Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase

Article abstract of DOI:10.1002/psc.1214

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors. Copyright

Full text of DOI:10.1002/psc.1214

Research Article
Received: 4 December 2009
Accepted: 10 January 2010
Published online in Wiley Interscience: 2 February 2010
(www.interscience.com) DOI 10.1002/psc.1214
Identification of small peptides mimicking the
R2 C-terminus of Mycobacterium tuberculosis
ribonucleotide reductase
Daniel J. Ericsson,a‡ Johanna Nurbo,b‡ Daniel Muthas,b Kalle Hertzberg,b§
b
b∗
a∗
´
Gunnar Lindeberg, Anders Karlen and Torsten Unge
Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium
tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller
RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding
affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described.
The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an
c
attractive candidate for further development of non-peptidic RNR inhibitors. Copyright ꢀ 2010 European Peptide Society and
John Wiley & Sons, Ltd.
Keywords: Mycobacterium tuberculosis; ribonucleotide reductase; peptide inhibitors; fluorescence polarization
Introduction
virus RNR systems [12] can compete for their R2 binding site
and thus inhibit RNR activity. It has also been shown that the
heptapeptide 1 (Figure 1) derived from the C-terminal end of Mtb
R2 can compete for binding to R1 [13]. With this compound as
a starting point, we have reported on the design and synthesis
of a set of N-acetylated heptapeptide inhibitors of Mtb RNR [14].
Unfortunately, our attempts to make shorter inhibitors resulted
in a loss of inhibitory potency. The N-acetylated pentapeptide
retained some inhibition, while the N-acetylated tetrapeptide
was inactive. Interestingly, structure activity studies on small
Fmoc-protected peptides and amino acids have shown that they
have significant activity against mammalian RNR [15–17]. To
investigate if Fmoc-protected peptides also have good binding
properties for Mtb, we have now prepared a series of analogues
and evaluated them using a competitive fluorescence polarization
(FP) assay.
Tuberculosis (TB), caused by the bacteria Mycobacterium tuber-
culosis (Mtb), is one of the most common infectious diseases in
the world, with an estimated 2 billion people infected. Despite a
fall in the incidence rate, TB-related deaths are still on the rise
[1]. The current treatment regimen is a course at least 6 months
long, leading to poor patient compliance. Added to the fact that
multidrug-resistant TB (MDR-TB) is present in virtually all surveyed
countries, with an even more resistant form denoted extensively
drug-resistant TB (XDR-TB) present in 45 of these, the urgent need
for novel drugs and drug targets is made clear [2].
All cellular organisms depend on access to a source of
deoxyribonucleotides for synthesis and repair of DNA. This
source is renewed by the reduction of ribonucleotides into
deoxyribonucleotides, a reaction that can only be carried out
in vivo by the enzyme ribonucleotide reductase (RNR) [3]. This
makes RNR an excellent target, e.g. for inhibiting de novo genome
synthesis, such as in cancer treatment [4]. In the case of Mtb, the
essential role of RNR is supported by a transposon mutagenesis
study [5]. Mtb utilizes a class Ib RNR, where the biologically active
unit is composed of a larger R1 homodimer and a smaller R2
homodimer, in an α2β2 complex. R1 and R2 are coded for by the
genes nrdE (Rv3051c) and nrdF2 (Rv3048c), respectively [5]. It was
shown first by Dawes et al. and later confirmed by Mowa et al. that
these genes are crucial for Mtb’s survival, also supporting RNR as a
promising antitubercular drug target [6,7].
∗
Correspondence to: Torsten Unge, Department of Cell and Molecular Biology,
Structural Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala,
Sweden, Sweden. E-mail: torsten.unge@icm.uu.se
´
Anders Karlen, Department of Medicinal Chemistry, Organic Pharmaceutical
Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden.
