Selective irreversible inhibition of fructose 1,6-bisphosphate aldolase from Trypanosoma brucei

Article abstract of DOI:10.1021/jm050237b

An irreversible competitive inhibitor hydroxynaphthaldehyde phosphate was synthesized that is highly selective against the glycolytic enzyme fructose 1,6-bisphosphate aldolase from Trypanosoma brucei (causative agent of sleeping sickness). Inhibition invo

Full text of DOI:10.1021/jm050237b

©
Copyright 2006 by the American Chemical Society
Volume 49, Number 5
March 9, 2006
Letters
Selective Irreversible Inhibition of Fructose
,6-Bisphosphate Aldolase from Trypanosoma
brucei
1
Chantal Dax, Francis Duffieux, Nicolas Chabot,†
†
‡,#
§
§
Mathieu Coincon, Jurgen Sygusch,
Paul A. M. Michels, and Casimir Blonski*
‡
,†
LSPCMIB, UMR-CNRS 5068, Groupe de Chimie Organique
Biologique, UniVersit e´ Paul Sabatier, Bat. IIR1, 118 Route de
Narbonne 31062, Toulouse Cedex 9, France, Department of
Biochemistry, UniVersity of Montreal, PaVillon Roger Gaudry, CP
6
128, Station Centre Ville, Montr e´ al, QC, Canada, H3C 3J7, and
Figure 1. Drugs currently in use to treat trypanosomiasis: 1,
pentamidine; 2, difluoromethylornithine; 3, melarsoprol; 4, nifurtimox;
5, suramin, sodium salt.
Research Unit for Tropical Diseases, Christian de DuVe Institute of
Cellular Pathology, and Laboratory of Biochemistry, UniVersite´
Catholique de LouVain, ICP-TROP 74-39, AVenue Hippocrate 74,
B-1200 Brussels, Belgium
by killing 3 million cattle per year. A number of drugs have
been used in the treatment of trypanosomal infections (Figure
ReceiVed March 15, 2005
1
).
Suramin, pentamidine, and some arsenical drugs often induce
Abstract: An irreversible competitive inhibitor hydroxynaphthaldehyde
phosphate was synthesized that is highly selective against the glycolytic
enzyme fructose 1,6-bisphosphate aldolase from Trypanosoma brucei
severe toxic side effects, and resistance against these drugs is
spreading. The drug most recently introduced (early 1990s)
2
(causative agent of sleeping sickness). Inhibition involves Schiff base
against sleeping sickness is (R,S)-2-difluoromethylornithine
formation by the inhibitor aldehyde with Lys116 followed by reaction
of the resultant Schiff base with a second residue. Molecular simulations
indicate significantly greater molecular geometries conducive for
nucleophilic attack in T. brucei aldolase than the mammalian isozyme
and suggest Ser48 as the Schiff base modifying residue.
3
(
DFMO, initially developed as an anticancer drug ). However,
this drug is only active against Trypanosoma brucei gambiense
(prevalent in west and central Africa) whereas T. b. rhodesiense
(
prevalent in east and southern Africa) is insensitive to this
4
compound. An additional problem is that large amounts of
DFMO are required to treat a patient. The need for broad
Among the many diseases that afflict humankind, those
caused by protozoan parasites occupy an important place
because of the large number of victims, the lack of efficient
therapy, and their continuing spread. The World Health Orga-
nization reported 55 000 deaths in 2002 out of 500 000 cases
for sleeping sickness in sub-Saharan Africa, caused by infection
5
spectrum, inexpensive, highly efficient, and nontoxic drugs
therefore remains a priority.
Among the potential metabolic targets considered for the
development of new drugs, glycolysis appears as a highly
promising pathway, since it is the only ATP-generating process
1
with Trypanosoma brucei. Trypanosomiasis also significantly
6
in the bloodstream form of T. brucei. It has been shown that
affects human nutrition through their impact on food animals
trypanosomal glycolysis contains several structural features
distinct from the corresponding process in mammalian cells,
which may allow for the design of selective compounds without
*
To whom correspondence should be addressed. Phone: 33561556486.
Fax: 33561556011. E-mail: blonski@cict.fr.
7
†
‡
#
liability in mammals. The approach we considered was to
Universit e´ Paul Sabatier.
