chapter structure-based design of sialic...
TRANSCRIPT
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Chapter 5
Structure-based design of sialic acid
analogues as inhibitors to Vibrio cholerae
neuraminidase.
5.1 Introduction
The computer design of novel potent small molecules as potential new drug is
now a reality. Advances in structural and molecular biology have allowed
modern drug discovery efforts to focus on finding molecules that specifically
interact with particular targets (Gozalbes et al., 2002). The revolution in
biology over the past two decades has resulted in radically new approaches
and opportunities for drug discovery. Most drugs have been discovered in
random screens or by exploiting information about molecular receptors
(Kuntz, 1992). There has been an incredibly rapid increase in the rate of
determination of three-dimensional structures of biomolecUles. Many of these
macromolecules are important drug targets and it is now possible to use the
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knowledge of three-dimensional structures as a good basis for drug design.
With increasing computational power and availability of simulation software,
computer modeling gives a handy tool for studying enzyme inhibitor
interactions.
Enzyme substrate interactions can be intricately analysed if the three-
dimensional structure of an enzyme is known together with its active site
residues. This can be used to design substrate analogues that can effectively
bind to the active site and interact with the enzyme in the place of the
substrate itself (Martin et al., 1991). Despite the immense wealth of
information and the recent positive results that have come from the structural
knowledge, structure based drug design is still in its infancy. The design of
tight inhibitors has been impressively accomplished but the more stringent
requirements that a drug must fulfill in order to treat patients such as bio-
availability, toxicity, metabolism, half life are more difficult to address. It has
been estimated (Vagelos, 1991) that on average in a trial and error procedure
some 10000 compounds will be screened, 10 will go forward to trials and 1
may become a prescription medicine.
Zanamivir is the first anti-Influenza virus drug to reach the marketplace and is
an example of successful application of structure-based drug design (Elliott,
2001; Colman, 1999; Dunn and Goa, 1999; von ltzstein et al., 1993; Wade,
1997; Cheer and Wagstaff, 2002). The target for the drug is the
neuraminidase enzyme, which is involved, in viral replication in influenza
(Varghese, 1999). The active site residues of neuraminidase is highly
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conserved. In particular the eleven key amino acid residues that line the
shallow pocket and interact directly with the natural substrate (sialic acid) are
identical in all known influenza subtypes. This has made it an attractive target
for structure based rational drug design. The carbocyclic pro-drug oseltamivir
is an orally active inhibitor of influenza virus neuraminidase with inhibitory
properties similar to those of zanamivir (Kim et al., 1999; Gubareva et al.,
2001).
Derivatives, analogues and glycosides of N-acetyl neuraminic acid are of
interest as substrates and inhibitors for sialidases or sialyltransferases, and
potential modifiers of cell-surface sialic acid. Several of the sialic acid
analogues have been synthesised by different research groups and the
inhibition properties have been studied either in vivo or in vitro (Zbiral et al.,
1989; Smalec and von ltzstein, 1995; Taylor et al., 1998; Bianco, 2001;
Abdel-Magid, 2001). In a very recent review, Kiefel and von ltzstein (2002)
elaborately discussed the synthesis of various sialic acid derivatives such as
glycosides, thiosialosides, sialylmimetics which have been potential
applications as inhibitors for various sialic acid binding proteins.
Substrate analogue compound can also be designed as inhibitors to mimic
the activity of the enzymes. Glucose analogue compounds have been
sucessfully designed, synthesised and tested against the glycogen
phosphorylase enzyme to minimize its activity (Martin et al., 1991). The
substrate analogue compounds usually occupy the site where the substrate
usually binds.
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The pathogenic bacteria V. cholerae releases neuraminidase which cleaves
the sialic acid from higher gangliosides to monoganglioside GMI and thereby
facilitates the binding of CT to its receptor ganglioside GMI (Galen et al.,
1992). Hence blocking the activity of this enzyme may reduce the number of
receptor sites (CMI) for CT and may reflect in the reduction of virulence of
Cholera.