E-mail: anders.karlen@orgfarm.uu.se
a
Department of Cell and Molecular Biology, Structural Biology, BMC, Uppsala
University, Box 596, SE-751 24 Uppsala, Sweden
Besides the two active sites where the reduction of all four
species of ribonucleotides takes place, the RNR holoenzyme also
contains two specificity sites [8]. These allow the enzyme to be
allosterically regulated [9]. In this study, rather than targeting
any of the nucleotide binding sites, an alternative approach is
used: disrupting the formation of the holoenzyme. Studies have
revealed that peptides corresponding to the C-terminal tail of
the R2 subunit from the E. coli [10], mammalian [11], and herpes
b
Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC,
Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
‡
§
D. J. Ericsson and J. Nurbo contributed equally to this work.
Deceased.
c
J. Pept. Sci. 2010; 16: 159–164
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

ERICSSON ET AL.
sequence of the dansylated peptide 17 (Gly-Ser-Gly-Glu-Asp-
Asp-Asp-Trp-Asp-Phe) was synthesized with a Symphony instru-
ment (Protein Technologies, Inc., Tucson, AZ, USA) followed
by manual dansylation. The starting polymer was H-L-Phe-2-
chlorotrityl resin. Removal of the Fmoc group was accomplished
by treatment with 20% piperidine in N,N-dimethylformamide
(DMF) for 2 + 10 min. Coupling was performed in DMF,
using N-[(1H-benzotriazole-1-yl)-(dimethylamino)-methylene]-N-
methylmethanaminium hexafluorophosphate N-oxide (HBTU) in
the presence of N,N-diisopropylethylamine (DIEA). Amino acids
with the following side chain protection were used: Asp(Ot-Bu),
Glu(Ot-Bu), Ser(t-Bu), Trp(Boc). All peptides prepared by solid
phase synthesis were cleaved by treatment of the resin with
TFA/H2O/triethylsilane (95 : 5 : 5) for 1.5 h. The TFA was evaporated
and the products were precipitated by addition of diethyl ether.
Dipeptides 7 and 16 were prepared in solution as described be-
low. The crude peptides were purified by preparative RP-HPLC and
selected fractions were pooled and lyophilized. The peptides were
analyzedbyHRMSandbytwodifferentanalyticalRP-HPLCsystems.
The peptide content was determined by amino acid analysis.
Figure 1. The N-acetylated heptapeptide corresponding to the R2
C-terminal end of Mtb RNR.
Materials and Methods
General Methods
LC-MS was performed on a Gilson–Finnigan AQA system in ESI
mode using a Chromolith SpeedROD RP-18e 4.6 × 50 mm column
(Merck) and a CH3CN/H2O linear gradient with 0.05% HCOOH.
Preparative RP-HPLC was carried out using a Zorbax SB-C8,
5 µm (21.2 × 150 mm) column. The purity of the compounds
was determined by analytical RP-HPLC in the following systems
(UV detection at 220 nm): (A) ACE 5 C18, 50 × 4.6 mm, H2O/MeCN
gradient with 25 mM NH4OAc, pH 6.3 and (B) Thermo Hypersil C4,
50 × 4.6 mm, 5 µm, H2O/MeCN gradient with 0.1% trifluoroacetic
acid (TFA). Amino acid analysis was conducted at the Department
of Biochemistry, Uppsala University, Sweden. Samples were
hydrolyzed with 6 M HCl at 110 ◦C for 24 h and analyzed with
ninhydrin detection. Exact molecular masses were determined
on a Micromass Q-Tof2 mass spectrometer equipped with an
electrospray ion source.
General Procedure for Preparation of Peptides 2–6
Peptides 2–6 were prepared starting with 500-µmol H-L-Phe-2-
chlorotritylresin.Couplingoftheappropriateaminoacids(3equiv)
was performed in DMF, using HBTU (3 equiv), in the presence of
DIEA (6 equiv), for 3 h. After each coupling step, part of the resin
was removed to give the Fmoc-protected peptides of the desired
lengths. The crude peptides were purified by preparative RP-HPLC
(MeCN/H2O gradient with 25 mM NH4OAc, pH 6.3). The overall
yields ranged from 25–46%. Analysis data are given in Table 1.
Peptide Synthesis
Preparation of Peptide 7
Peptides 2–6 and 8–10 were prepared manually on a solid
phase in a disposable syringe equipped with a porous polyethy-
lene filter using standard Fmoc/t-Bu-protection. The peptide
Dipeptide 7 was prepared in solution from the pentafluorophenyl
(Pfp) ester derivative, Fmoc-L-Asp(Ot-Bu)-Pfp on a 100-µmol scale.