Universit e´ Catholique de Louvain.
Present address: Groupe Sanofi Aventis, Protein Production and
inhibit a glycolytic enzyme with a compound targeting its active
site. We chose the fourth enzyme in the cascade as the target,
i.e., class I fructose 1,6-bisphosphate aldolase (EC 4.1.2.13).
Engineering, 13, Quai Jules Guesde, BP14 94403 Vitry-sur-Seine Cedex,
France.
§
8
University of Montreal.
On the basis of the literature and previous work from our
1
0.1021/jm050237b CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

1
500 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Letters
Scheme 1. Synthesis of T. brucei Aldolase Inhibitor 9a
Figure 2. Scheme for the interaction kinetics of 9 with T. brucei
aldolase.
Table 2. Selective Inhibition of Class I Aldolases by 9
enzyme (0.2 mg/mL)
residual activity (%)
Conditions: 100 µM 9, 15 min
rabbit muscle
human liver
100
100
<0.5
15
Trypanosoma brucei
Leishmania mexicana
Plasmodium falciparum
a
Reagents: (a) biphenylformamidine, acetone, 82% (6; see Supporting
30
Information); (b) H2SO4, H2O/ EtOH, 96%; (c) (EtO)3P, I2, pyridine,
CH2Cl2/THF, 31%; (d) Me3SiBr, CH2Cl2, H2O, NaOH, 90%.
Table 3. In Vitro Study of T. brucei Aldolase Protection against 9
Table 1. Interaction of TBK 1 with Rabbit Muscle (RM) and T. brucei
Inhibition by Its Substrates and Competitive Inhibitorsa
(Tb) Class I Aldolase
residual
activity (%) ( 5% at
incubation
time (min)
enzyme
(0.2 mg/mL)
residual
activity (%)
9 (mM)
compd
concn (mM)
0.250
5 min
15 min
5
1
RM
RM
Tb
80
80
100
100
<20
0
none
DHAP
15
56
0
48
1
1
1
1
5
5
5
5
5
5
5
0.1
0
1
1
1
1
1
0
1
.500
63
60
80
80
100
100
100
100
44
52
70
51
0.5
0
FBP
0.02
Tb
75
50
0
89
hexitol 1,6-bisphosphate (HBP)
100
100
100
.1
9
naphthyl 1,6-bisphosphate (NBP)
laboratory, we envisaged mimics of Schiff base formation
iminium ion) by use of phosphorylated naphthalene derivatives.
a
(
T. brucei aldolase, 0.2 mg/mL.
Such derivatives feature an aldehyde group for Schiff base
formation and whose resulting iminium ion is stabilized by the
presence of an OH group at the ortho position. Permutation
of the three functionalities (phosphate, aldehyde, and hydroxyl)
on the naphthalene skeleton allows for the design of lead
compounds with different functional topologies.
A number of such compounds were synthesized and assayed
on both T. brucei and rabbit muscle aldolase. Synthesis of the
most promising compound, 9 (TBK1) likely to form a Schiff
base with one of the three lysyl groups of the active site (Lys116,
Plasmodium falciparum) were inhibited, with the strongest effect
on T. brucei aldolase.
10
Compound 9 appears to be a very selective inhibitor of T.
brucei aldolase. Further analyses (see Supporting Information)
yielded inhibition constants K ) 23.03 ( 2.31 µM and k )
i
3
-
1
0.39 ( 0.04 min with k ≈ 0, values indicating a quasi-
4
irreversible behavior by 9 (Figure 2). We therefore concentrated
our efforts on T. brucei aldolase.
(2) To determine the inhibition mode of 9, we analyzed the
protection of T. brucei aldolase against inhibition by its
-156, and -239 in T. brucei aldolase), is shown in Scheme 1.