Bacterial sialidases represent important colonization or virulence factors. The
development of a rational basis for the design of antimicrobials targeted to
sialidases requires the knowledge of the exact roles of their conserved amino
acids. Since the interactions of sialyloligosaccharide substrate at the active
site of V. cholerae neuraminidase have been known as has been discussed in
the forgoing chapters, new inhibitors of the enzyme V. cholerae
neuraminidase have been designed on the basis of the knowledge of enzyme
structure and the mode of binding of substrate. Molecular dynamics
simulations can act as a tool in the computer-aided drug design projects
(Hansson et al., 2002).
5.2 Materials and methods
5.2.1 Structure of sialic acid analogues
The substrate analogue molecules that were selected for modeling in to the
active site of V. cholerae neuraminidase have been shown schematically in
Figure 5.1. The substituents at the C2 position were based on the work of
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0 \01
C1__H12
C1113
2
/:euc__z'/
o2_l2_fi3_oi3C6 06
C3I12 H13
C501
21112
,C2C12_13_N1_N2_N3
C6 /NeuNAc
112
/C4 C3
C5
01
1112 H13
3C2- 12L3 -013
C6 06/ H12 H13NeuNAc
,C4
C501 - 01
1112 013A H14
I I IC2_ Cl2-013-S13-c14-H14
0C6 6/NeuNAc
H12 13B
C3C4
C501 - 01" * H12 1113 1114Cl
©5C2 - C12-C13 -N14 -H14
//111C6 -- 06 H12 H13 H14/ NeuNAc /I C4 C3
Figure 5.1 Sialic acid analogue compounds
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Martin et al., (1991). These substituents are used as a probe to identify
potential drug targets for glycosylation phosphorylase enzyme, associated
with the diabetes, where they form the part of glucose molecule. The
numbering of compounds and the substituents at C2 position are given in
Table 5.1.
The coordinates for Vibrio cholerae neuraminidase were obtained from
Protein Data Bank; PDB ID: 1 KIT (Berman et al., 2000). The coordinates of
sialic acid as well as the analogue side chains were generated by using the
standard internal geometry and the structures were optimized using
HYPERCHEM, a molecular modeling software for windows environment. The
structure of the compounds obtained are further used for the docking studies,
which makes use of the modeling software INSIGHT II running on SGI 02.
5.2.2 Modeling/docking of sialic acid analogues into the active site
The protocol adopted to dock the substrate analogue molecules into the
active site of substrate-free V. cholerae neuraminidase may be as follows.
The knowledge from allowed orientations explored for sialic acid at the active
site of V. cholerae neuraminidase discussed in Chapter 4, have been used as
a primary step to dock the sialic acid analogues into the active site of V.
cholerae neuraminidase.
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Table 5.1
Sialic acid analogues
Compound Compound Substituents at C2no position
2-hydroxyethyl--D-siaIic acid
13-OCH2CH20H
2 -azidoethyl-2-deoxy sialic acid
13-CH2CH2N3
3 -hydroxyethyl-2-deoxy sialic acid 3-CH2CH20 H
4 -mesylate-2-deoxy sialic acid f3-CH20SO2C H3
5 3-aminoethyl-2-deoxy sialic acid -CH2CH2NH3+
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5.2.3 Molecular mechanics and Molecular dynamics of the V. cholerae
neuraminidase-sialic acid analogue complexes
Molecular dynamics simulations plays an important role in computer-aided
drug design (Hansson et al., 2002; Karplus and McCammon, 2002). The
complexed structures of Vibrio cholerae neuraminidase-sialic acid analogue
were further subjected to energy minimization. The molecular dynamics is
performed for the minimized structures over a period of lOOps at 300K.
Forcefield parameter used was AMBER (Weiner et al., 1984).
Crystallographic water molecules are included and a distance dependent
dielectric constant of 4*r, is used in all the computations. From the dynamics
trajectory, structures were collected systematically for every I Ops and are
further energy minimized. The energy minimized complexes thus obtained are
for the analysis of atomic level interactions
5.3 Results and discussion
5.3.1 Vibrio cholerae neuraminidase-com pound I complex
The simulated structure of the V. cholerae neuraminidase-compound I
complex is shown in Figure 5.2. It is obvious from the Figure 5.2 that the siaIic
acid in compound 1 has moved into the open channel (away from the active
site center) and its orientation is also changed by pushing the carboxylic acid
group towards the 3-channel (the position where the second residue of the
sialyloligosaccharide occupies). This has led to loss of few original
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ASP 250
ASN 318 ARG 245
ARG 224
PHE 638
A637
SER 760
TYR 740
ARG 635 THR 680
ARG 712
Figure 5.2 Compound I at the active site of Vibrio cholerae
neuram in idase
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interactions. However, the loss is compensated by forming hydrogen bonds
with other residues (Table 4.11 and Table 5.2). This gives an indication that
this compound can act as an inhibitor.
