INSILICO CHARACTERIZATION OF CD59 LIKE SNAKE VENOM PEPTIDE INHIBITORS TO TREAT AUTO IMMUNE DISORDERS

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INSILICO CHARACTERIZATION OF CD59 LIKE SNAKE VENOM PEPTIDE INHIBITORS TO TREAT AUTO IMMUNE DISORDERS

ABSTRACT

Present work is aimed for the treatment of auto immune diseases with special reference for the treatment of Rheumatoid Arthritis (RA) which is commonly seen all over the world. One such cause for the auto immune disease is the cascade activation of complement system which in turn activates CD59 which is called MAC inhibitory protein. This CD59 resembles the structure of snake venom neurotoxin (three finger toxin). Docking studies of various neurotoxins from Indian cobra has revealed that engineered neurotoxins can be targeted towards C8 and C9 which binds irreversibly and is very specific in nature.

INTRODUCTION

Autoimmune diseases are the third most common category of disease in the United States after cancer and heart disease (NIH2002). The National Institutes of Health (NIH) estimates that it affects approximately 8% of the US population (or 23.5 million) and the prevalence is rising every year. Rheumatoid Arthritis (RA) is one of the major variant of auto immune disorders. According to WHO 1% of world population and 3% of Indian population is suffering from RA.

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The complement system is part of both innate and adaptive immunity, made up of a large number of distinct plasma proteins that react with one another to opsonize pathogens and induce a series of inflammatory responses that help to fight infection. However, it may cause substantial injury when activated inappropriately and leads to autoimmune diseases (Kuby, 2000). In humans, complement system is well controlled by the host and is mainly mediated by complement regulatory proteins. One such regulatory glycoprotein called CD59, also known as MAC-inhibitory protein (MAC-IP), is a cell-surface molecule that protects host cells from complement-mediated lysis, by binding to and preventing the normal functioning of the complement proteins C8 and/or C9 which form part of a membrane penetrating assembly called the membrane attack complex (MAC). CD59 also has limited sequence homology to snake venom neurotoxins (Harrison, 1993), which are members of three-fingered proteins’ (TFPs) superfamily. Hence, toxins from snake venom sharing similar structure with CD59, which are members of three-fingered proteins, could become possible therapeutic peptides to treat complement mediated immune disorders.

Currently available drugs to treat complement mediated immune system disorders like corticosteroids (prednisone) and non-steroid drugs such as azathioprine, cyclophosphamide, mycophenolate, sirolimus or tacrolimus are often prescribed to control or reduce the immune system's response (as immunosuppressive agents) (Niethemmer 1999). Monoclonal antibodies based treatments are expensive and their prolonged usages have proven side effects (Trevor T. Hansel 2010). Hence there is an immediate need for alternative efficacious and selective therapy.

The number of venom components in venomous animals like snake, scorpion or cone snail ranges from 50-200 toxins (Tan et al., 2003). The natural library of toxins is thus estimated to contain millions of different toxins and variants. Since there is growing number of identified snake venom neurotoxin sequences, it is very difficult to study them by experimentation only. Detailed bioinformatics analysis offers a convenient methodology for efficient in silico preliminary analysis of possible function of toxins.

The common name ‘Cobra’ is applied to about 30 species of snakes in 7 genera within the family Elapidae. Following are the genera Boulengerina, Hemachatus, Naja, Ophiophagus, Aspidelaps, Pseudohaje and Walterinnesia. Naja comprises approximately 25 species and is the most widespread. Snakes are equipped with venomic armory to tackle different prey and predators in adverse natural world. The composition of snake’s venom is a cocktail of active proteins and polypeptides and non-enzymatic polypeptide like cytotoxins and short neurotoxin. These two components structurally resemble to three-finger protein superfamily specific scaffold.

Neurotoxins are the chemical or natural or synthetic agents which disrupt the transmission of signals betweenNeurons, causing numerous problems. Neurotoxins can affect the cell at any step of neural transmission i.e., presynaptic or postsynaptic. Present work is concentrated on the α-neurotoxins from the Indian cobra belonging to Naja genera. The α-neurotoxins from Indian cobra are categorized to be postsynaptic neurotoxin which occurs on the receiving end of a discharge across the synapse. These neurotoxins mimic the shape of the acetylcholine molecule and fit into the acetyl choline receptors block the acetyl choline flow resulting in numbness and paralysis. Depending on their amino acid sequence and tertiary structures, α-neurotoxins can be classified into short chain α-neurotoxins, long chain α -neurotoxins, which have significant sequence homology and share the same three-dimensional structure, but differ in association or dissociation with the receptor. A neurotoxin can be called strong when its effect on its receptor is rapid and weak when its effect is slow.

  • Short Type: Contain four disulfide bridges; composed of 60-62 amino acids.
  • Long Type: Contain five disulfide bridges;composed of 66-75 amino acids.
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CD59 is a membrane bound protein present in various cells which binds to C8 and C9 of the terminal complement system. CD59 is a small glycoprotein made of 77 amino acids and has a molecular weight of 18 to 25kD. CD59 is also called as the Membrane Attack Complex Inhibitory protein. The less common function of CD59 is to influence the proliferation capacity of T cells and their ability to produce cytokines, influencing T cells response to a given antigen that enters the bloodstream. CD59 works in the both innate immune system and also in the adaptive immune system.

