Inhibition of the exo-β-d-glucosaminidase CsxA by a glucosamine-configured castanospermine and an amino-australine analogue

Article abstract of DOI:10.1039/b913235j

The synthesis of amino-derivatives of castanospermine and australine and their characterisation as inhibitors of the exo-β-d-glucosaminidase CsxA through enzyme kinetics and X-ray structural analysis is described.

Full text of DOI:10.1039/b913235j

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Inhibition of the exo-b-D-glucosaminidase CsxA by a glucosamine-configured
castanospermine and an amino-australine analogue†
b
c
c
b
a
Benjamin Pluvinage, Mariana G. Ghinet, Ryszard Brzezinski, Alisdair B. Boraston and Keith A. Stubbs*
Received 6th July 2009, Accepted 4th August 2009
First published as an Advance Article on the web 14th August 2009
DOI: 10.1039/b913235j
The synthesis of amino-derivatives of castanospermine and
australine and their characterisation as inhibitors of the
exo-b-D-glucosaminidase CsxA through enzyme kinetics and
X-ray structural analysis is described.
mechanism of the enzyme and the basis for the enzyme’s specificity
for its substrate, chitosan.
21
CsxA, like other members of GH2, uses a catalytic mechanism
involving a glycosyl-enzyme intermediate with the overall reaction
proceeding in two ordered steps, each involving inversion of stere-
ochemistry at the anomeric centre such that the reaction proceeds
with overall retention of stereochemistry (Fig. 1A). One key
feature of the active site was the unique negatively charged pocket
that specifically accommodates the nitrogen of non-reducing
end D-glucosamine residues and which allows this enzyme to
discriminate between D-glucose, D-glucosamine and N-acetyl-D-
glucosamine. This feature was of interest to us as it formed a
potential starting point for the rational design of inhibitors of
CsxA. Another important consideration in the inhibitor design
was the choice of inhibitor scaffold. Multiple crystal structures
Polysaccharides are well known for their roles in the storage of
energy, in the provision of structural integrity and most notably, for
their complexity. Due to the nature of their overall complex struc-
tures there are a wide variety of enzymes that are involved in their
biosynthesis and degradation. The polysaccharides chitin and chi-
tosan, for example, are widely distributed in living organisms, in-
cluding insects, crustaceans, and fungi. These two macromolecules
are similar in that chitin is a linear polysaccharide composed
almost exclusively of b-1,4-N-acetyl-D-glucosamine residues while
chitosan is a partly or totally N-deacetylated chitin derivative.
Similarly, the enzymes that process these two polysaccharides
are also abundant. Endo-chitinases and exo-chitinases (including
chitobiohydrolases and exo-b-N-acetyl-D-glucosaminidases),
22–24
have been determined for enzymes in GH2
(other enzymes
in this family include b-glucuronidases, b-mannosidases and b-
galactosidases) both in the native form as well as in complex with
25,26
1
–3
several inhibitors.
From this information it was decided that an
are involved primarily in the degradation of chitin,
with
4–7
appropriately designed imino-sugar would be a good candidate for
an inhibitor of CsxA. These compounds have proven to be good in-
hibitors of a wide range of enzymes, not only in GH2 but other GH
families as well. We felt a stable inhibitor of CsxA could be based
endo-chitosanases (found in organisms including bacteria and
fungi ) and exo-chitosanases (exo-b-D-glucosaminidases)
8–10
11–14
acting primarily to degrade chitosan.
Due the biological importance of these enzymes in the break-
down of these polysaccharides it would be of interest to design
small molecule inhibitors to be used as tools for establishing the
role these enzymes have in biological processes. For the chitinases,
there are examples of compounds that have been prepared that
27,28
on the indolizidine imino-sugar castanospermine 1
(Fig. 1B)
with the choice of this scaffold being made for several reasons.
Castanospermine 1 is a natural product readily obtained in large
27
quantities from the seeds of the Moreton Bay Chestnut tree, and
29
3,15–18
it is a potent inhibitor of retaining b-glucosidases. Furthermore,
polyhydroxylated pyrrolizidine alkaloids such as australine 2
are inhibitors of both the enzymes that act on chitin,
but
quite surprisingly little work has gone into the development of
compounds as inhibitors for chitosanases, especially the exo-b-D-
glucosaminidases. One reason for this is, in part, due to the lack of
crystallographic information available that describes the molecular
basis of substrate recognition by this class of enzyme. Recently
an exo-b-D-glucosaminidase from Amycolatopsis orientalis, CsxA,
(Fig. 1B), another potential inhibitor scaffold for CsxA, can
be readily obtained synthetically from castanospermine 1. In
consideration of this scaffold, we synthesized the novel castano-
spermine and australine analogues 3 and 4 (Fig. 1C) respectively
30
from the known triacetate 5 (Scheme 1).
