Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that regulates fibrinolysis. It functions by inhibiting tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PAI-1 exists in both an active conformation that can inhibit tPA/uPA and a latent inactive conformation. Several small molecule inhibitors have been developed that target the active site of PAI-1 and accelerate its conversion to the latent state. These inhibitors typically contain both carboxylic acid and aromatic groups important for binding to PAI-1's hydrophobic cleft near residues Phe114 and Val121. However, no P

1. Plasminogen Activator
Inhibitor -1

2. Plasminogen Activator Inhibitor -1
•
Name and origin of the protein
– Protein name: Plasminogen activator inhibitor-1
– Synonyms PAI-1, Endothelial plasminogen activator inhibitor, PAI,
serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen
activator inhibitor type 1)
– Gene name SERPINE1, PAI1, PLANH1
• FUNCTION: This inhibitor acts as "bait" for tissue plasminogen
activator, urokinase, and protein C. Its rapid interaction with t-PA may
function as a major control point in the regulation of fibrinolysis.
• SUBUNIT: Interacts with VTN. Binds LRP1B; binding is followed by
internalization and degradation
•
SUBCELLULAR LOCATION: Secreted.
• SIMILARITY: Belongs to the serpin family (Serine Proteases). Homology
of PAI-1 only 35% with other serpins (antithrombin, antiplasmin,
antitrypsin, ovalbumin, and angiotensinogen).

3. Plasminogen Activator Inhibitor -1
• DISEASE: High concentrations of PAI-1 have been associated
with thrombophilia;an autosomal dominant disorder in which
affected individuals are prone to develop serious spontaneous
thrombosis. Defects in serpine1 are the cause of plasminogen
activator inhibitor-1 deficiency (pai-1 deficiency). PAI-1
deficiency is characterized by abnormal bleeding due to PAI1defect in the plasma.
• MISCELLANEOUS: PAI-1 is inactivated by proteolytic attack of
the urokinase-type (u-PA) and the tissue-type (TPA), cleaving the
369(R)-370(M) bond. Two types of PAI have been identified.
PAI-1 is an acid-stable glycoprotein found in plasma and platelets
and in endothelial, hepatoma, and fibrosarcoma cells. Vascular
endothelial cells may be the primary site of synthesis of plasma
PAI.

4. Plasminogen Activator Inhibitor -1
•
Sequence Information:
* extracted from Swiss-sprot DB
– Length: 402 Amino acids , 45060 Da
•
Features of sequence
– SIGNAL
1 to 23
– CHAIN
24 to 402
Plasminogen activator inhibitor-1.
– SITE
369 to 370
2 Reactive bond.
– CARBOHYD
232- 232
N-linked Glycosylation site
– CARBOHYD
288 - 288
N-linked Glycosylation site
– CARBOHYD
352 - 352
N-linked Glycosylation site
– VARIANT
15- 15
A -> T (SNP)
– VARIANT
17- 17
V -> I (SNP)
– VARIANT
25 - 25
H -> P (SNP)
– VARIANT
209 - 209
R -> H (SNP)
– VARIANT
255 - 255
T -> N (SNP)

6. PAI-1 is the natural inhibitor of uroplasminogen activators. It's the cognate inhibitor of
both tissue type plasminogen activator (t-PA) and the urokinase type plasminogen
activator (u-PA) in plasma and in tissues. This is present throughout the mammalian
systems. The relative balance between t-PA and PAI-1 in blood or in tissues to a large
extent determines the net activity of this system. This system is best known for its role in
dissolving clots. But it also plays other important roles in tissue and in the blood vessel
wall. It remodels the extracellular matrix. It's a "housekeeping" system. It also promotes
cellular migration.

7. Serpin-1 structure->Conformations of PAI-1
The serpin plasminogen activator inhibitor-1 (PAI-1) folds into an active structure
then converts slowly to a more stable, but low activity, "latent" conformation.The
structure of active PAI-1 has to be different from the binding regions in latent and
cleaved PAI-1. Crystal structures available in all conformations without any
known ligand.

