2
R2
n
R1
Br
n
R1
R1
R2
n
i
ii
v
7
5
6
O
O
O
6a,b, n = 2
HN
2
6c
, n = 2, R
=
8a, n = 2, R2
=
R1
R1
=
=
7a
N
N
, n = 3,
OBn
OH
R1 = -PO(OEt)2
N
O
7b,c, n = 2, R2 = -PO(OEt)2
=
2
H
5a
8b c
- , n = 2, R
-PO(OH)2
O
Br
O
NH 5b
1
n = 3, R2
=
7d,
O
R
=
O
N
N
-PO(OEt)2
Ph
O
8d-f, n = 3, R2
=
-PO(OH)2
iii
7e
n = 3, R2
=
O1
, R = N
N
N
-PO(OEt)2
iv
O
O
n = 3, R2
-PO(OEt)2
=
, R1 = N
7f
Scheme 2. Synthesis route for the compounds 8a–8f. Reagents and conditions: (i) 6a: HBr 47%,
DMF, 50 °C; 7b and 7c: TEP, ; 7d: 6-bromo-oxazolo[4,5-b]pyridin-2(3H)-one, NaOEt, DMF, rt; (iii) phenylboronic acid, Pd(PPh3)4, TEA, DMF, MW, 120 °C; (iv) H2, 10% Pd/C,
MeOH, rt; (v) 8a: H2, 10% Pd/C, MeOH, rt; 8b–8f: TMSBr, DCM, rt.
D; 6b; (a) TEA, MsCl, DCM, rt; (b) LiBr, THF, rt; (ii) 7a: BnONHCOCH3, NaH,
D
CO2Et
CO2Et
CO2Et
CO2Et
i
ii
iii
N
O
N
O
COCH3
N
O
COCH3
N
O
NHOBn
N
N
N
O
OBn
OH
9
10
11
12
iv
CO2H
N
O
COCH3
OH
8g
Scheme 3. Synthesis route for inhibitor 8g. Reagents and conditions: (i) (a) O-Benzylhydroxylamine hydrochloride, pyridine, EtOH, rt; (b) NaCNBH3, MeOH, HCl (12 M), rt; (ii)
TEA, AcCl, DCM, rt; (iii) H2, 10% Pd/C, MeOH; (iv) NaOH (10 M), MeOH, rt.
the whole-cell Mt bacteria because of the lack of uptake through
the highly complex mycobacterial cell wall. This is probably due
to the inherent polar and charged nature of this class of phos-
phonic acid/hydroxamic acid inhibitors.4

M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5403–5407
5405
O
P
O
P
OH
N
O
P
OEt
OEt
OEt
OEt
O
N
Br
N
OEt
OEt
O
N
O
O
iii
ii
i
16
8i
13
14
15
iv
OH
N
O
P
OH
OH
O
Scheme 4. Synthesis route for the inhibitor 8i. Reagents and conditions: (i) diethyl vinylphosphonate, Pd(OAc)2, t-Bu4NOAc, KCl, K2CO3, DMF, 90 °C; (ii) H2, 10% Pd/C, MeOH,
rt; (iii) (a) mCBPA, DCM, rt; (b) AcCl, MeOH, rt; (iv) TMSBr, DCM, rt.
O
P
O
P
O
P
O
O
O
O
ii
HO
HO
OBn
OBn
i
Ph
Ph
OH
OH
Ph
Ph
8j
17
18
Scheme 5. Synthesis route for the inhibitor 8j. Reagents and conditions: (i) n-BuLi, THF, ꢀ78 °C, 1 h, 3,4-dibenzyloxybenzaldehyde, ꢀ78 °C-rt, 4 h, t-BuONa, ꢀ78 °C, 10 min;
(ii) H2, 10% Pd/C, MeOH/cat. AcOH, rt.
In a drug discovery program aiming to identify less polar MtDXR
inhibitors that can penetrate the Mt cell wall and yield whole-cell
minimum inhibition concentration (MIC) activity, we decided to
prepare and test FR900098 analogs carrying only one of the origi-
nal phosphonic acid or the hydroxamic acid groups. Thus, the de-
sign rationale of these compounds involved the substitution of
either the phosphonic acid (strategy A) or the hydroxamic acid
(strategy B) of the FR900098 scaffold with alternative acidic or me-
tal-coordinating functionalities (Fig. 1). Several bioisosters of the
phosphonate group and the hydroxamate group have been previ-
ously evaluated for activity on DXR.5–7 In order to investigate both
strategies we re-synthesized previously reported DXR inhibitors
4a, 4b, 8d, 8e, 8f, 8h, 8j and developed new synthetic routes to ob-
tain the novel structures 4c, 4d, 8a, 8b, 8c, 8g, 8i for evaluation in
the MtDXR inhibition assay and to measure the in vitro anti-Mt
whole-cell activity.
