Dr. Girish Sahni

Dr. Girish Sahni
INSTITUTE OF MICROBIAL TECHNOLOGY
(CSIR) CHANDIGARH CHANDIGARH, INDIA

Design g of Target g Specific p Clot
Busters
by
Protein Engineering Approaches

CSIR, IMTECH
&
the h microbial i bi lworld ld

Prevalence of Coronary Artery Disease
Coronary artery disease has been the number
one killer in the western world.
As life-styles change, it has began to to affect
millions across the worldwide.

* Th The discovery di of f clot l t dissolver di l drugs d has h led l d to t the th
emergence of thrombolytic therapy.
* Thrombolytic agents are mostly plasminogen
activators.
ti t

Schematic representation of the fibrinolytic system
Plasminogen activators
Plasminogen g activator
Inhibitor
Plasminogen g Plasmin
Fibrin
SK
UK
t-PA t PA
APSAC
α α22- Antiplasmin tpas
Fibrin degradation
product

SK SK:PG PG complex l
Restored blood
supply
Fibrinogen
Clotting factors
Blood factors
PG

All of the clot-buster clot buster drugs used in India
and in most of the developing world
were iimported t d till a few f years ago….
Average cost of treatment for MI
* Streptokinase : US $ 100 per dose
• tPA : US $ 1200 per dose

Highlight
Process developed developed at 300 litre fermentation fermentation scale
scale
• Negotiated & transferred in 2001
• Commercialized by licensee in 2001
• Recombinant form transferred in 2003
clinical trials near completion, currently
approaching commercialization
• EEngineered i d( (clot-specific) l t ifi ) form f
developed. Awaiting commercialization
Average cost: US $ 20 !
IMTECH

The challenge of constructing novel clotdissolver
proteins with enhanced fibrin clot
specificity p y

Acute need for second-generation Streptokinase
derivatives with
* increased plasma half-life
* decreased immunogenicity
* increased clot specificity

Molecular properties desired in a clot-specific
Plasminogen g activator drug g
Tight fibrin binding
Localized plasminogen activation
* Fibrin dependent activation
* Time-delayed action
* Plasmin dependent “molecular switch”

Structure-function studies on mechanism of
hhuman plasminogen l i activation i i leading l di to the h
design of clot-specific SK.
Our fundamental studies over the last decade has
revealed a unique mechanism for SK action at the
molecular level that has enabled us to design
improved versions of this life life-saver saver drug.

SK and PG form a very er tight binary binar complex comple
SK PG

Our strategy to explore PG-binding
domains in SK
Streptokinase
CnBr/Trypsin/Pepsin/V8 Protease
“Large chunks”
Probe b ffor PG-binding G bi di function/s
f i /

Limited Proteolysis of Streptokinase with Trypsin
1 59 143 293 414
Nihalani et al., BBRC 1995; 217:1245
Nihalani et al., Protein Sci. 1997; 6:1284
Small peptides

Functional map of the plasminogen-interacting
regions g in SK
16 51 234 293 414
SITE 1 SITE 2
Region in SK that interacts with substrate PG
Region in SK that interacts with partner PG to form a tight
Region in SK that interacts with partner PG to form a tight
binary complex

SK-α
μPN
SK-β
SK- γ
Crystal structure of SK in complex with the
catalytic domain of HPN, solved at 2.9 Å
Wang et al; Science (1998) 281: 1662

EK 281-282
EK 272-273
Ch dh t l 1999 P t i S i 8 2791
Chaudhary et al., 1999., Protein Sci. 8: 2791;
Yadav et al., 2008, BBA Proteins & Proteomics 1784: 1310
Aneja et al., 2009., J Biol. Chem. 284:32642
RE 248 248-249 249
KK 256-257 256 257

Docking of kringle 5 of PG with the beta domain
of SK
Dhar et al., J. Biol. Chem. 2002; 277: 13257;
Sundram et al 2003; J Biol Chem 2003; vol. 278: 30569

SK is a protein cofactor that modulates the
substrate substrate preferance preferance of of plasmin plasmin by by providing providing Exosites Exosites
SK
PN Substrate PG
Product PN
Physiological functions

