Signal transducing machinery as targets for potential drugs. Drugs:- a). Diclofenac- for treating cholera toxin b). Fasentin- for treating insulin signalling

1. Assignment of Molecular cell
Biology
On the topic of signal transducing
machinery as targets for potential
drugs
Submitted to :- Dr.Sulekha chahal
Submitted by :- Sahil
Roll-No. :- 28
M.Sc biotechnology 1st sem….
Kurukshetra university, kurukshetra

2. Cell Signaling :-
Cell signaling is the fundamental process by
which specific information is transferred from the cell surface to the
cytosol/ and ultimately to the nucleus, leading to changes in gene
expression.
It includes ligand and receptor which finally leads to a response/ gene
expression.
Signal Transduction :-
It is a transduction/path through
which a ligand or enzyme ,peptide or hormone enter the cell through
receptor and finally reached to the nucleus for response.

3. Signalling through GPCR receptor :-
GPCR –
 It is 7 time pass transmembrane (Serpentine) protein or cell
surface receptors with 3 domain-
a). N-terminal domain (which having ligand/ peptide/ hormon/
chemical binding site) on extracellular site.
b). Transmembrane domain (which act as a channel for the signal).
c). C-terminal domain/cytosolic domain (a catalytic domain which
further activate AC (adenylyn cyclase) by conformational change in
it.
 known as G-protein coupled receptors because receptor on the
other hand is associated with G-protein (Guanine nucleotide binding

4. protein).
 known as G-protein coupled receptors because receptor on
the other hand is associated with G-protein (Guanine
nucleotide binding domain) in cytosolic site/c-terminal
domain.
 Mainly Gs (which is stimulatory in nature) is used by body
for a particular response.
 It involve cAMP (cyclic adenosine 3,5 monophosphate) as
secondary massengers & also involves phosphorelay system
as secondary massengers.Both help ligand’s signal to reach to
nucleus.

5. Importants GPCR’s ligands
Ligands
o Hormons- Epinephrin, ADH,
glucogen etc.
o Chemicals- Acetylcholin
o Blood factor- Thrombin
o Mediators- Chemokines,
prostaglandins, leukotrins
etc.
Responces
o Smell, vision, taste.
o Immune cell activation.
o H2O reabsorption.
o Alpha-amylase secretion.
o Blood pressure.
o Glycogen breakdown.
o Immune cell migration.
o Thyroxin secretion.

6. Systematic GPCR signalling :-

7.  N-termini end of GPCR is extracellular and contain ligand binding
site and C-termini in cytosolic associated with G-proteins.
 In absence of ligand/signal, G-protein is trimeric and GDP bound
(inactive).
 Ligand binding induces conformational change in the receptor
and activates GEF (usually associated with receptor).
 GEF exchanges GDP with GTP and G-protein becomes monomeric
and active.
 Alpha-submit is catalytic and further regulates membrane bound
enzymes AC (Adenylyn cyclase) and ions channels by conformation
change.

8.  Adenylyn cyclase then converts ATP into cAMP and cAMP
activates PK-A by recruite itself on negative regulatory subunit (R
subunit) of PK-A.
Then catalytic subunit of PK-A is free from its negative regulatory
subunit and performs its function and run further phosphorelay
system as secondary massenger.
 The end phosphorylated protein Having nuclear localization
signal as NLS then go into nucleus and recruite transcription factor
and other accessory protein which help in gene regulation.
Finally response will generate.

9.  During termination, endogenous GTPase activity of G-protein
causes GTP hydrolysis but in presence of GAP (GTPase activating
protein) or RGS (Regulator of G-protein signalling).

10. Cholera toxin:-
 An endotoxin produced by bacteria vibrio cholera.
Causes intestinal infection.
It is more prominent in new born.
Belongs to AB toxin family – having 5 identical B subunit + 1
catalytic A subunit.
 B subunit recognizes carbohydrates chain on extracellular
surface of cell e.g.,Gangliosides which are receptor for AB toxin.

11. Cholera toxin infection :-

12.  B- subunit intract with GM1 (e.g. of ganglioside) chain present
on the apical surface of intestinal epithelial cells.
 A subunit in the cytosol is protolytically cleaved to produce A1
and A2 fragments
A1 fragment intract with ARF-6 and ARF-6 causes ADP-
ribosylation on argnine residue to active Gs aplha.
ADP-ribosylated Gs2 can no longer intract with GAPs, result in
constitutivelly on of Gs2 signalling.
 Gs2 constitutivelly on to AC which continuously converts ATP
into cAMP and cAMP activates PK-A by recruite itself on negative
regulatory subunit (R subunit) of PK-A.
 Hyperactive PK-A phosphorylate and active CFTR chloride ion
channel and sodium-hydrogen ion exchanger.

13.  Loss of salt occurs and inside become hypotonic
condition.Therefore large amount of water loss occures result in
shrinkage of the cell which ultimately leads to diarrohea.