Synthesis of 9 required, as starting material, 2,6-dihydrox-
substrates fructose 1,6-P (FBP) and dihydroxyacetone-P (DHAP)
2
ynaphthalene formylated at position 1 using diphenyl forma-
midine11 followed by hydrolysis of the resulting Schiff base
under acidic conditions to yield the aldehyde derivative 7. The
key step was selective monophosphorylation at position 6 of 7
to obtain the ester derivative 8 without a protecting group at
position 2. This was performed using the triethyl phosphite/
pyridine/iodine procedure.12 Compound 9 was then obtained by
the transesterification reaction of 8 using silyltrimethyl bromide
and followed by routine hydrolysis.
and by two strong competitive inhibitors, namely, hexitol-1,6-
P (HBP, an FBP analogue resulting from the reduction of the
2
1
3
FBP carbonyl group, K ) 0.45 µM) and naphthalene-2,6-P2
(NBP, K ) 0.28 µM). Compound 9 (100 µM) was incubated
i
8
i
with T. brucei aldolase and in the presence of different
concentrations of substrates (FBP, DHAP) or competitive
inhibitors (HBP, NBP). The residual activity was determined
after 5 and 15 min of incubation. The results are summarized
in Table 3 and are consistent with active site binding. FBP and
DHAP only partially protected the enzyme against inhibition.
By contrast, both competitive inhibitors, which cannot undergo
a retro aldolization reaction, totally protected the enzyme.
(3) To investigate the molecular process by which inhibition
occurs, we used UV-vis difference spectroscopy to probe the
aldolase-9 interaction. The spectrum corresponding to the
interaction of inhibitor with the T. brucei enzyme is shown in
Figure 3. Compared to the spectrum of 9 reacting with a lysine
mimic, ꢀ-aminocaproic acid (Supporting Information), the
spectrum for the reaction with the enzyme is different. In both
cases, however, the presence of isosbestic points in the spectra
indicates that complex formation proceeds without accumulation
of intermediates or side reactions.
The effect of 9 on aldolase can be summarized in six points.
(1) The inhibitory effect of 9 was investigated on mammalian
and parasite aldolases. Standard conditions were employed for
kinetic constant determination (see Supporting Information). A
time-dependent inhibition of the T. brucei enzyme was observed
at low concentration (20 µM) (Table 1) which could not be
reversed upon dialysis (see Supporting Information).
Rabbit muscle aldolase by comparison was not inhibited or
only very weakly at high inhibitor concentration (1 mM).
Compound 9 selectivity was further explored using aldolases
from other sources, and the results are summarized in Table 2.
In each case, 9 (100 µM) was incubated with enzyme at 0.2
mg/mL. No effect was observed on the mammalian enzymes
(
rabbit muscle and human liver), whereas the aldolases from
We hypothesized that 9 inhibition involves initial Schiff base
formation with enzyme followed by a nucleophilic attack by a
three protozoan parasites (T. brucei, Leishmania mexicana, and

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1501
Figure 3. UV-vis difference spectrum of 9. Reaction of T. brucei
aldolase (0.2 mg/mL) and 9 (50 µM) in TEA buffer, pH 7, ∆t ) 2
min. Absorption spectrum has λmax ) 278 and 356 nm with isosbestic
point at 262 nm.
second active site residue. Such a reaction sequence was reported
for naphthalene dicarboxaldehyde, which upon Schiff base
formation reacts with a cysteine residue.14 Similarly in the
1
5
reaction of pyridoxal phosphate with ethane-1,2-diamine, the
resulting iminium ion undergoes an intramolecular nucleophilic
attack by the second amino group to form a cyclic geminal
diamine. To explore such a possibility, we examined the reaction
of 9 with model compounds that are mimics of amino acid
residues: ethanolamine as a serine analogue, cysteamine as a
cysteine mimic, and ethane-1,2-diamine as a second lysyl group.
The difference spectrum of the reaction with 9 by ethanolamine
resembled closely the spectrum obtained with the enzyme that
is shown in Figure 3; resultant absorption maxima (λmax at 270
and 350 nm) and isosbestic point (λ ) 255 nm) were only
slightly shifted (∼7 nm) toward the UV. We therefore concluded
that the amino acid reacting with the intermediate iminium ion
formed between the enzyme and 9 is most likely a serine residue.
Further support for this conclusion is not inconsistent with the
observation below.
Figure 4. Michaelis complex formed in the T. brucei aldolase active
site with 9 inhibitor. The solvent-accessible surface of the protein is
depicted in gray. Dotted green lines represent hydrogen bonds or
electrostatic interactions, and active site residues proximal to 9 are
labeled. The red arrow shows the presumed trajectory corresponding
7
to nucleophilic attack by Lys116 Nz on the electrophilic C aldehyde
carbon. Lys116 is oriented by two hydrogen bonds made by its Nz
atom to carboxylate and backbone oxygens of Asp43 (one is hidden
by the red arrow) and that is pointing its free electron pair in the
direction of the 9 C
of Ser48 to the C
http://pymol.sourceforge.net/).