8. The three forms of PAI-1
When PAI-1 is synthesized in endothelial cells and
released into blood, it is in a functionally active form (Lindahl and
Wiman 1989), which is the native conformation and has the
inhibitory activity towards its target proteases.
In the in vitro experiments at physiological conditions,
active PAI-1 spontaneously undergoes a conformational change
and decay to a latent form with a half-life of about 2 h (Levin and
Santell 1987, Lindahl et al 1989).
Normally, in plasma, PAI-1 is completely bound to another
protein, vitronectin (Wiman et al 1988, Declerck et al 1988) that
stabilizes the active form of PAI-1 and prolongs the half-life to 4
h.

9. J Thromb Haemost, Vol 2, Issue 2, pp. 289-297
PAI-1 activity can be inhibited by at least 3
mechanisms:
•prevention of the formation of the PAI-1/t-PA
complex
•acceleration of the PAI-1 latency conversion
•inducing a substrate behaviour of PAI-1

10. Small Molecule Active Site of PAI-1
Small molecule binds to which conformer- cleaved, active or
latent?
The active form of PAI-1 is structurally unstable with a
plasma half-life of 1 hour, it is the conformation that is
inhibitory toward tPA and uPA and therefore the target for small
molecule interaction. (J. Med. Chem. 2004, 47, 3491).
Small molecule binding site -> Arg 76, Arg 115 and Arg
118. The binding sites are the regions of the molecule where the
most dramatic structural changes take place in Active, cleaved
and latent conformations of PAI-1. (J. Mol. Biol. 2000, 297,
683).
CLB-2C8 (residues 110- 145) binding epitope of PAI-1
constitutes the binding site for the low molecular weight
inhibitor (Biochemistry, 1998, 37, 1227)

11. Where Do they Go?

12. Three-dimensional structure of PAI-1
in the active form
The active form a large
pocket is formed by a-helices
D, E, F and β-strand s1A.
In the other PAI-1 conformers
this pocket is much shallower,
due to the translational
rearrangements involving the
above secondary structural
elements, but also on account
of the side-chain reorientation
of Y79.
Plasminogen Activator Inhibitor 1. Structure of the
Native Serpin, Comparison to its Other Conformers
and Implications for Serpin Inactivation
. Mol. Biol. (2000) 297, 683 -695

13. Elucidation of the binding regions of PAI-1 neutralizing antibodies
using chimeric variants of human and rat PAI-1.
Thromb Haemost. 2001 May;85(5):866-74
Three segments in human PAI-1 containing each at least one
site involved in the neutralization of PAI-1 activity could be
identified, i.e.
(1) the segment from residue 81 to residue 187 (comprising
alpha-helices hD, hE and hF, beta-strands s4C, s3A, s2A and
s1A and the loops connecting these elements).
(2) the segment between residues 277 and 327 (hI, thIs5A,
s5A and s6A) and
(3) the region C-terminal from amino acid 327, including the
reactive site loop.
The current data. together with previous data, indicate that
PAI-1 contains at least four different regions that could be
considered as putative targets to modulate its activity.

14. A Regulatory Hydrophobic Area in the Flexible Joint
Region of Plasminogen Activator Inhibitor-1,
Defined with Fluorescent Activity-neutralizing
Ligands
JBC;Vol. 276, No. 16, pp. 13077–13086, 2001
•An important perspective is the possibility of utilizing the
hydrophobic area as a target for anti-cancer and anti-thrombotic
drugs.
•The strong binding to serum albumin common to all the
compounds must be avoided.
•Since PAI-1 is expected to be bound to vitronectin in vivo,
pharmacologically potentially interesting molecules must have a
high affinity not only to free PAI-1 but also to PAI-1 in its
vitronectin-associated state.
•Other serpins have similar hydrophobic areas, and the specificity
of PAI-1 neutralizers of potential interest for in vivo use must
therefore be ensured.