19,20
The triacetate 5 was prepared from castanospermine 1 accord-
which is a member of family 2 (GH2)
of the glycoside
30
ing to a known procedure. Reaction of 5 with methanesulfonyl
chloride in pyridine afforded cleanly the mesylate 6. Treatment
hydrolases, was crystallized in complex with a reaction product
and a natural substrate, which has allowed insight into the catalytic
30
of 6 with NaN in DMSO, using a known procedure, gave a
3
separable mixture of the desired azido-castanospermine 7 and the
azido-australine analogue 8. The preparation of the desired amino-
a
Chemistry M313, School of Biomedical, Biomolecular and Chemical
Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley,
WA, Australia 6009. E-mail: kstubbs@cyllene.uwa.edu.au; Tel: +61 8 6488
30
castanospermine 3 followed a literature procedure but instead
of removing the acetyl protecting groups from 9 with methanolic
sodium methoxide solution, we found that in our hands a saturated
solution of ammonia in methanol gave a far better result. For the
amino-australine derivative 4, care had to be taken due to the
known acetyl migration from nitrogen to oxygen that occurs when
2
725
b
Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN
CSC, Victoria, BC, Canada V8W 3P6
c
Centre d’ E´ tude et de Valorisation de la Diversit e´ Microbienne, D e´ partement
de Biologie, Universit e´ de Sherbrooke, Sherbrooke, QC, Canada J1K 2R1
†
Electronic supplementary information (ESI) available: Experimental de-
30
tails, NMR spectra of new compounds used in assays and crystallographic
statistics table. See DOI: 10.1039/b913235j
8 is placed under reducing conditions. Removal of the acetyl
protecting groups first with a saturated solution of ammonia in
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Fig. 1 (A) CsxA uses a catalytic mechanism involving a glycosyl–enzyme intermediate. Both steps of the reaction occur with inversion of stereochemistry
at the anomeric centre such that the overall reaction proceeds with net retention of stereochemistry. (B) Structures of the known glycosidase inhibitors
castanospermine 1 and australine 2. (C) Structures of the corresponding amino analogues 3 and 4, relevant to the work described here.
3 2 3 2
Scheme 1 a) i. MsCl, pyridine; b) NaN , DMSO; c) H , Pd/C, toluene; d) NH , MeOH; e) H , Pd/C, MeOH.
methanol yielded 10, which was then reduced using an atmosphere
of hydrogen to give 4 in excellent yield.
To gain a more detailed understanding of the molecular basis for
these differences, we determined the three-dimensional structure
˚
Next we prepared a known substrate for exo-b-D-glucosaminid-
ases, 4-methylumbelliferyl 2-amino-2-deoxy-b-D-glucopyrano-
of CsxA in complex with 3 and 4. At 2.3 and 2.4 A resolutions
the electron densities of 3 and 4, respectively, were unambiguous,
allowing the inhibitors to be easily modelled into the active site
of CsxA (Fig. 3A and B). Notably, the structure of 4 with CsxA
is, to our knowledge, the first X-ray complex of a derivative of
australine with any enzyme.
31
side, and evaluated the inhibitors 3 and 4 against CsxA. We found
in both cases a clear pattern of competitive inhibition (Fig. 2A
and B). To our knowledge these compounds are the first rationally
designed inhibitors of exo-b-D-glucosaminidases. We found 3 to
be a potent competitive inhibitor of CsxA, with a K
2 nM (Fig. 2A). The amino-australine derivative 4 on the other
hand was only a modest inhibitor of CsxA with a K value of
75 ± 8 mM. Complementing this data was the fact that neither 1
nor 2 were capable of inhibiting the enzyme at a concentration of
i
value of 610 ±
A comparison of these inhibitor complexes with the previously
determined complex of CsxA with D-glucosamine revealed
a virtually identical set of protein–carbohydrate interactions
(Fig. 3C). Within the error limitations imparted by the resolutions
of these structures, there are no apparent large differences in
the inter-atomic distances. Indeed, all of these compounds also
1
i
1
1
mM.