Table 1. Analytical data of compounds 2–16
HPLC
HRMS
purity
[M-H]−
(%)a
Compound
Amino acid analysis
Calcd
Found
A
B
2
3
Asp 3.72, Glu 1.01, Phe 0.99, Trpb
1161.3689
1032.3263
917.2994
802.2724
687.2455
503.1818
786.2775
788.2932
712.2619
386.1392
462.1705
425.1501
296.0923
354.0978
469.1247
1161.3651
1032.3256
917.3000
802.2730
687.2450
503.1815
786.2773
788.2916
712.2634
386.1388
462.1717
425.1506
296.0918
354.0984
469.1235
99
>99
>99
>99
>99
>99
>99
>99
>99
>99
98
98
>99
99
Asp 3.81, Phe 1.00, Trpb
4
Asp 2.79, Phe 1.00, Trpb
5
Asp 1.89 Phe 1.00, Trpb
>99
>99
>99
>99
>99
>99
>99
97
6
Asp 1.01, Phe 0.99, Trpb
7
Asp 0.99, Phe 1.01
8
Asp 1.66, Phe 1.00, Trpb
9
Asp 1.61, Phe 1.00, Trpb
10
11
12
13
14
15
16
Asp 1.68, Phe 1.00, Trpb
–
–
–
>99
99
>99
>99
>99
>99
–
–
>99
>99
Asp 2.00
a The purity of the compounds was determined by analytical RP-HPLC in the following systems (UV detection at 220 nm): (A) ACE 5 C18, 50 × 4.6 mm,
H2O/MeCN gradient with 25 mM NH4OAc, pH 6.3 and (B) Thermo Hypersil C4, 50 × 4.6 mm, 5 µm, H2O/MeCN gradient with 0.1% TFA.
b Not determined.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 159–164

SMALL PEPTIDES TARGETING M. TUBERCULOSIS RIBONUCLEOTIDE REDUCTASE
Coupling with H-L-Phe-Ot-Bu (1.1 equiv) was performed in DMF,
in the presence of DIEA (2.1 equiv), for 30 min. EtOAc was added
to the reaction mixture and the organic layer was washed with
1.0 M aqueous HCl, 5% aqueous NaHCO3, dried with MgSO4 and
concentrated by rotary evaporation. The residue was treated with
TFA/H2O/triethylsilane (95 : 5 : 1) for 30 min. The crude dipeptide
was purified by preparative RP-HPLC (MeCN/H2O gradient with
25 mM NH4OAc, pH 6.3). Selected fractions were pooled and
lyophilized. The yield was 23%. Analysis data are given in Table 1.
was concentrated to 100 µM, with some batch variation. The
protein could then be stored for at least one month at −20 ◦C
withoutanynoticeablelossofactivity.Proteinconcentrationswere
determined by taking the average of measurements at 280 nm
on an Eppendorf Biophotometer and a Thermo Scientific 1000
Nanodrop Spectrophotometer.
Fluorescence Polarization Assay
All lyophilized compounds were solubilized in 100% dimethyl
sulfoxide (DMSO) to a stock concentration of 5–25 mM and stored
at −20 ◦C. The assay buffer contained 5 mM HEPES pH 7.0; 0.01%
Nonidet P-40 and 100 nM dansylated peptide (17). For evaluating
the dissociation constant between probe and R1, the protein was
serially diluted from a starting concentration of 10 µM down to
0.004 µM. For compound screening, compound concentrations
were serially diluted from 100 µM down to 0.01 µM or lower. R1
was added to a final concentration ranging from 0.8 to 1.5 µM.
All final assay volumes were 50 µl and set up in Perkin Elmer
384 F OptiPlates. Reactions were set up at room temperature in
PCR-strips, mixed by inversion, and allowed to reach equilibrium
during 5 min of incubation. Measurements were done on triplicate
reactions on a Perkin Elmer Envision 2104 Multilabel Reader using
340 and 535 nm filters for excitation and emission, respectively.
General Procedure for Preparation of Peptides 8–10
Peptides8–10weregeneratedona50-µmolscale. Couplingofthe
appropriate carboxylic acid derivative (3 equiv), 9-fluoreneacetic
acid, 3,3-diphenyl propionic acid or 3-phenyl propionic acid, to
H-Asp(Ot-Bu)-Trp(Boc)-Asp(Ot-Bu)-Phe-2-chlorotrityl resin, were
performed using HBTU (3 equiv) in the presence of DIEA (6 equiv),
for 3 h. The crude peptides were purified by preparative RP-HPLC
(MeCN/H2O gradient with 25 mM NH4OAc, pH 6.3). The total yields
ranged from 15 to 25%. Analysis data are given in Table 1.
