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“Look Deep into nature then you will understand everything better”

-Albert Einstein

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• There are multiple strategies opted by creatures to catch their prey.

• Spiders possess neurotoxic venom which either can paralyse or kill the prey.

• Very potent neurotoxin TTX comes from pufferfish and porcupinefish.

*image source: Google and Wikipedia unless specified.

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Pharmacology of Spider Venoms

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Voltage gated Sodium channel (Nav 1.7)

• Nav 1.7 is predominantly expressed in dorsal root ganglia and plays a key role in nociception

• Loss-of-function mutations in Nav1.7 lead to Congenital insensitivity to pain (CIP)

• Patients with CIP have a complete insensitivity to pain without impairment of any other sensory modalities except a loss of smell (anosmia)

Name of presentation Month 2008Genus: Selenotypus

Genus: Phlogillus

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Fluorescence assay

• Dye 1: Binds to Ca+2

• Dye2: Masking dye

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• 102 samples from various species. • SHS5Y5 cells expresses Nav 1.7, 1.5, 1.2• Tetrodotoxin blocker

FLIPR™ Assay

• Veratridin activator

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1.C18 Column2.Eluted with step gradient of ACN

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Whole cell patch assay

*Image source: Biolin Scientific

Dose Response

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Dotted black line: Max concentration of venomSky blue: Min concentration of venomExperiment done on HEK cells which express only Nav1.7Observations:1.At concentration of 1 µM of venom, more than 75% activity of voltage gated Na channels is blocked.2.Fraction 30 of Phogilus (F2$) species blocks Sodium channels even strongly and rapidly than TTX. 3.Fraction 16 of Selenotypus (A3) species doesn’t block as strongly and rapidly as TTX.

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MALDI-TOF MS

2999.0 3603.6 4208.2 4812.8 5417.4 6022.0Mass (m/z)

373.3

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC[BP = 3925.2, 373]

3923

.215

639

29.2

517

3956

.168

9

3882

.248

3

4865

.585

4

5131

.596

2

5080

.119

1

3917.0 3920.6 3924.2 3927.8 3931.4 3935.0Mass (m/z)

373.3

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC[BP = 3925.2, 373]

392

3.2

156

392

9.2

517

3923

.215

3929

.251

Selenotypus fraction 16

Matrix: α-cyano-4-hydroxy-cinnamic acid(CHCA)

Name of presentation Month 20082999.0 3603.6 4208.2 4812.8 5417.4 6022.0Mass (m/z)

163.8

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC[BP = 3970.1, 164]

3967

.132

639

90.1

208

3911

.131

3

4491

.743

2

5507

.340

3

4623

.038

6

5131

.680

7

4869

.210

0

5772

.439

9

5221

.831

1

3959.0 3963.8 3968.6 3973.4 3978.2 3983.0Mass (m/z)

163.8

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC[BP = 3970.1, 164]

3967

.132

6

3973

.111

1

Phogilus fraction 30

Matrix: α-cyano-4-hydroxy-cinnamic acid(CHCA)

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De novo sequencing

Phogilus fraction 30

C-xx-R-xx-U—T/C-Y-xx-N—G—xx

Matrix: 1,5-diaminonaphthalene(1,5 DAN)

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ACKNOWLEDGMENT

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Thank you!