2
˚
In light of these results we considered that the amino-australine
result in the burial of ~107 A of surface area in the active
derivative 4 may not bind as favourably to CsxA due to the
aminomethyl arm not fully utilizing the negatively charged pocket
that in the case of the natural substrate, chitosan, specifically
accommodates the nitrogen atom of D-glucosamine. Alternatively,
the conformational rigidity imposed by the ring system may not
allow 4 to adopt a conformation that maximises adventitious
interactions with the enzyme.
site, which is divided roughly 1:1 between polar to apolar
surface area. In particular, the amino groups of 3 and 4 are
found in virtually identical positions in the acidic pocket
formed by E394, E591, and D649 of the CsxA active site. This
interaction is thought to be particularly important in substrate
recognition, which is supported by our observation that 1
21
and 2 were incapable of inhibiting the enzyme. Given the very
4
170 | Org. Biomol. Chem., 2009, 7, 4169–4172
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similar binding modes of 3 and 4 and the capacity of the
amino groups of these compounds to make a potentially key
charge–charge interaction with the active site it is somewhat
surprising that the two compounds bind with an approximate
difference of 3.5 kcal/mole in their change in Gibbs free energy.
However, closer examination of the interactions made by the
amino groups on 3 and 4 provided some insight into the selectivity
CsxA shows between these two inhibitors. Though the amino
groups of 3 and 4 are positioned similarly, only the amino group
+
of 3 has the appropriate geometry in its protonated NH
3
form to
make three hydrogen bonds with all of the residues in the acidic
pocket (Fig. 3D). This is directly analogous to the key interaction
noted between CsxA and a D-glucosamine substrate that sits in the
21
-
1 subsite. In contrast, due to the bend in the aminomethyl arm
of 4, the geometry of the amino group is only suitable for making
a single hydrogen bond with E394 (Fig. 3E). The aminomethyl
arm of 4 has the potential for some flexibility, possibly allowing
it to make numerous transient interactions in the acidic pocket.
Analysis of the B-factor of this nitrogen atom revealed it to be
very similar to that of all of the atoms in 4 and the surrounding
atoms of the protein, indicating only one major conformation of
4
in the active site. These observations imply that charge–charge
complementarity in the acidic pocket is not the major driving force
between the interaction of substrates and inhibitors with CsxA and
that the primary difference in the effectiveness of 3 and 4 lies in
their different hydrogen bonding patterns in the acidic pocket of
the active site. We cannot, however, rule out the possible influences
that the different ring structures of 3 and 4 and the flexibilities
imparted therein might have on the energetic contributions to
their recognition by CsxA. Nevertheless, our observations here are
Fig. 2 (A) Inhibition of CsxA catalyzed hydrolysis of UMB-GlcN by 3
shows a pattern of competitive inhibition. The concentrations of 3 (mM)
used were 5.5 (ꢀ), 1.9 (♦), 1.0 (ꢀ), 0.4 (ꢁ), 0.2 (᭹), and 0.0 (᭺). Inset,
graphical analysis of K
i
from plotting values of K
M
apparent against
concentration of 3. (B) Similarly for 4, a pattern of competitive inhibition
is observed. The concentrations of 4 (mM) used were 2.5 (ꢀ), 1.4 (♦), 0.9
(
ꢀ), 0.4 (ꢁ), 0.2 (᭹), and 0.0 (᭺).
Fig. 3 Electron density of (A) 3 and (B) 4 shown with the aromatic amino acids in the CsxA active site in stick representation. The
-
˚
3
blue mesh shows maximum-likelihood/s
maximum-likelihood/s -weighted Fobs-Fcalc electron density maps at 3s (0.13 e /A for 3 and 0.18 e /A for 4) produced prior to modelling the
a
-weighted 2Fobs-Fcalc electron density maps at 1s (0.35 e /A for both inhibitors). Green mesh shows
-
˚
3
-
˚
3
a
inhibitors. (C) A divergent stereo view of an active site overlap of the complexes of 3 (green), 4 (blue), and glucosamine (orange; PDB ID 2VZS).
Hydrogen bonds are shown as dashed lines. The catalytic nucleophile is E541 and the acid/base D469. (D) Interactions between the amino group of 3
and the acidic pocket of CsxA. (E) Interactions between the amino group of 4 and the acidic pocket of CsxA. Hydrogen bonds are shown in panels D
and E as dashed lines and distances are shown in A˚ .
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consistent with our previous supposition that the unique hydrogen
bond accepting acidic pocket in the active site of CsxA and the
distinctive hydrogen bonding donor capacity of protonated amine
sugars are critical components of substrate, and now inhibitor,
recognition by CsxA.
In conclusion, we have developed two compounds one of which
is a potent inhibitor of CsxA, the exo-b-D-glucosaminidase from
A. orientalis. Collectively, the results obtained here suggest that
compounds having a suitably placed amino moiety, which take
full advantage of the unique, negatively charged pocket found in
these enzymes, will potentially also be potent inhibitors. Overall,
these inhibitors and further derivatives will be useful molecules for
studying exo-b-D-glucosaminidases.
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