Fmoc-Protected Amino Acids 11–15
The Fmoc-protected amino acids 11–14 were all used as
obtained from the suppliers. Fmoc-Asp (15) was generated from
Fmoc-L-Asp(Ot-Bu)-OH, by treatment with TFA/H2O/triethylsilane
(95 : 5 : 3) for 30 min, and was purified by preparative RP-HPLC
(MeCN/H2O gradient with 0.1% TFA). All Fmoc-protected amino
acids were analyzed by HRMS and by two different analytical
RP-HPLC systems. Analysis data are given in Table 1.
Results and Discussion
In our previous study based on the N-acetylated heptapeptide
1, we monitored the reduction of ribonucleotides to the
corresponding deoxyribonucleotides by estimating the reduction
of 3H-labeled cytidine 5ꢁ-diphosphate (CDP). We have now
adopted a more high-throughput binding assay based on FP,
which we use in the present study. We have previously shown
Preparation of Peptide 16
Dipeptide 16 was prepared in solution from Fmoc-L-Asp(Ot-
Bu)-Pfp on a 100-µmol scale. Coupling with H-L-Asp(Ot-Bu)-
Ot-Bu (1.1 equiv) was performed in DMF, in the presence
of DIEA (2.1 equiv), for 30 min. EtOAc was added to the
reaction mixture and the organic layer was washed with 1.0
M aqueous HCl, 5% aqueous NaHCO3, dried with MgSO4 and
concentrated by rotary evaporation. The residue was treated with
TFA/H2O/triethylsilane (95 : 5 : 1) for 30 min. The crude dipeptide
was purified by preparative RP-HPLC (MeCN/H2O gradient with
0.1% TFA). Selected fractions were pooled and lyophilized. The
yield was 74%. Analysis data are given in Table 1.
Table 2. Binding affinity of compounds 1–16
Com-
pound
KD2 SD
(µM)a
Sequence
r2b
1c
2
Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe
Fmoc-Glu-Asp-Asp-Asp-Trp-Asp-Phe
Fmoc-Asp-Asp-Asp-Trp-Asp-Phe
Fmoc-Asp-Asp-Trp-Asp-Phe
Fmoc-Asp-Trp-Asp-Phe
Fmoc-Trp-Asp-Phe
8.3 0.3 0.988
0.7 0.1 0.857
2.3 0.1 0.990
7.6 0.1 0.985
3
4
5
19
3
0.978
Preparation of Peptide 17
6
102 10 0.849
7
Fmoc-Asp-Phe
60
29
84
1
2
9
0.956
0.977
0.833
–
The dansylation of the H-Gly-Ser(t-Bu)-Gly-Glu(Ot-Bu)-Asp(Ot-Bu)-
Asp(Ot-Bu)-Asp(Ot-Bu)-Trp(Boc)-Asp(Ot-Bu)-Phe-2-chlorotrityl
resin was performed manually in a disposable syringe on a
75-µmol scale. The polymer was treated with a solution of dansyl
chloride (4 equiv) and DIEA (3 equiv) in DMF and was allowed
to react for 3.5 h. The dansylated peptide was cleaved and
precipitated as described above and was purified by preparative
RP-HPLC (MeCN/H2O gradient with 25 mM NH4OAc, pH 6.3) to
give the dansylated peptide 17 in 17% yield.
8
9-Fluoreneacetyl-Asp-Trp-Asp-Phe
3,3-Diphenylpropanoyl-Asp-Trp-Asp-Phe
3-Phenylpropanoyl-Asp-Trp-Asp-Phe
Fmoc-Phe
9
10
11
12
13
14
15
16
>150
>150
–
Fmoc-Dif
51
3
0.932
0.995
–
Fmoc-Trp
12.4
1
Fmoc-Gly
>150
>150
>150
Fmoc-Asp
–
Fmoc-Asp-Asp
–
a KD2 values are the average of three wells with the associated standard
deviation. Values >150 µM are estimates only.
Cloning, Expression and Purification of Mtb RNR R1
b The correlation index, used as the parameter to be maximized during
thenonlinearcurvefit[20–22]. ForKD2 values>150 mM, nomeaningful
fit is possible.
The cloning, expression and purification of the Mtb R1 subunit
was performed as previously described by Nurbo et al. [14].