14. Transduction for the drug “Diclofenac” is given
below:-

15. Drug transduction mechanism
For diclofenac :-

16. 1) Diclofenac salt is a hydrophobic in nature.
1) It pass plasma membrane by simple diffusion.
2) Declofenac is a inhibitor of cAMP signalling and its subtrate is
negative regulatory subunit (R-subunit) in PK-A.
3) It goes and directly binds to its substrate and result is the agonist
activation of C-subunit in PK-A.
4) Now, C-subunit starts to activate phosphorelay system as
secondary massengers.
5) The final phosphorylated protein with the help of NLS in its
sequence goes to nucleus.

17. 6) Then it phosphorylate and activate some other tanscriptions
factore with other accessory proteins.
7) Finally,gene is expressed owing to which closure of CFTR chloride
and sodium, hydrogen ion channels will occure.
---Drug’s side effects---
• Declofenac cause closure of CFTR chloride and sodium,
hydrogen ion channels.
•Overuse of declofenac can cause different sort of chronic
complication like chronic muscular spasm etc.

18. • It is a type of enzymes linked tyrosine kinase receptor.
• Receptors are auto catalytic in function.
• It is a one time pass cell surface receptor.
• It is a monomeric structure with 2 subunits.
a) Alpha - Extracellular domain having ligand binding site.
b) Beta – Transmembrane domain which associates with catalytic
C-terminal domain in cytosolic site.
• It only phosphorylate tyrosine residue of itself C-termini and
further protein as well.

19. • Upon binding, ligand induces receptor dimerization.
• C-termini of receptor undergoes auto-transphosphorylation.
• Ras-MAPK pathway is important in the signalling activates
phosphorelay system which function as secondary massenger.

20. Important ligands
Ligand
• Insulin.
• EGF (Epidermal growth
factor).
• IGF (Insulin like growth
factor).
• PDGF (Platelets derived
growth factor).
Response
• Carbohydrate utilization,
cellular translation.
• Stimulating growth ,
proliferation and
differentiation of epi &
subepidermal cell
• Growth and development
of muscles and bones.
• Proliferation and migration
of immune cells during
injury.

21. • NGF (Nerve growth
factor).
• FGF (Fibroblast growth
factor).
• M-CSK (Macrophage
colony stimulating
factor)
• Growth and survival of
neurons.
• Growth and survival of
fibroblast cell.
• Differentiation of
monocytes into
macrophages.

22. Insulin signalling

23. l. Insulin is a peptide hormone which interact with receptors mainly
expressed on muscle and adipose tissues,liver and kidney.
ll. Insulin receptor is made of smaller alpha subunit and larger beta
unit.
lll. Alpha unit- Insulin binding site.
lV. Beta unit- Transmembrane domain and catalytic domain.
V. Insulin binding causes receptor dimerization and beta unit
undergoes trans-autophosphorylation on Tyrosine residue.
VI. SH-2 containing IRS-1 interact with phosphorylated receptor and
receptor causes phosphorylation on Tyrosine residue of IRS-1.
VII. Phosphorylated IRS-1interact with an adapter protein R/S Grb-2.
IIX. Grb-2 contain SH-2 and SH-3 domain.
IX. SH-3 domain of Grb-2 interact with a GEF-(SoS).
X. SoS replaces GDP with GTP and activates a membrane lipid linked
protein-Ras.
XI. Ras protein interact with 'N' termini of kinase and activates the
enzyme by confirmational change.

24. XII. Raf phosphorylated and activates MEK and MEK further
phosphorylated and activates ERK.
XV. ERK enters nucleus- phosphorylated of SRF.SRF interacts with
ELK to induce gene expression e.g glycolytic genes, glycogen
synthase.
XVI. During Insulin signalling ,GLUT-4 is also recruited to plasma
membrane for mediating large influx of glucose.

25. Drug transduction mechanism
For fasentin :-

26.  When there is hyperactivation of insulin signalling.It causes
hypoglycemia ( a condition in which blood sugar (glucose) level is
lower than normal.
 For treating/rescue, a salt is used known as “FASENTIN”.
 Fasentin is a hydrophobic salt which cross the mammalian
plasma membrane by simple diffusion.
 It goes and directly bind to the ERK-1/2 (extracellular signal-
regulated kinases) and inhibit the kinases activity of MEK ( mitogen-
activated protein kinase kinase ) on ERK.
 Unphosphorylated ERK is then unable to direct itself into the
nucleus even in the presence of NLS in it.

27.  Unphosphorylated ERK is unable to direct itself into the nucleus
even in the presence of NLS in it.
 Finally gene expression of GLUT-4 will not occure which caused
high influx of glucose from blood streams.
 It also inactivate GLUT-4 transporter in the membrane which are
active by its phophatases activity.
Finally, the condition of hypoglycemia recover after afew hours of
taking diet.