5.3.2 Vibrio cholerae neuraminidase-com pound 2 complex
The dynamics simulated and energy minimized structure of compound 2
bound to the V. cholerae neuraminidase is shown in Figure 5.3. This
compound has moved considerably out of the binding pocket, lies on the
surface of the neuraminidase. It was able to make very few hydrogen bonding
interactions (Table 5.3) and hence it can not act as an inhibitor.
5.3.3 Vibrio cholerae neuraminidase-compound 3 complex
In this complex, the substrate analogue tends to make good number of
profound interactions (hydrogen bonds) with the active site of V. cholerae
neuraminidase. The oxygen atom of the substituent group contribute two
additional hydrogen bonds with Arg224, a highly conserved active site residue
in neuraminidases. The structure of compound 3 bound to the V. cholerae
neuraminidase is shown in Figure 5.4. The favourable interactions it makes
with the active site is given in Table 5.4. There is also a water mediated
interaction that bridges N5 and ODI of Asp250. Analysis of the structures
revealed that the interactions mentioned in Table 5.4 are consistent in most of
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Table 5.2
The hydrogen bonding interactions observed for Compound I at the active
site of Vibrio cholerae neuraminidase
Inhibitor atoms Protein atoms Distance Protein atoms Distance(A) holding the water (A)
09 OH TYR 740 2.8
OHOH 964 2.9 NH2ARG 245 2.9
ODI ASP 250 2.7
08 OHTYR74O 2.9
N5 0D2 ASP 250 2.8
01S NHIARG224 2.8
NH2ARG224 2.8
OlD NH1ARG712 2.7
NI-12 ARG 712 2.9
OH TYR 740 3.3
02 NH2ARG712 2.8
013 OTHR 680 2.5
OG1THR680 2.9
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ASP 250
r
ARO 224
SER 760
ASN 318
ARG 245
THR 80
ARG 635
TYR 740
ARG 712
Figure 5.3 Compound 2 at the active site of Vibrio cholerae
neu ram in id ase
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Table 5.3
The hydrogen bonding interactions observed for Compound 2 at the active
site of Vibrio cholerae neuraminidase
Inhibitor atoms Protein atoms Distance Protein atoms Distance(A) holding the water (A) -
04 0 HOH 1458 2.8 0D2 ASP 250 2.8OIS NH1ARG224 2.9OlD NH1ARG7I2 2.7
NH2ARG712 3.3Ni OG SER 760 3.2N3 OG SER 760 3.3
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ASN
ASP 250 ARG 245
r -,,z0^-- ^^\\ PHE 638
ARO 224
ASP 637
SER 760
ARG 635
ARG 712
Figure 5.4 Compound 3 at the active site of Vibrio choleraeneuraminidase
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Table 5.4
The hydrogen bonding interactions observed for Compound 3 at the active
site of Vibrio cholerae neuraminidase
Inhibitor atoms Protein atoms Distance Protein atoms Distance(A) holding the water (A)
09 OGI THR 680 2.808 ODI ASP 637 2.7
NI-12 ARG 635 2.804 NEARG224 3.1
0D2 ASP 250 2.7N5 0 HOH 938 2.9 ODI ASP 250 2.8
010 OD2 ASP 25O 3.5OIS NH2 ARG 635 2.8
OH TYR 740 3.5OlD NHIARG7I2 2.8
NH2ARG7I2 2.8013 NHIARG224 2.9
NH2ARG224 2.9
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the structures collected during dynamics. This compound 3 can act as an
inhibitor better than compound 1.