7
atom. The white dotted line indicates the proximity
7
carbon of 9. The drawing was made with PyMOL
(
simulations16 using the minimized aldolase complexes as the
starting point. Simulations sought to identify configurations of
9 in the active site that are consistent with incipient Schiff base
formation, using a protocol previously described to analyze the
reactivity of a 9 isomer, HNA-P, with rabbit muscle aldolase.10
For all simulations, the reactive lysine residue was modeled in
its nucleophilic form consistent with a pKa of ∼8 for Lys107
(4) The formation of enzyme-inhibitor complexes was
studied by electrospray mass spectrometry with the recombinant
L. mexicana aldolase, which exhibits the same inhibition pattern
as the T. brucei enzyme (see point 1). Mass spectrometric
analyses with the recombinant Trypanosoma protein were
unsuccessful. The results obtained with the recombinant L.
mexicana aldolase corresponded to an observed ∆m/z of 255
between the free enzyme and the 9-enzyme complex (see
Supporting Information) and indicate a single inhibitor molecule
bound per subunit of enzyme. These data are not inconsistent
with the formation of the iminium ion concomitant with the
loss of a water molecule and that of the putative internal adduct
because both have the same mass as the iminium ion intermedi-
ate.
1
0
in the mammalian aldolase. Energy minimizations yielded a
stable Michaelis complex with 9, free of close contacts, filling
the entire active site cleft and necessitated the expulsion of only
several water molecules in the crystal structure. Simulations of
5 ns duration for the parasite and mammalian aldolases showed
the phosphate moiety to be persistently bound in its binding
site, making identical electrostatic and hydrogen-bonding in-
teraction with active site residues in both isozymes. We then
analyzed the trajectories generated by the simulation for
coordinate frames whose geometries are conducive for nucleo-
philic attack in T. brucei and rabbit muscle enzymes.10 Only
3% of the frames analyzed displayed geometries consistent with
nucleophilic attack by Lys116 in the T. brucei enzyme (Sup-
porting Information). A representative frame is shown in Figure
4.
(5) To determine which residue was involved in Schiff base
formation, point mutations were made of active site lysine
residues in the recombinant T. brucei enzyme. Active site lysine
residues were mutated to methionine, yielding constructs
K116M, K156M, and K239M. Compound 9 was incubated with
each construct, and the corresponding UV-vis difference spectra
were compared to that obtained with the wild-type (Figure 3).
For mutants K156M and K239M, the spectra were identical to
that of the wild-type enzyme. By contrast, K116M yielded a
different spectrum, indicating that Lys116 is the active-site
residue responsible for Schiff base formation. This result was
corroborated by the observation that the K116M mutant was
not inhibited by 9.
Notable is the reaction geometry in Figure 4 that places
Lys116 nearly perpendicular to the aldehyde of 9 at a distance
of 3.36 Å. This orientation is favorable for Schiff base formation
in the first step of the inhibition mechanism. A similar
percentage of frames (1.8%) suitable for nucleophilic attack by
Lys107 was noted in the HNA-P Michaelis complex with
1
0
(6) Finally, to gain insight into the preferential reactivity of
mammalian aldolase. Moreover, the intermolecular hydrogen
bond between the hydroxyl and the aldehyde on the naphthalene
skeleton of 9 (not shown in Figure 4) stabilizes the carbonyl
group orientation for nucleophilic attack by Lys116. In model
systems, the presence of a hydroxyl ortho to the aldehyde is
9
toward parasite aldolases, we docked it into the crystal-
lographic structures of rabbit muscle and T. brucei aldolases,
minimized the interaction energies of resulting complexes, and
then compared the trajectories of the molecular dynamics

1
502 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Letters
important for formation and Schiff base stabilization.10 Surpris-
ingly, no frame was found suitable for nucleophilic attack in
the rabbit muscle aldolase simulation of the Michaelis complex
with 9 and is consistent with poor reactivity by 9 in the
mammalian isozyme. Although active sites differ in one residue
(4) Sjoerdsma, A.; Schechter, P. J. Chemotherapeutic implication of
polyamine biosynthesis inhibition. Clin. Pharmacol. Ther. 1984, 35,
287-300.