Prior to concentrating R1, urea and glycerol were added to final
concentrations of 1 M and 50%, respectively. The R1 subunit
c Synthesis and characterization reported earlier by Nurbo et al. [14].
c
J. Pept. Sci. 2010; 16: 159–164 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ERICSSON ET AL.
that at least a hexapeptide was required to achieve micromolar
RNR inhibition. In the present study the aim was to investigate if
smaller analogues of peptide 1 with the Fmoc protecting group
could compete for binding to the Mtb R1 subunit. The compounds
evaluated are shown in Table 2.
Synthesis of Peptides
The peptides 2–6 were generated manually using standard
Fmoc/t-Bu solid-phase peptide synthesis. The starting polymer
was H-Phe-2-chlorotrityl resin, HBTU was used as the activating
agentandDIEAwasusedasthebase.Thedipeptide7wasprepared
in solution from the activated ester derivative Fmoc-Asp(Ot-Bu)-
PfpandH-Phe-Ot-Bu.Peptides8–10weregeneratedfrompartially
protectedH-Asp-Trp-Asp-Phe-2-chlorotritylresinbycouplingwith
the appropriate carboxylic acid derivative. After completion of the
syntheses, a mixture of TFA, water and triethylsilane were used
to cleave the target peptides from the resin. The crude products
were precipitated in diethyl ether and purification by RP-HPLC
gave homogeneous products. The Fmoc-protected amino acids
11–14wereallobtainedfromcommercialsuppliersandwereused
without further purification. Fmoc-Asp (15) was produced from
Fmoc-Asp(Ot-Bu)-OH by treatment with TFA and the dipeptide 16
waspreparedinsolutionfromFmoc-Asp(Ot-Bu)-PfpandH-Asp(Ot-
Bu)-Ot-Bu. AlltargetcompoundswereanalyzedbyHRMSandgave
ions consistent with their molecular masses. According to analyses
by two different HPLC systems, the target compounds were at
least 97% pure. The fluorescent peptide used for displacement
measurements in the FP assay, Dansyl-Gly-Ser-Gly-Glu-Asp-Asp-
Asp-Trp-Asp-Phe (17), was synthesized as described for the other
peptides synthesized on solid phase, followed by removal of the
terminal Fmoc protecting group and derivatization with dansyl
chloride in the presence of DIEA.
Figure 3. A plot of the direct binding of 100 nM dansylated heptapeptide
to increasing concentration of R1. Fitting the data to Equations (6) and (39)
fromRoehrlet al.[19]yieldedthesolidcurvewiththefollowingparameters:
Q = 3.1; AB = 0.124 0.0004; AF = 0.011 0.001; KD1 = 2.2 0.02 µM.
The dotted curves enclose a 95% confidence interval of the fit.
Figure 4. Displacement of dansylated heptapeptide by compounds 1,2
and 13 according to the complete competitive model.
Competitive Fluorescence Polarization Assay
The affinity of compounds 1–16 as binders to the Mtb R1
subunit was evaluated by use of a competitive FP assay. The
method has the advantage of acting on systems in equilibrium,
so there is no need for time measurements to determine
initial velocities. Furthermore, since it is a method for studying
binding behavior, no catalysis has to take place. The use of
fluorescence polarization for screening small-molecule inhibitors
of protein–protein interactions is exhaustively detailed in two
back-to-back publications by Roehrl et al. [18,19]. As a first step,
the direct binding model (Figure 2(a)) was used to calculate a
dissociation constant for the interaction between the dansylated
heptapeptide (17) and R1. The KD1 was evaluated to be
2.2 0.02 µM. Figure 3 illustrates the sigmoidal curve produced by
increasing concentrations of R1 while keeping the concentration
of probe constant. With KD1 determined, it is possible to determine
KD2 values for each compound, i.e. the dissociation constant of the
interaction between a compound and R1. This is done by applying
the complete competitive model, as shown in Figure 2(b). KD2
values for the evaluated compounds are presented in Table 2.
Dissociation curves for compounds 1, 2 and 13 are shown in
Figure 4.