15. Active Site

16. Tiplaxtinin docked in active PAI-1

17. Wyeth inhibitor docked in active PAI-1

18. The standard small molecule
IC50 12 µm
•Structure-function relationship analysis suggested that the acidic
function in AR-H029953XX was important for PAI-1 inhibition.
•Interactions of AR-H029953XX with PAI-1-> A salt bridge between
carboxylate group of AR-H029953XX with one of 3 arginine residues
(76, 115 and 118) and a hydrophobic interaction between the
dichlorophenyl moiety of AR-H029953XX and a lipophilic assembly of
side chains in the vicinity of Val 121 and Phe 114.
•The most likely site for binding inhibitor appeared to be a hydrophobic
cleft near residues Phe 114 and Val 121 flanked by a basic surface
region containing 3 arginine residues (76, 115 and 118).
(Biochemistry, 1998, 37, 1227).

19. Plasminogen activator inhibitor-1 polymers, induced by
inactivating amphipathic organochemical ligands
Biochem. J. (2003) 372, 747–755
The binding of inactivating amphipathic compounds was much stronger to active
PAI-1 than to the RCLinserted forms.
The hydrophobic cavity binds a variety of structurally diverse compounds,
including ANS (8-anilinonaphthalene-1-sulphonic acid), AR-H029953XX {N-[N(3,4-dichlorophenyl)benzamide] anthranilic acid}, bis-ANS (4,4-dianilino-1,1bisnaphthyl-5,5-disulphonic acid), and 1-dodecyl sulphuric acid (1-DS), with Kd
values between 0.5 and several hundred micromolar.
The negatively charged organochemical compounds induce substrate behaviour,
followed by conversion of PAI-1 to inactive, polymeric forms
Tiplaxtinin ?

20. PAI-1 Inhibitors Small molecules
IC50 13 µm
IC50 not known
•None of the compounds have
shown in vivo activity.
•Structurally,
all
PAI-1
compounds contain carboxylic
acid
and
bulky
aromatic
functions.. It is possible that both
acidic and aromatic groups play
an important role in the binding
of small molecules to human
PAI-1.

21. Xenova Limited
Evaluation of a low molecular weight modulator of human
plasminogen
activator
inhibitor-1
activity.
Thromb Haemost. 1996 May;75(5):808-15.
The evaluation of three diketopiperazine-based low molecular weight
inhibitors of PAI-1 activity XR334, XR1853 and XR5082.
Biochemical mechanism of action of a diketopiperazine
inactivator of plasminogen activator inhibitor-1
Biochem. J. (2003) 373, 723–732
XR5118, positively charged compound is a novel modulator of
plasminogen activator inhibitor-1 (PAI-1).

22. Biochemical mechanism of action of a diketopiperazine
inactivator of plasminogen activator inhibitor-1
Biochem. J. (2003) 373, 723–732
Effector sites for XR5118, displayed on a ribbon and wireframe presentation
of a part of the structure. Substitution of the red residues resulted in an IC50
value above 250 µM for XR5118 inactivation of PAI-1. Substitution of the
yellow residues resulted in an IC50 value below 250 µM for XR5118
inactivation of PAI-1, but significantly different from the IC50 value for PAI-1
XR5118 binds laterally to β-sheet A.
wt.

23. Mapping of the epitope of a monoclonal antibody
protecting plasminogen activator inhibitor-1 against
inactivating agents
Eur. J. Biochem. 270, 1672–1679 (2003)
A few organochemical compounds able to inactivate PAI-1
have been indentified, including a group of negatively
charged amphipathic compounds like bis-ANS and the
diketopiperazine derivative XR5118. Their exact
binding sites in PAI-1 remain to be established, but all
available evidence is in agreement with these compounds
having overlapping, but not identical, binding sites in the
flexible joint region. VitroNectin protects PAI-1 from
inactivation by bis-ANS and XR5118. These compounds do
not bind to relaxed PAI-1. Thus, there is bidirectional
communication between the flexible joint region and the
movements of the RCL.