5.3.4 Vibrio cholerae neuraminidase-compound 4 complex
It has been earlier reported that the thiosialosides shows resistant to
enzymatic degradation by V. cholerae neuraminidase based on NMR studies
(Suzuki et al., 1990; Wilson et al., 1999). In thiosialosides, at the C2 position
sulphur atom is present instead of 02 in sialic acid. However, in compound 4,
at the C2 position of sialic acid the attached substituent group, -CH2-0-S02-
CH3 consists of a sulphur atom. Analysis of the interactions of this long group
with the active site reveals that the conserved active site residues Arg224 and
Asp250 are involved in direct hydrogen bonding interactions with 012 and
013A. The potentially strong hydrogen bond formed between 013A and the
main chain N of Asp250 is found to be consistent. 013B is involved in water
mediated interaction with Asp250 or Arg712, however this interaction is
observed for a short duration. Some of the favourable interactions it makes
with the active site is given in Table 5.5. The structure of compound 4 bound
to the V. cholerae neuraminidase is shown in Figure 5.5. Based on the
interactions it is having with the active site, this compound is proposed to act
as an inhibitor. Compound can be a better substrate than compound I and
compound 3.
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Table 5.5
The hydrogen bonding interactions observed for Compound 4 at the active
site of Vibrio cholerae neuraminidase
Inhibitor atoms Protein atoms Distance Protein atoms Distance(A) holding the water (A)
08 0D2 ASP 637 3.107 ND2ASN 318 2.804 NI-12 ARG 245 2.8
0 HOH 964
2.9N5
0E2 GLU 619
2.7
0 HOH 935
2.8ols
OH TYR 740
2.9
NI-12 ARG 712
2.7LIH]
NH1 ARG 712
2.8
NI-12 ARG 224
2.9012 NH1ARG224 2.9
S013A NASP 250 3.1
OD1 ASP 292 2.8
OG SER 291 2.9
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24
kSER 760
48
ASP 63
ARG 245ASN 318
Figure 5.5 Compound 4 at the active site of Vibrio cholerae
neu rami nidase
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5.3.5 Vibrio cholerae neuraminidase-compound 5 complex
The structure of compound 5 bound to the V. cholerae neuraminidase is
shown in Figure 5.6. Since the nitrogen is smaller than sulfur there may be
difficulties in maintaining the key interactions of compound 5 with the
protein. However, the nitrogen N14 at the substituent group is having an
interaction with 0D2 of Asp637. Initially this particular interaction is between
N14 and Asp250. The anticipated interactions haven not been maintained
during dynamics. Analysis of the dynamics trajectory reveals that this
compound can not be accommodated in the active site, because it has been
moved out to the surface and takes unusual orientations and hence may not
be able to act as a suitable inhibitor. Only a few interactions have been
observed (Table 5.6).
5.4 Conclusion
Sialic acid analogue compounds have been designed as inhibitors to Vibrio
cholerae neuraminidase enzyme using modeling tools such as molecular
mechanics and molecular dynamics. The compounds modeled are 2-
hydroxyethy3-D-sialic acid (1), 3-azidoethyI-2-deoxy sialic acid (2), 13-
hydroxyethyl-2-deoxy sialic acid (3), 13-mesylate-2-deoxy sialic acid (4) and 13-
aminoethyl-2-deoxy sialic acid (5). The analysis of the favourable interactions
and the stability of the structure at the active site during dynamics enable us
to envisage the order of inhibitory potency as compound 4 > compound 3>
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PHE638
j
ASP 250 ARG 245
RG 224
ER 760
TYR 740
I- MI,
ARG 635
ASP 637 4^
THR
ARG 712
Figure 5.6 Compound 5 at the active site of Vibrio cholerae
neurami n idase
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Table 5.6
The hydrogen bonding interactions observed for Compound 5 at the active
site of Vibrio cholerae neuraminidase
Inhibitor atoms Protein atoms Distance(A)
09 0ASN318 3.2
08 NGLY319 3.0
04 0D2 ASP 250 2.7
N5 0D2 ASP 250 3.0
OlD ND2ASN 318 3.0
N14 0D2 ASP 637 2.7
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compound I > compound 2 > compound 5. The ideal candidature for
synthesising and testing will be compound 4 and compound 3.