Truncation of Fmoc-Protected Peptides
To investigate the effect of replacing the N-acetyl group with
Fmoc, a series of N-terminally truncated peptide analogues
were synthesized and evaluated (Table 2). The N-acetylated
heptapeptide 1 was first reanalyzed using the FP assay and the
KD2 value evaluated to be 8.3 0.3 µM. The corresponding Fmoc-
protected peptide 2 showed a 10-fold improvement in binding
affinity with KD2 = 0.7 0.1 µM. Encouraged by this result, we
prepared and evaluated the truncated Fmoc-protected peptides
3–7. Although each deleted amino acid resulted in a 3–5-fold
Figure 2. (a) Direct binding model. (b) Complete competitive model. The
following definitions are used: R, free R1; L, free unlabeled ligand (the
tested compound); LS, free labeled ligand (the probe, i.e. the dansylated
heptapeptide); KD1, dissociation constant of the interaction between free
R1 and the probe; KD2, dissociation constant of the interaction between
free R1 and unlabeled ligand. This definition is identical to that of Ki which
is used elsewhere in the literature.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 159–164

SMALL PEPTIDES TARGETING M. TUBERCULOSIS RIBONUCLEOTIDE REDUCTASE
an increase in binding affinity. This effect was also seen for
shorter peptides and truncation down to single Fmoc-protected
amino acids was tolerated. The identification of Fmoc-Trp as
a potential lead compound, with binding affinity comparable
to the acetylated heptapeptide, is especially promising. This
small molecule represents an excellent starting point for the
development of non-peptidic inhibitors of Mtb RNR.
Acknowledgements
We acknowledge Dr Aleh Yahorau, Department of Pharmaceutical
Biosciences, Uppsala University, for help with HRMS analyses.
Dr Luke Odell is acknowledged for linguistic advice. We also
express our gratitude to the Swedish Foundation for Strategic
Research (SSF), the Swedish Research Council (VR) and the EU
Sixth Framework Program NM4TB CT:018923 for financial support.
Figure 5. N-terminal groups investigated in this study.
loss in affinity, the dipeptide 7 (KD2 = 60 1 µM) had surprisingly
good binding affinity. Thus, small Fmoc peptides were capable of
competing for binding to R1.
References
1 http://www.who.int/entity/tb/publications/2009/tbfactsheet 2009
one page.pdf [Last accessed October 2009].
Effect of Introducing Different N-terminal Groups
2 http://www.who.int/entity/tb/features archive/drs factsheet.pdf
[Last accessed October 2009].
3 Thelander L, Reichard P. Reduction of ribonucleotides. Annu. Rev.
Biochem. 1979; 48: 133–158.
4 Cerqueira NM, Fernandes PA, Ramos MJ. Ribonucleotide reductase:
a critical enzyme for cancer chemotherapy and antiviral agents.
Recent Pat. Anticancer Drug Discov. 2007; 2: 11–29.
5 Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial
growth defined by high density mutagenesis. Mol. Microbiol. 2003;
48: 77–84.
6 Dawes SS, Warner DF, Tsenova L, Timm J, McKinney JD, Kaplan G,
Rubin H, Mizrahi V. Ribonucleotide reduction in Mycobacterium
tuberculosis: function and expression of genes encoding class Ib
and class II ribonucleotide reductases. Infect. Immun. 2003; 71:
6124–6131.
7 Mowa MB, Warner DF, Kaplan G, Kana BD, Mizrahi V. Function and
regulation of class I ribonucleotide reductase-encoding genes in
mycobacteria. J. Bacteriol. 2009; 191: 985–995.
Next, we were interested to see if this positive binding effect
was associated only with the Fmoc group. Thus, we decided
to investigate other N-terminal groups. The tetrapeptide (5)
was chosen as the model compound. Three tetrapeptides were
acylated with different N-terminal groups to give peptides 8–10
(Figure 5). Peptide 8, acylated with 9-fluoreneacetic acid, turned
out to have only slightly less affinity than the corresponding
Fmoc-protected tetrapeptide 5 (KD2 = 19 3 µM), with a KD2
value of 29 2 µM. Making the N-terminal ring system less rigid
as in peptide 9 (KD2 = 84 9 µM) gave a drop in affinity, whereas
introduction of a smaller ring system (10, KD > 150 µM) resulted
in a more dramatic loss of binding affinity. These results showed
that the Fmoc group could be substituted for other similar groups
with almost retained affinity.
8 Kolberg M, Strand KR, Graff P, Andersson KK. Structure, function,
and mechanism of ribonucleotide reductases. Biochim. Biophys. Acta
2004; 1699: 1–34.
Fmoc-Protected Amino Acids
9 Reichard P. Ribonucleotide reductases: the evolution of allosteric
regulation. Arch. Biochem. Biophys. 2002; 397: 149–155.
