Chapter Structure-based design of sialic...

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

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

107

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.

108

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

109

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

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.

11n

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+

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

111

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

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

112

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

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

Table 5.3

The hydrogen bonding interactions observed for Compound 2 at the active


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

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

Table 5.4




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

the structures collected during dynamics. This compound 3 can act as an

inhibitor better than compound 1.


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.

113

Table 5.5




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

24

kSER 760

48

ASP 63

ARG 245ASN 318


neu rami nidase


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>

PHE638

j

ASP 250 ARG 245

RG 224

ER 760

TYR 740

I- MI,

ARG 635

ASP 637 4^

THR

ARG 712


neurami n idase

Table 5.6



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

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

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

117

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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