A new generation of specific Trypanosoma cruzi trans-sialidase inhibitors

Article abstract of DOI:10.1002/anie.200705435

(Chemical Equation Presented) A substitution for inhibition: The incorporation of anaryl substituent at C9 of 3-fluorosialyl fluorides provides specificity and dramatically slows the reactivation of the glycosylphosphatidylinositol-anchored surface protein Trypanosoma cruzi trans-sialidase (TcTS) by transglycosylation (see picture). X-ray crystallographic analysis of the trapped intermediate has provided a structural rationale for this behavior.

Full text of DOI:10.1002/anie.200705435

Communications
DOI: 10.1002/anie.200705435
Enzyme Inhibitors
A New Generation of Specific Trypanosoma cruzi trans-Sialidase
Inhibitors**
Sabrina Buchini, Alejandro Buschiazzo, and Stephen G. Withers*
The protozoan parasite Trypanosoma cruzi is the causative
agent of human American trypanosomiasis, also known as
Chagasꢀ disease, which in its chronic form can lead to severe
debilitation and ultimately death.[1] According to World
Health Organization (WHO) estimates, 16 to 18 million
people were infected in Latin America in 2005. Only two
approved drugs (nifurtimox and benznidazole) are currently
used for the treatment of the infection, but both display low
efficacy and are associated with severe undesired side
effects.[2] The whole genome sequence of Trypanosoma
cruzi, completed in 2005, contains genes predicted to
encode almost 23000 proteins.[3] The identification of essen-
tial proteins specific to the parasite will hopefully provide
attractive new drug targets. Among such targets, the Trypa-
nosoma cruzi trans-sialidase (TcTS) has been extensively
studied. TcTS is a glycosylphosphatidylinositol-anchored
surface protein that is differentially expressed during the
infective developmental stage of the parasite.[4] It belongs to
the glycoside hydrolase family 33 (http://afmb.cnrs-mrs.fr/
CAZY) and catalyzes the a-(2,3) transfer of sialic acid
residues from host glycoconjugates to the terminal galactosyl
units of mucin-like glycoproteins on the surface of the
parasite.[5] Extensive sialylation of the T. cruzi surface is
pivotal for the establishment of a chronic infection. The
crystal structure of TcTS was recently solved, and a two-step,
double-displacement mechanism involving a covalent sialyl–
enzyme intermediate was demonstrated (Scheme 1).[6] Tyr342
was identified as the catalytic nucleophile by the use of 3-
fluorosialyl fluoride 1 as a substrate analogue, which forms a
trapped intermediate species (Scheme 1).[7] The fluorine atom
at C3 inductively destabilizes the positively charged oxocar-
benium ion like transition states, thereby slowing both the
formation and the hydrolysis of the covalent intermediate.
However, the presence of a good leaving group at C2 allows
glycosylation to proceed rapidly and results in the trapping of
Scheme 1. Mechanism of action of T. cruzi trans-sialidase.
the sialyl–enzyme intermediate. Compounds of this class
therefore have the potential to act as anti-trypanosomal
agents if they can be made selective for TcTS over human
sialidases and if they form long-lived intermediates.
[*] Dr. S. Buchini, Prof. Dr. S. G. Withers
Department of Chemistry
University of British Columbia
2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 (Canada)
Fax: (+1)604-822-8869
3-Fluorosialyl fluoride 1 was shown previously to inacti-
vate wild-type TcTS in a time-dependent manner according to
the kinetic model illustrated in Scheme 2.[8] However, after
removal of excess inactivator, the enzyme rapidly recovered
full activity by transglycosylation when incubated with the
natural acceptor lactose. Such rapid transglycosylation would
render these compounds useless as anti-trypanosomal agents.
Inspection of the three-dimensional structures of TcTS
and the only human sialidase as to yet be characterized
crystallographically, Neu2,[9] revealed that the TcTS site is
more spacious and hydrophobic in the region around C9 of
E-mail: withers@chem.ubc.ca
Dr. A. Buschiazzo
Laboratory of Structural Biology
PasteurInstitute of Montevideo
2020 Mataojo, Montevideo, 11400 (Uruguay)
[**] We thank the Canadian Institutes forHealth Research and the Swiss
National Science Foundation forfinancial support. We also thank
Soichi Wakatsuki fora sample of Neu2.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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commercially available N-acetylneuraminic acid aldolase
(Neu5Ac aldolase, EC: 4.1.3.3) to yield a mixture of 9-
azido-3-fluorosialic acids (axial/equatorial 5:1 to 10:1), which
were separated after protection. Selective deprotection of the
anomeric site with hydrazine acetate yielded the hemiacetal 5
which, upon treatment with DAST followed by ZemplØn
deacetylation and saponification, was converted into 6.
Catalytic hydrogenation afforded the corresponding 9-
amino derivative 7, which was coupled to several activated
esters to give 8, 9, and 10. The configurations at C2 and C3
were assigned on the basis of 1H and 19F NMR coupling
constants.