10 Climent I, Sjo¨berg BM, Huang CY. Carboxyl-terminal peptides as
probes for Escherichia coli ribonucleotide reductase subunit
interaction: kinetic analysis of inhibition studies. Biochemistry 1991;
30: 5164–5171.
11 Yang FD, Spanevello RA, Celiker I, Hirschmann R, Rubin H,
Cooperman BS. The carboxyl terminus heptapeptide of the R2
subunit of mammalian ribonucleotide reductase inhibits enzyme
activity and can be used to purify the R1 subunit. FEBS Lett. 1990;
272: 61–64.
12 Gaudreau P, Michaud J, Cohen EA, Langelier Y, Brazeau P. Structure-
activity studies on synthetic peptides inhibiting herpes simplex virus
ribonucleotide reductase. J. Biol. Chem. 1987; 262: 12413–12416.
13 Yang F, Curran SC, Li LS, Avarbock D, Graf JD, Chua MM, Lu G,
Salem J, Rubin H. Characterization of two genes encoding the
Mycobacterium tuberculosis ribonucleotide reductase small subunit.
J. Bacteriol. 1997; 179: 6408–6415.
14 Nurbo J, Roos AK, Muthas D, Wahlstro¨m E, Ericsson DJ, Lundstedt T,
Unge T, Karle´n A. Design, synthesis and evaluation of peptide
inhibitors of Mycobacteriumtuberculosis ribonucleotide reductase. J.
Pept. Sci. 2007; 13: 822–832.
Furthermore, inspired by the retained affinity of dipeptide 7,
we decided to investigate single Fmoc-protected amino acids.
Interestingly, truncation down to single Fmoc-Phe (11) was not
tolerated. However, the use of amino acids with larger side chains
as in Fmoc-protected 3,3-diphenylalanine (12) and Fmoc-Trp (13)
was more successful, with KD2 values of 51 3 µM and 12.4 1 µM,
respectively. Fmoc-Gly (14) and Fmoc-Asp (15) which are smaller
and less hydrophobic were not able to compete for binding to R1.
TheFmoc-protecteddipeptide16wasalsopreparedtoinvestigate
if additional charged residues might be favorable for binding.
However, the affinity was too low for a dissociation constant to
be determined. Surprisingly, Fmoc-Trp (13) has a binding affinity
comparable with the acetylated heptapeptide 1 and is therefore
an interesting compound for further discovery of small inhibitors
targeting Mtb RNR.
Conclusions
15 Pender BA, Wu X, Axelsen PH, Cooperman BS. Toward a rational
design of peptide inhibitors of ribonucleotide reductase: structure-
function and modeling studies. J. Med. Chem. 2001; 44: 36–46.
16 Gao Y, Liehr S, Cooperman BS. Affinity-driven selection of tripeptide
inhibitors of ribonucleotide reductase. Bioorg. Med. Chem. Lett. 2002;
12: 513–515.
An assay suitable for screening potential Mtb RNR inhibitors
has been developed. Furthermore, we have demonstrated
that the introduction of an N-terminal Fmoc group in the
heptapeptide, corresponding to the C-terminus of Mtb R2 gives
c
J. Pept. Sci. 2010; 16: 159–164 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ERICSSON ET AL.
17 Tan C, Gao Y, Kaur J, Cooperman BS. More potent linear peptide
inhibitors of mammalian ribonucleotide reductase. Bioorg. Med.
Chem. Lett. 2004; 14: 5301–5304.
18 Roehrl MH, Kang S, Aramburu J, Wagner G, Rao A, Hogan PG.
Selective inhibition of calcineurin-NFAT signaling by blocking
protein-protein interaction with small organic molecules. Proc. Natl.
Acad. Sci. USA 2004; 101: 7554–7559.
20 Bowen WP, Jerman JC. Nonlinear regression using spreadsheets.
Trends Pharmacol. Sci. 1995; 16: 413–417.
21 Brown AM. A step-by-step guide to non-linear regression analysis
of experimental data using a Microsoft Excel spreadsheet. Comput.
Methods Programs Biomed. 2001; 65: 191–200.
22 Motulsky HJ, Ransnas LA. Fitting curves to data using nonlinear
regression: a practical and nonmathematical review. FASEB J. 1987;
1: 365–374.
19 Roehrl MH, Wang JY, Wagner G.
A general framework for
development and data analysis of competitive high-throughput
screens for small-molecule inhibitors of protein-protein interactions
by fluorescence polarization. Biochemistry 2004; 43: 16056–16066.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 159–164