(5) Van Nieuwenhove, S. Clinical evaluation of eflornithine in Trypa-
nosomiasis. Trans. R. Soc. Trop. Med. Hyg. 1985, 79, 692-696.
(6) (a) Visser, N.; Opperdoes, F. R. Glycolysis in Trypanosoma brucei.
Eur. J. Biochem. 1980, 103, 623-632. (b) Verlinde, C. L.; Hannaert,
V.; Blonski, C.; Willson, M.; Perie, J. J.; Fothergill-Gilmore, L. A.;
Opperdoes, F. R.; Gelb, M. H.; Hol, W. G.; Michels, PAM.
Glycolysis as a target for the design of new anti-trypanosome drugs.
Drug Resist. Updates 2001, 4, 50-65.
(Gly302 in the mammalian enzyme being replaced by a bulkier
Ala312 in T. brucei aldolase), analysis of dynamical trajectories
did not reveal conformational differences in the positioning of
9
with respect to CR atoms of these residues, suggesting that
this amino acid change may not account for the observed
reactivity differences. Reaction of 9 with the malarial aldolase
whose active site composition is identical to that of mammalian
aldolase would corroborate this conclusion.
(7) Opperdoes, F. R.; Baudhuin, P.; Coppens, J.; De Roe, C.; Edwards,
S. W.; Weijers, P. J.; Misset, O. Purification, morphometric analysis
and characterization of the glycosomes of the protozoan hemofla-
gallate Trypanosoma brucei. J. Cell Biol. 1984, 98, 1178-1184.
(8) Shu, B.; Barker, R. Fluorescence studies of the binding of alkyls
The reactant configuration in Figure 4 places the γ-OH of
Ser48 within van der Waals contact of the 9 carbonyl.
Intriguingly, Ser48 oriented opposite and nearly perpendicular
to the aldehyde would be well-positioned to interact with the
nascent iminium ion and could be the residue that reacts with
the Schiff base, leading to the final intermediate in the slow
binding inhibition mechanism.
In conclusion, we have synthesized a selective time-dependent
inhibitor of T. brucei aldolase that does not significantly inhibit
mammalian aldolase activity. We identified Lys116 as being
responsible for Schiff base formation. Molecular dynamics
suggest that differences in stabilization of reaction geometries
are responsible for the differential reactivity of 9 with T. brucei
aldolase compared to rabbit muscle aldolase and that Ser48 is
most likely the reactive residue in the second step of the
inhibition mechanism. MS experiments performed on L. mexi-
cana aldolase support these conclusions. Compound 9 and its
and aryl phosphates to rat muscle aldolase. J. Biol. Chem. 1971, 246,
7041-7045.
(
9) (a) Blonski, C.; Gefflaut, T.; P e´ ri e´ , J. Effects of chirality and
substituents at carbon 3 in dihydroxyacetone-phosphate analogues
on their binding to rabbit muscle aldolase. Bioorg. Med. Chem. 1995,
3, 1247-1253. (b) Gefflaut, T.; Blonski, C.; Perie, J. Slow reversible
inhibitions of rabbit muscle aldolase with substrate analogues:
synthesis, enzymatic kinetics and UV difference spectroscopy studies.
Bioorg. Med. Chem. 1996, 4, 2043-2054. (c) Gefflaut, T.; Blonski,
C.; P e´ ri e´ , J.; Willson, M. Class I aldolases: substrate specificity,
mechanism, inhibitors and structural aspects. Prog. Biophys. Mol.
Biol. 1995, 63, 301-340.
(
10) (a) Blonski, C.; Moissac, D.; Perie, J.; Sygusch, J. Inhibition of rabbit
muscle aldolase by phosphorylated aromatic compounds. Biochem.
J. 1997, 323, 71-77. (b) Dax, C.; Coincon, M.; Sygusch, J.; Blonski,
C. Hydroxynaphthaldehyde phosphate derivatives as potent covalent
Schiff base inhibitors of fructose-1,6-bisphosphate aldolase. Bio-
chemistry 2005, 44, 5430-5443.