Incubation of TcTS with different concentrations of each
compound resulted in time-dependent decreases in the
enzyme activity, as expected for mechanism-based inhibitors
acting according to Scheme 2, and as shown for 8 in
Figure 1A. Saturation behavior was not observed, as inacti-
vation of the enzyme at high concentrations of the inactiva-
tors was too fast to allow reliable rate measurements.
Individual values of ki and Kd could not therefore be
determined. However, the second-order rate constant ki/Kd
was calculated from the slope of the plot of the apparent first-
order rate constants versus
Scheme 2. Kinetic scheme forinhibition. I-F: inhibitorfluoride,
E: enzyme, E-I: sialyl–enzyme intermediate.
the sialic acid than is Neu2. Incorporation of a larger
substituent, such as an aromatic group, at C9 might therefore
confer specificity. In addition, such a substitution might well
affect the positioning of the nearby Tyr119 group, which
moves upon binding of sialic acid and appears to be important
in the binding of lactose.[6,7] This could alter transglycosyla-
tion rate constants.
A second generation of 3-fluorosialyl fluoride derivatives
bearing various modifications at C9 was thus developed by
following a similar synthetic pathway to that used previously
for 1 (Scheme 3).[10] 6-Azido-6-deoxy-N-acetylmannosamine
(3)[11] was coupled with 3-fluoropyruvic acid using the
the concentration of 8 (Fig-
ure 1B).
As
summarized
in
Table 1, with the exception
of 7, the ki/Kd values for the
compounds tested are all of
the same order of magni-
tude, with 6 and 10 being the
most efficient in the series.
The presence of aryl sub-
stituents (8,9,10 ) had no
deleterious effect on the
inactivation, and in one
case improved it, while
clearly the presence of the
charged ammonium sub-
stituent at C9 (7) did.
Samples of TcTS that
had been inactivated with
each reagent and from
which excess inactivator
had been removed by gel
filtration were incubated in
the presence of different
concentrations of lactose
and the reactivation of
TcTS monitored. No reacti-
vation was observed in the
absence of lactose; further-
more, much higher concen-
trations of lactose were nec-
essary to promote significant
reactivation by transglycosy-
lation than was the case for 1
(Figure 1C, Table 1). Satu-
Scheme 3. Synthesis of the 3-fluorosialyl fluorides. a) TsCl, Py, 4 h, 08C, then Ac2O, 4 h, RT, 69%; b) TMS-N3,
TBAF, CH3CN, 9 h, 908C, 82%; c) NaOMe, MeOH, 2 h, 08C, 85%; d) F-pyruvate sodium salt, Neu5-aldolase
(23 Umgꢀ1), H2O, 10–20 h, RT, 93%–quantitative; e) Amberlite IR-120H, MeOH, overnight, RT, 80–88%;
f) Ac2O, Py, 20 h, 08C!RT, 62%; g) hydrazine acetate, CH2Cl2/MeOH, 6 h, 08C, 60–80%; h) DAST, CH2Cl2,
1 h, ꢀ308C, 60–80%; i) NaOMe, MeOH, 3 h, RT, then NaOH, 40 min, RT, quantitative; j) cat. Pd/C, MeOH,
H2, 7 h, RT, quantitative; k) N-(benzoyloxy)succinimide, Et3N, DMF, 5 h, RT, 68%; l) pentafluorophenyl-4-(tert-
butoxycarbonylamino)butanoate, Et3N, DMF, 4 h, RT, 83%; m) N-(benzoyloxy)succinimide, Et3N, DMF, 6 h,
RT, 52%; n) N-succimidyl-7-hydroxycoumarin-3-carboxylate, sodium carbonate buffer pH 8.5, DMF, 14 h, RT,
42%. Ts=toluene-4-sulfonyl, Py=pyridine, TMS=trimethylsilyl, TBAF=tetrabutylammonium fluoride,
DAST=diethylaminosulfur trifluoride.
ration
behavior
was
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Communications
space that would otherwise
accommodate the glucose moiety
of the lactose acceptor and inter-
acts hydrophobically with the
phenyl side chain of Tyr119,
thereby locking it in the confor-
mation seen when the lactose
binding site is filled (Figure 2B).
Interestingly a molecule of 4-(2-
hydroxyethyl)-1-piperazine-etha-
nesulfonic acid (hepes) is bound in
the galactose site in this case. The
9-benzoyl substituent therefore
seriously impairs the binding of
lactoside acceptors, drastically
reducing reactivation rates with-
out any deleterious effect on the
inactivation by the sialyl fluoride.
Figure 1. Time-dependent inactivation and reactivation of wild-type
TcTS by 8. The enzyme was incubated with the indicated concentra-
tions of 8, and aliquots assayed with 0.5 mm trifluoromethylumbelli-
*
feryl sialic acid substrate. A) Inactivation by 8 at 0.1 mm ( ), 1 mm
~
^
(&), 3 mm ( ), 5 mm ( ), 10 mm (N), and 20 mm (”). B) Plot of
pseudo-first-order inactivation kinetic constants (kiobs) from (A) versus
concentration of 8. C) Reactivation of TcTS in the presence of lactose
~
!