Some possible configurations for the design of FBD(4,5)-SK chimeric proteins.
A. All the four FBD’s at the C-terminal of SK Fusion construct
B. All the four FBD’s at the N-terminal of SK
CFBD[1 C. FBD [1,2] 2]ffused dat the h CC-terminal i l of f SK
D. FBD [1,2] fused at the N-terminal of SK
E. FBD [4,5] fused at the C-terminal of SK
F. FBD [4,5] fused at the N-terminal of SK
GFBD[4 G. FBD [4,5] 5] fused f dat tb both thC C as well llas N-terminals Nt i l of f SK
H. FBD [4,5] fused with in the body of SK
SK-FBDs
11,2,4&5 2 4&5
FBD 1,2,4&5-
SK
SK-FBD 1,2
FBD1,2- SK
SK-FBD4,5
FBD4,5-SK
FBD4,5-SK-
FBD4,5
SK-FBD4,5
SK-FBD4,5-
SK.

Schematic representation of the engineered intergenic sequences
of hybrid construct , FBD (4,5)- SK.
TG site
FBD
4 5
N
C
V- Q- A- Q- Q- I- V
SK

SSchematic h ti representation t ti of f th the gene – bl blocks, k iincluding l di the th
engineered intergenic sequences in the hybrid construct,
FBD(4,5)– SK -FBD(4,5).
TG site it FBD SK Gl Gly li linker k TG site it FBD
4 5 4 5
N C
V-Q-A-Q-Q-I-V
G-G-G
QQ-A-Q-Q-I-V A Q Q I V

γ
α
γ
β
α
β
FBD
FBD
FBD
FBD
PN
Design of a targeted “clot-buster” drug
CLOTT
PG No activation
FBD
FBD
γ
α
β
PN
Degraded
Clot

Plasmin-dependent activation of clot-specific
streptokinase t t ki
nSK
Increasing PN
PN depleted

Mechanism of delayed action
Unlike native SK, protein engineered, clot-specific
SK is activated in the presence of plasmin.
Since the clot is plasmin-rich, and the blood plasma
is plasmin-poor p p ( (due to its rapid p removal by yalpha-2 p
Antiplasmin), the property of clot-specificity is
iintroduced t d dautomatically. t ti ll

F
E
D
C
B
A
Schematic diagram of Circulatory plasma loop model system used
for radioactive Fibrin clot lysis studies of SK, FBD(4,5)- FBD(4,5) SK and
FBD(4,5)- SK- FBD(4,5).
A. Magnetic stirrer, B. Magnetic bead, C. Reservoir containing plasma, E .Out let, F. Inlet into Reservoir, G
.Peristaltic pump, H. Permeable Chamber containing Radiolabeled fibrin clot.
H
G

Clot dissolution with Native Streptokinase p
Clot lyssis,
%
100
80
60
40
20
0
0 30 60 90 120 150 180 210 240 270 300
Time, min

Clot-specific p Engineered g Streptokinase p
Clot C lysis, %
100
80
60
40
20
0
0 30 60 90 120 150 180 210 240 270 300
Time, min

Non-specific fibrinogenolysis by different
proteins
Plasma fibrinogen
conc. (relative)
100
80
60
40
20
0
1 2
3 4
0 30 60 90 120 150 180 210 240 270 300
5
Time, min
Curve 1 …. SK
Curve 2 …. Constr. 1
Curve 3 …. TPA
Curve 4 …. Constr. 2
Curve 5 …. Constr. 3
Fibrinogenolysis in plasma milieu with different PG activators under conditions resulting in
comparable lysis of clot but exhibiting varying circulating fibrinogen degradation due to “non-specific
systemic” plasminogen activation. Note, however, the markedly high “fibrinogen protection” exhibited
by the novel, clot-specific engineered SK (‘Construct 3’; curve 5).

Advantages of clot-specific clot specific streptokinase
* Reduced systemic reactions (less side-effects)
* Localized plasminogen activation
* Decreased doses/economic benefits
(greater efficacy at low doses)

CSSK Licensing to Nostrum Inc., USA in July 2006

The Group at IMTECH
Abhay Pande K. Rajagopal
Deepak Nihalani i i K L Dikshit i i
Jagpreet Singh Vasudha Sundram
Jayeeta Dhar Rajesh Kumar
Amitabh Chaudhury Mahavir Yadav
Paramjit j Kaur Nandita Garg g
Anita Chaudhary
S S Komath
G P S Raghava

THANK YOU