O
P
COOH
HO
HO
N
O
COCH3
N
N
The synthetic route used for the preparation of the first set of
compounds 4a–4d is outlined in Scheme 1. Treatment of ethyl 4-
bromoacetate (1a) with N-(benzyloxy)acetamide8 in the presence
of sodium hydride as base and DMF as solvent led to the formation
of ethyl 4-(N-(benzyloxy)acetamido)butanoate (2a). The genera-
tion of N-(benzyloxy)-N-(3-bromopropyl)acetamide (2b) was per-
formed in two steps according to the procedure previously
reported in the literature.9 The sodium 3-(N-(benzyloxy)acetam-
ido)propane-1-sulfonate compound (2c) was obtained as the so-
dium salt by nucleophilic substitution of the corresponding
bromide 2b with sodium sulfite and basic work-up.10 Next, hydro-
lysis of ester 2a with 1 M sodium hydroxide yielded the expected
carboxylic acid (3a). The latter was activated by 1,10-carbonyldiim-
idazole (CDI) in THF at 60 °C, followed by reaction with methane-
sulfonamide to achieve the corresponding acyl sulfonamide (3b).
Treatment of 3a with gaseous ammonia and CDI at 0 °C furnished
the primary amide, which was subsequently reacted with triethyl-
amine (TEA) and trifluoroacetic acid (TFAA) to afford the corre-
sponding nitrile compound 3c. Tetrazole 3d was synthesized
according to the procedure previously reported in the literature,
employing sodium azide and microwave heating.11 The final inhib-
itors were obtained by catalytic hydrogenation of the intermedi-
ates 2c, 3a, 3b and 3d, providing a test set of phosphonic acid-
free compounds 4a–4d.
OH
8i HO
8g
O
Figure 2. (I) Docking pose of compound 8g (in blue) bound to MtDXR (2JCZ). (II)
Docking pose of compound 8i (in grey) docked to MtDXR (2Y1F). The absence of
Trp203 gives greater room for ligand 8i. In both images MtDXR and NADPH are
shown in green, the metal ion in purple, and hydrogen bond interactions in orange.
phosphonic acid has been replaced and five compounds in which
the hydroxamic acid moiety has been replaced by alternative me-
tal-binding functionalities (8b–8f) (Scheme 2). Reflux of 5a12 in
47% HBr gave direct formation of the alkyl bromide 6a. It is note-
worthy that the bromination of the related compound 5b13 re-
quired
a two-step procedure: mesylation of the hydroxy
functionality followed by substitution using LiBr. The attachment
of the hydroxamic acid part to the alkyl chain of 6a, affording 7a,
was achieved by the addition of sodium hydride and
N-(benzyloxy)acetamide in DMF. The synthesis of the phosphonate
compounds 7b and 7c was performed by refluxing 6a and 6b in
excess triethyl phosphite (TEP). Furthermore, N-alkylation of 6-
bromo-oxazolo[4,5-b]pyridin-2(3H)-one14 with diethyl (3-bromo-
propyl)phosphonate (6c) yielded compound 7d, which was
transformed into compound 7e by a microwave-assisted Suzuki
reaction employing phenylboronic acid as the coupling part-
ner.15–17 Classical hydrogenation of compounds 7a and 7d led to
The next part of the project concerned the synthesis of inhibitor
structures 8a–8f, representing one compound (8a) in which the

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M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5403–5407
compounds 8a and 7f. The TMSBr-mediated hydrolysis of the phos-
phonate ester in 7b–7f furnished the corresponding phosphonic
acid products 8b–8f.
crystal structures were used; one with fosmidomycin (2JCZ)3 and
one with the recently reported MtDXR inhibitor
a-3,4-dichloro-
phenyl-fosmidomycin bound to the active site (2Y1F).22,23 Both
crystal structures were used in the docking study as the change
in position of especially tryptophan 20322 allowed differently sized
inhibitors to fit into the enzyme.
Next series of FR900098 analogs prepared was 8g–8j. Inhibitor
8h was essentially synthesized according to the procedure re-
ported by Perruchon et al.5 In contrast, the novel compounds 8g
and 8i required the development of the synthetic routes presented
in Schemes 3 and 4. Regarding isoxazole carboxylic acid, 8g, we
decided to start the synthesis with aldehyde 9.18 Treatment of 9
with O-benzylhydroxylamine hydrochloride followed by reduction
with sodium cyanoborohydride gave 10. As expected, acetylation
of O-benzylhydroxylamine 10 with acetyl chloride in the presence
of triethylamine yielded acetamide 11. Finally, catalytic hydroge-
nation of 11 followed by alkaline hydrolysis afforded test com-
pound 8g.