Material and Method

Below mentioned sequences and structures were obtained from databases like Uniprot and PDB. Protein – protein docking was carried out by online tool ClusPro and the analysis was done using tools PYMol and SPDB Viewer. Multiple sequence alignment of neurotoxins was carried out by ClustalW and Superimposition studies were done by SPDB Viewer.

Sl No.

Neurotoxin

(ligand)

PDB ID

Length

Molecular Weight

(in Dalton)

1

Long Neurotoxin 1

2CTX

71

7847

2

Weak Neurotoxin 5

1LN7

62

6943

3

Weak Neurotoxin 6

1LN9

65

7568

4

Weak Neurotoxin 7

1LQ3

65

7637

5

Weak Neurotoxin 8

1LMG

65

7581

Table 1: Details of Neurotoxins used for Docking studies

Sl. No

Name of Receptor

PDB ID

Length

Molecular Weight

(in Dalton)

1

CD59

1CDQ

77

14177

2

C8

2QQH

334

65163

3

C9

Theoretical Model

559

4

Acetyl Choline Receptor

4D01

218

Table 2: Details of different receptor targets for Rheumatoid Arthritis and Natural Targets of Neurotoxins

Protocol for the work is illustrated in the Flowchart below Fig 1.

""

Similarity studies among neurotoxins were done using ClustalW for the sequence similarity and SPDBViewer was used to find the structural similarities among the neurotoxins.

""

Fig 2: Multiple Sequence Alignment result using ClustalW

2CTX - LN2

1LN7 -WN5

1LN9

-WN6

1LQ3-WN7

1LMG -WN8

2CTX – LN2

-

1.17Å

49 Atoms

1.25 Å

49 Atoms

1.31 Å

49 Atoms

1.29 Å

51 Atoms

1LN7 – WN5

-

-

1.09 Å

61 Atoms

1.21 Å

59 Atoms

1.08 Å

57 Atoms

1LN9-WN6

-

-

-

1.03 Å

64 Atoms

1.13 Å

63 Atoms

1LQ3-WN7

-

-

-

-

1.13 Å

56 Atoms

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Table 3: Superimposition Studies between ligands for C-α Atoms

2CTX - LN2

1LN7 -WN5

1LN9

-WN6

1LQ3-WN7

1LMG -WN8

2CTX – LN2

-

1.28Å

196 Atoms

1.27 Å

196 Atoms

1.26 Å

192 Atoms

1.32 Å

204 Atoms

1LN7 – WN5

-

-

1.1 Å

248 Atoms

1.24 Å

236 Atoms

1.11 Å

228 Atoms

1LN9-WN6

-

-

-

1.08 Å

256 Atoms

1.18 Å

268 Atoms

1LQ3-WN7

-

-

-

-

1.16 Å

228 Atoms

Table 4: Superimposition studies between ligands for all atoms

Number of Interactions within 5Å

2CTX – Long Neurotoxin

1LN7 – Weak Neurotoxin 5

1LN9 – Weak Neurotoxin 6

1LQ3 – Weak Neurotoxin 7

1LMG – Weak Neurotoxin 8

Acetyl Choline Receptor

8

7

7

7

8

CD59

9

5

9

11

9

C8

7

11

8

10

13

C9

14

13

10

22

19

Table 5: Interaction table of Neurotoxins with different targets

With the above interaction chart for 2CTX and LIGPLOTs for 1LN7, 1LN9, 1LQ3 and 1LMG we found that Proline 7 plays integral role in binding to its natural receptor Acetyl Choline Receptor hence we modified the structure of all neurotoxins and removed amino acid 1 to Proline 7 from every neurotoxin and docked it again and found the interactions which is been tabulated below.

Number of Interactions within 5Å

Modified 2CTX – Long Neurotoxin

Modified 1LN7 – Weak Neurotoxin 5

Modified 1LN9 – Weak Neurotoxin 6

Modified 1LQ3 – Weak Neurotoxin 7

Modified 1LMG – Weak Neurotoxin 8

Acetyl Choline Receptor

8

7

9

18

11

CD59

8

6

10

10

9

C8

8

11

12

17

14

C9

17

16

17

15

15

Table 6: Interaction table of modified Neurotoxins with different targets

Results and Discussion

The above interaction tables do give a clear picture that modified long neurotoxin with PDB ID 2CTX and weak neurotoxin 6 with PDB ID 1LN9 can be used to target C9 and hence can inhibit the polymerization of C9. This inhibition of polymerization of C9 will inhibit the formation of Membrane Attack Complex which is responsible for the destruction of self-cells. So the modified neurotoxins can be used as CD59 mimicking agent and these neurotoxins are readily soluble in the blood hence they can reach their targets with higher efficiency.

References

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