11) (a) Shoesmith, J. B.; Haldane, J. Condensation of diphenyl forma-
midine with phenol. Part I. A new synthesis of â-resorcy-aldehyde.
J. Chem. Soc. 1923, 123, 2704-2707. (b) Shoesmith, J. B.; Aldane,
J. Condensation of diphenyl formamidine with phenol. Part II. The
general nature of the reaction. J. Chem. Soc. 1924, 125, 2405-2407.
12) (a) Stowell, J. K.; Wildlanski, T. S. A new method for the
phosphorylation of alcohols and phenols. Tetrahedron Lett. 1995,
(
“
prodrug” analogues will be tested in in vitro cultures of T.
brucei to determine if it causes growth retardation.
(
Acknowledgment. This work was funded by the European
Commission, through its programs INCO-DC (Contract IC18-
CT97-0220) and INCO-DEV (Contract ICA4-CT-2001-10075)
to C.B. and P.A.M.M., and by a grant from the National
Scientific Research and Engineering Council (NSERC) of
Canada to J.S. We thank the R e´ seau Qu e´ b e´ cois de Calcul de
Haute Performance (RQCHP) for computational resources for
the molecular dynamical simulations. The authors are grateful
to Professor J. Poupaert, Universit e´ Catholique de Louvain,
Brussels, Belgium, for helpful discussions.
36, 1825. (b) Ladame, S.; Claustre, S.; Willson, M. Selective
phosphorylation on primary alcohols of unprotected polyols. Phos-
phorus, Sulfur Silicon Relat. Elem. 2001, 174, 37-47.
(
13) Ginsburg, A.; Mehler, A. H. Specific anion binding to fructose
bisphosphate aldolase from rabbit muscle. Biochemistry 1966, 5,
2623-2634.
(
14) McCurdy, C. R.; Le Bourdonnec, B.; Metzger, T. G.; El Kouhen,
R.; Zhang, Y.; Law, P. Y.; Portoghese, P. S. Naphthalene dicarbox-
aldehyde as an electrophilic fluorogenic moiety for affinity label-
ling: application to opioid receptor affinity labels with greatly
improved fluorogenic properties. J. Med. Chem. 2002, 45, 2887-
2890.
1
(15) (a) Tobias, P. S.; Kallen, R. G. Kinetics and equilibria of the reaction
of pyridoxal 5′-phosphate with ethylenediamine to form Schiff bases
and cyclic geminal diamines: evidence for kinetically competent
geminal diamine intermediates in transimination sequences. J. Am.
Chem. Soc. 1975, 97, 6530-6539. (b) McQuate, R. S.; Leussing, D.
L. Kinetic and equilibrium studies on the formation of zinc(II)
salicylaldehyde Schiff base derived from ethylenediamine and 1,3-
diaminopropane. J. Am. Chem. Soc. 1975, 97, 5117-5125.
Supporting Information Available: Synthetic protocols, H
and 13C NMR shifts, MS data and elemental analysis results of
synthesized compounds, molecular biology and enzymology pro-
cedures, and molecular modeling. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(
1) World Health Organization report on Tropical Diseases Research
TDR) program (2002).
(16) (a) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio,
C.; Alagona, G.; Profeta, S. J.; Weiner, P. A. New force field for
molecular mechanical simulation of nucleic acids and proteins. J.
Am. Chem. Soc. 1984, 106, 765-784. (b) Weiner, S. J.; Kollman, P.
A.; Nguyen, D. T.; Case, D. A. An all atom force field for simulation
of proteins and nucleic acids. J. Comput. Chem. 1986, 7, 230-252.
(
(
2) (a) Howells, R. E. The modes of action of anti-protozoal drugs.
Parasitology 1985, 90, 687-703. (b) Neujean, G. Chemotherapy and
chemoprophylaxis of sleeping sickness caused by Trypanosoma
gambiense. ReV. Med. Liege 1959, 14, 5-13.
(
3) Milord, F.; Pepin, J.; Loko, L.; Ethier, L.; Mpia, B. Efficacy and
toxicity of eflornithine for treatment of Trypanosoma brucei gam-
biense sleeping sickness. Lancet 1992, 340, 652-655.
JM050237B