&
^
*
at 0 mm ( ), 1 mm ( ), 10 mm ( ), 30 mm ( ), 50 mm ( ), and
100 mm (<). D) Plot of the first order reactivation kinetic constants
(krobs) from (C) versus concentration of lactose.
observed only for the 9-hydroxy and 9-azido (1 and 6)
derivatives and in these cases individual values for the kinetic
parameters for reactivation by lactose (krLact) and KdLact could
be measured. For the other sialic acids, in the absence of
Lact
saturation, an accurate second-order rate constant krLact/Kd
was derived from the plot of the apparent rate constants
versus the lactose concentration (Figure 1D). Astonishingly
the presence of an aromatic functionality at C9 (in 8, 9, and
10) led to more than a 1000-fold decrease in the second-order
rate constants for reactivation by lactose. Although it was not
possible to accurately measure the individual kinetic param-
eters for reactivation (krLact and KdLact) it seems probable that
the major effect is on the binding of lactose (KdLact), since the
Km value for lactose with wild-type TcTS is about 40 mm (data
not shown) while the KdLact value in this system is likely over
100 mm.
Insight into the structural basis for the effect of the aryl
substituent at C9 was provided by X-ray crystallographic
analysis of the trapped glycosyl–enzyme intermediate (for
technical details, see the Supporting Information). The
structure measured at a resolution of 1.7 Š reveals that the
presence of the benzoyl substituent at C9 induces a reori-
entation of the whole glycerol side chain compared to the
unsubstituted covalent complex (Figure 2A).[7] This effect is a
direct consequence of the pocket (W120, T121, Q195, V203)
being too small to accommodate the bulky benzoyl group.
The final position of the glycerol group of the sialic acid
closely resembles that found in the Michaelis complexes of
TcTS with sialyl–lactose (SL) or 4-methylumbelliferyl a-d-
sialoside (MU-NANA).[7] The benzoyl group occupies the
Figure 2. X-ray structure of the covalent intermediate formed between
TcTS and BFN (8). 3D superpositions with related complexes are
shown to highlight the induced structural rearrangements as described
in the text. A) TcTS–BFN structure (colored according to atomic
elements) superimposed onto TcTS-3F-NANA (2AH2, in green). Note
the variation of the glycerol group of NANA. The final refined
2 mFobs-DFcalc Fourier map is contoured at 1.2s, only drawn around
the BFN and the hepes moieties for clarity. B) TcTS–BFN superim-
posed on to the TcTS–sialyl-2,3-lactose Michaelis complex (1SOI, in
violet), in the same orientation as in (A). Note the single conformation
of Tyr119, equivalent in the two structures. The benzoyl substituent in
BFN blocks the binding of lactose. BFN=9-benzoyl-3-fluoro-N-acetyl-
neuraminic acid.
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Table 1: Kinetic parameters for the reaction of wild-type TcTS with
Keywords: bioorganic chemistry · enzymes · inhibitors ·
kinetics · structure elucidation
.
3-fluorosialyl fluorides bearing substituents at C9.[a]
Compd
ki/Kd ”10ꢀ3
krLact ”10ꢀ3
[minꢀ1
KdLact [mm]
(ki/Kd)Lact ”10ꢀ3
[minꢀ1 mmꢀ1
]
]
[minꢀ1 mmꢀ1
]
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1
6
7
8
9
10
8.0ꢁ0.4
14ꢁ0.8
N.D.
2.9ꢁ0.2
3.2ꢁ0.5
14ꢁ1.8
120ꢁ16
23ꢁ5
N.D.
N.D.
N.D.
0.09ꢁ0.02
0.05ꢁ0.02
–
1330ꢁ345
460ꢁ210
–
0.8ꢁ0.05
0.3ꢁ0.02
0.3ꢁ0.02
>200[b]
>500[b]
>150[b]
N.D.
[a] All the experiments were carried out in 20 mm tris(hydroxymethyl)-
aminomethane (Tris)/30 mm NaCl buffer, pH 7.6 containing 5% bovine
serum albumin (BSA). [b] Extrapolated from the highest lactose
concentration. N.D.=not detectable.
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This is an ideal characteristic for trans-sialidase inactivators.
Furthermore, the ability to also incorporate a fluorescent
moiety may allow the monitoring of labeled trypanosomes
in vivo. Encouragingly, preliminary studies show that these
C9-substituted compounds inactivate the human sialidase
Neu2 some 150 times more slowly than do the parent
inactivators, as desired. With this proof of principle having
been established, current efforts are focused upon improving
inherent affinities (Kd) of the reagents to allow their use at
lower concentrations as potential anti-trypanosomals.
Received: November 28, 2007
Published online: February 25, 2008
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