For N-hydroxy-pyridinone-containing 8i, the synthetic proce-
dure started with the Heck reaction of 2-bromo-6-methoxypyri-
dine with diethyl vinylphosphonate under phase-transfer
conditions (Scheme 4).18,19 The arylated olefin, 14, was hydroge-
nated with Pd/C in methanol to give the saturated product, 15. Oxi-
dation of the pyridine ring in 15 was conducted with mCBPA in
DCM. Subsequent treatment of the intermediate with acetyl chlo-
ride followed by addition of methanol led to the formation of the
pyridinone N-hydroxy ring system. Hydrolysis of compound 16
gave the desired inhibitor 8i.
All synthesized compounds retain either the phosphonic acid or
hydroxamic acid part of the fosmidomycin molecule. This simpli-
fies the evaluation as one can directly compare the docking poses
with the binding mode of fosmidomycin. Docking poses of com-
pounds 4a, 4b, 4d, 8g, 8c and 8i present convincing binding pat-
terns, exemplified by the docked conformations of structures 8g
and 8i in Figure 2. It is worth noting that the docking pose of 8g
shows almost perfect overlay with fosmidomycin at both the metal
binding site and at the phosphonic acid binding site, with an al-
most fully retained hydrogen bond network.
All 14 compounds were screened for inhibitory activity against
MtDXR at 100 lM (Table 1). Next, the IC50 values were determined
for inhibitors with more than 35% inhibition at 100 lM (4b, 8g, 8i,
and 8j).24,25 Only one of the seven analogs with phosphonic acid
replacements, the isoxazole carboxylic acid 8g, showed MtDXR
inhibition (IC50 = 151 lM). Interestingly, it has been indicated (pre-
liminary data) that monoester 8h has some activity against TB.10
Synthesis and biological evaluation of the molecules with a
modified hydroxamic part (8b–8f, 8i–8j) led to two new active
fosmidomycin analogs (8i and 8j). Heterocyclic structure 8i inhib-
Finally, it was of interest to replace the hydroxamate function-
ality with a catechol group.7 Thus, compound 8j was prepared by
the condensation20 of dibenzyl methylphosphonate and 3,4-diben-
zyloxybenzaldehyde followed by the hydrogenation using a Pd/C
catalyst (Scheme 5).
ited MtDXR at 53
lM. Catechol-containing phosphonic acid 8j has
been reported to inhibit Escherichia coli DXR with an IC50 of
4.5
41
l
M.7 In the case of MtDXR, 8j inhibited it with an IC50 of
lM. Although active in the enzyme assay, 8g, 8i and 8j did
Possible binding modes of the synthesized compounds were
not provide MIC value below 512
ously shown to have inhibitory activity in a whole-cell assay of
l
g/mL. Compound 8e was previ-
predicted using the docking software Glide.21 Two different MtDXR
Table 1
Evaluation of FR900098/fosmidomycin analogs
Modification of the phosphonic acid part
Modification of the hydroxamic acid part
%Inh at 100
lm
IC50
(
lm)
%Inh at 100
lm
IC50 (lm)
O
O
HO
HO
P
COR
O
NH
O
N
P
0.08 0.02
0.16 0.03
HO
O
8
0
6
N
H
OH
HO
8b
8c
Fosmidomycin , R = H
FR900098
, R = CH3
COCH3
O
P
HO
N
HO
O
OH
0
O
N
H
4a
HO
O
O
Br
COCH3
S
ONa
N
36 14
29 12
>100
OH
N
O
P
4b
HO
HO
7
4
O
O
N
O
H
COCH3
S N
N
8d
O
O
OH
4c
N
COCH3
Ph
N
N
OH
NH
N
4d
N
O
P
HO
HO
0
11
11
6
7
N
O
O
O
8e
8f
O
N
O
P
COCH3
HN
HO
HO
N
13
4
O
N
O
N
H
OH
8a
COOH
O
P
OH
N
N
O
COCH3
HO
HO
O
N
41
23
1
7
151 22
61
78
5
3
53 13
41 10
OH
8i
8j
8g
O
O
P
HO
HO
HO
P
COCH3
OH
OH
N
O
OH
8h
Ph
IC50 values22 were determined for inhibitors with more than 35% inhibition at 100
lM. Only 8e showed an MIC value below 512 lg/mL (256 lg/mL).

M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5403–5407
5407
19. Datta, G. K.; von Schenck, H.; Hallberg, A.; Larhed, M. J. Org. Chem. 2006, 71,
3896.
20. Harmata, M.; Rayanil, K.-o.; Espejo, V. R.; Barnes, C. L. J. Org. Chem. 2009, 74,
3214.