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Conclusions
The major conclusions arrived from the present investigation are outlined
below. The global minimum energy conformations of the disaccharide
fragments of sialyloligosaccharides NeuNAca(2-3)Gal, NeuNAca(2-6)Gal,
NeuNAca(2-8)NeuNAc, and NeuNAca(2-9)NeuNAc arrived using molecular
mechanics and molecular dynamics simulation reveals that, the glycosidic
torsion angles of these disaccharides share a common conformational space
(region B) in the steric map (Ramachandran plot or (,ii) plot). These
disaccharide fragments have local minima in the other conformational region
A. In the case of NeuNAac(2-9)NeuNAc, in addition to region A, a local
minima occurs in region C and it is a high energy region and this
conformational structure is accessible for a relatively short duration during
dynamics simulations. A highly conserved water mediated hydrogen bonding
between the sugar residue in the disaccharide fragments also helps to
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maintain these structures in a common conformational space. The
conformational preference of these disaccharides in a common
conformational space irrespective of the type of linkage leads to a structural
similarity among them. This structural similarity is accounted for
neuraminidases lacking linkage specificity.
The influenza virus N9 neuraminidase was able to accommodate the sialic
acid in a highly restricted spatial orientation in the Eulerian space at the active
site. The percentage of allowed Eulerian space for sialic acid at the active site
is less than 1. Molecular mechanics and molecular dynamics calculations of
neuraminidases with NeuNAco(2-3)Gal, NeuNAca(2-6)Gal, NeuNAca(2-
8)NeuNAc and NeuNAca(2-9)NeuNAc at the active site reveals that the
enzyme can accommodate these disaccharide fragments without any
stereochemical clash but with good number of hydrogen bonds between the
protein atoms and the substrate atoms. These disaccharides at the active site
of influenza virus N9 neuraminidase predominantly prefer a common
glycosidic conformational region. This common glycosidic conformation
among these disaccharide fragments leads to a structural similarity, perhaps
is an essential requirement for the neuraminidase enzyme to recognize and
cleave sialic acid irrespective of the type of linkage.
The active site of Salmonella typhimurium LT2 neuraminidase and Vibrio
cholerae neuraminidase was able to accommodate the sialic acid in a highly
restricted spatial orientation. The percentage of allowed Eulerian spaces are
1.7 and 0.3 respectively for sialic acid at the active site of Salmonella
typhimurium LT2 and Vibrio cholerae neuraminidases. These enzymes have
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enough space to accommodate the disaccharide fragments of
sialyloligosaccharides NeuNAcc(2-3)Gal, NeuNAca(2-6)Gal, Neu NAca(2-
8)NeuNAc and NeuNAca(2-9)NeuNAc. These enzymes can accommodate
the second residue in the 13-channel. The molecular mechanics calculations
indicated that NeuNAcc,(2-3)Gal is a better substrate than NeuNAcc(2-6)Gal
for both the neuraminidases and are also substantiated by molecular
dynamics calculations. The many fold cleavage of NeuNAcx(2-3)Gal
substrate by Salmonella typhimurium LT2 neuraminidase than NeuNAccr(2-
6)Gal has been accounted for the partial hydrophobic interaction between
galactose and His278 and to the movement of Tyr307. The molecular
mechanics and molecular dynamics calculations reveal that NeuNAca(2-
9)NeuNAc is a better substrate than NeuNAca(2-8)NeuNAc for Salmonella
typhimurium LT2 neuraminidase and Vibrio cholerae neuraminidase. The
lacking of linkage specificity of various neuraminidases is accounted for the
preference of common glycosidic conformation leading to a structural
similarity between these substrates.
Sialic acid analogue compounds have been designed as inhibitors to Vibrio
cholerae neuraminidase enzyme using modeling tools such as molecular
mechanics and molecular dynamics. The compounds modeled are 2-
hydroxyethyl-13-D-Sialic acid (1), 13-azidoethyl-2-deoxy sialic acid (2), 1 3
-hydroxyethyl-2-deoxy sialic acid (3), 13-mesylate-2-deoxy sialic acid (4) and f3-
aminoethyl-2-deoxy sialic acid (5). The analysis of the favourable interactions
and the stability of the structure at the active site during dynamics enable us
to envisage the order of inhibitory potency as compound 4 > compound 3 >
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