Catharanthus roseus ajmalicine and the activity of the compound
was proposed to be due to DXR inhibition.13 The fact that this com-
pound did not show activity against MtDXR, but still exhibited an
21. Schrödinger LCC: New York, NY, 2009.
MIC of 256 lg/mL, might indicate that 8e acts on another target.
22. Andaloussi, M.; Henriksson, L. M.; Wie˛ckowska, A.; Lindh, M.; Björkelid, C.;
Larsson, A. M.; Suresh, S.; Iyer, H.; Srinivasa, B. R.; Bergfors, T.; Unge, T.;
Mowbray, S. L.; Larhed, M.; Jones, T. A.; Karlén, A. J. Med. Chem. 2011, 54, 4964.
23. Deng, L.; Diao, J.; Chen, P.; Pujari, V.; Yao, Y.; Cheng, G.; Crick, D. C.; Prasad, B. V.
V.; Song, Y. J. Med. Chem. 2011, 54, 4721.
24. Kuzuyama, T.; Takahashi, S.; Watanabe, H.; Seto, H. Tetrahedron Lett. 1998, 39,
4509.
25. Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. Proc. Natl. Acad. Sci. USA
1998, 95, 9879
In summary, 14 FR900098/fosmidomycin-derived inhibitors
substituted with either acidic or metal coordinating moieties were
prepared and evaluated against MtDXR. The results showed that it
is possible to replace both the phosphonic acid group and the
hydroxamic acid group in FR900098 and to achieve active inhibi-
tors. Further development of these inhibitors may be worthwhile
in an attempt to improve both MtDXR binding and Mt whole-cell
activity.
Preparation of compound 8j: To a solution of dibenzyl methylphosphonate 17
(138 mg, 0.5 mmol) in THF (2 mL) was added n-BuLi (0.25 mL, 0.62 mmol,
2.5 M in hexanes) at ꢀ78 °C under N2 atmosphere and stirred at ꢀ78 °C for 1 h.
Then added a solution of 3,4-dibenzyloxybenzaldehyde (159 mg, 0.5 mmol) in
THF (2 mL) at ꢀ78 °C and the reaction mixture was brought to room
temperature over 4 h. The reaction was cooled to ꢀ78 °C and a solution of
NaOtBu (58 mg, 0.6 mmol) in THF (2 mL) was added and stirred for 10 min. The
reaction was quenched with saturated aq NH4Cl and extracted with EtOAc to
obtain the crude compound 18. Compound 18 (107 mg) was taken in methanol
(5 mL) and added 10% Pd/C (40 mg) and glacial acetic acid (0.05 mL). The
contents were stirred under the atmosphere of hydrogen for 16 h at room
Acknowledgments
We acknowledge the Swedish Foundation for Strategic Research
(SSF), the Swedish Research Council (VR) and the EU Sixth Frame-
work Program NM4 TB CT:018 923 for financial support.
temperature. The reaction mixture was filtered on
a pad of celite and
References and notes
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(J = 3.9 Hz), 28.8 (J = 131.9 Hz), 115.8, 116.2, 120.2, 134.5 (J = 17.5 Hz), 142.0,
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Inhibition of MtDXR-activity was measured in
a spectrophotometric
assay3,24,25 by monitoring the NADPH-dependent rearrangement and
reduction of DXP to form MEP using the absorption of NADPH at 340 nm
wavelength. Assay reactions had a final volume of 50
lL and contained 50 mM
HEPES-NaOH pH 7.5, 100 mM NaCl, 1.5 mM MnCl2, 0.2 mM NADPH, 0.19
lM
MtDXR, 0.2 mM DXP and inhibitory compound at various concentrations.
Initial screening for inhibition was performed with an inhibitor concentration
of 100
l
M. IC50-measurements were performed using six reactions with
M. Reactions were
inhibitor concentrations ranging between 0.01 and 1000
l
initiated by adding DXP and followed simultaneously in a 96-well plate (UV-
Star, Greiner) at 22 °C with a spectrophotometer (Envision 2140 Multilabel
Reader, PerkinElmer). Absorbance at 340 nm was measured every 5 s during a
500 s period. The slope of the linear phase of each reaction was used to
calculate the initial velocity. This was compared to the velocity of the
uninhibited reaction and used to calculate enzyme activity. Enzyme activities
were plotted against the corresponding inhibitor concentration and data points
HiꢀLo
50
were fitted to (equation 1), Y ¼ Lo þ 1þ
where Hi is the estimated highest
X
IC
enzyme activity at zero inhibitor concentration, Lo is the estimated lowest
enzyme activity at infinite inhibitor concentration, X is the concentration of
inhibitor and Y is the measured enzyme activity. IC50-values presented are the
average value of three independent experiments.