24. The effect of fibrates
on PAI-1 production
appeared
to
be
compound specific and
concentration
dependent.
Structurally, all fibrates
contain a carboxylate
and
functional
importance of this
acidic group in the
inhibition of PAI-1
production
remains
determined.

25. All benzothiophene derived compounds contain a carboxylic
acid and bulky aromatic groups which play an important role
in the binding of small molecules to human PAI-1.

26. Tiplaxtinin, a Novel, Orally Efficacious Inhibitor of PAI-1
J. Med. Chem. 2004, 47, 3491
F3C
O
A lipophilic assembly of
side chains in the vicinity
of Val 121 and Phe 114.
O
N
COOH
Arginine residues
(76 / 115 / 118)
PAI- 039
IC50 2.5 µm
• Identified with the HTS of wyeth’s compound libraries.
• Existence of a carboxylic acid or an acid bioisostere which is
common to most PAI-1 inhibitors.

27. Design and Synthesis of Novel Oxime-Based PAI-1 Inhibitors
-Wyeth Research
The oxime moiety was introduced to address patent and stability issues. The
oxime replaces one of the ether linkages in compound 1 . The substituted
benzofuran moiety was found to be effective to add structural distinction to the
series and to impart PAI-1 activity in the binding assay.
Compared to an aryl-oxime series produced, the SAR of the oxime-benzofuran
series showed this series required less lipophilicity. Compound 2 retains in vitro
potency, is efficacious in vivo @ 5 mpk, po and is an inhibitor of PAI-1.

28. Design, Synthesis and In Vitro Evaluation of Potent, Novel,
Small Molecule Inhibitors of Plasminogen Activator Inhibitor1
Bioorganic & Medicinal Chemistry Letters 12 (2002) 1063–
1066
IC50 0.20 µm
Poor pKa
Xenova Limited focused on the diketopiperazine nucleus. It was speculated
that the biological activity of these compounds was exerted through one of
diketopiperazine ring amides existing in the enol tautomer, whilst the other
remained as a carboxamide.
Bioorganic & Medicinal Chemistry Letters 12 (2002) 2367–2370

29. Synthesis and Biological Evaluation of Menthol-Based
Derivatives as Inhibitors of Plasminogen Activator Inhibitor1 (PAI-1)
3365
Bioorganic & Medicinal Chemistry Letters 13 (2003) 3361–
IC50 0.38 µm
Berlex Biosciences have explored structure–activity relationships around the novel
PAI-1 inhibitor 1 by modification of the B- and C-segments.
The B-segment could be replaced with a variety of aromatic diamines without loss
of potency, but aliphatic diamines were not tolerated.
Efforts to optimize the C-segment resulted in the identification of a series of novel
diphenyl ether carboxylic acid derivatives having improved potency, moderate to
good functional activity, and oral bioavailability.

30. Synthesis and biological evaluation of piperazine-based
derivatives as inhibitors of plasminogen activator inhibitor-1
(PAI-1)
Bioorganic & Medicinal Chemistry Letters 14 (2004) 761–765
No improvement was seen in the modification of the
A segment -Berlex Biosciences

31. Design and synthesis of oxadiazolidinediones as
inhibitors of plasminogen activator inhibitor-1
Bioorganic & Medicinal Chemistry Letters 14 (2004) 3477–3480
IC50 5.29 µm
Wyeth
explored the structure–activity relationships around the novel
oxadiazolidinedione based PAI-1 inhibitor 5 by modifying regions 1 and 2.
Initial optimization of the region 1 indicates the preference for lipophilic groups
in that region and seems to be flexible in terms of steric requirements
accommodating bulky substituents.
Preliminary studies in region 2 indicated that the linker need not be a rigid
double bond and is flexible with respect to the stereochemistry as well. As far as
the substituents of the linker, a lipophilic group like phenyl is favored over
smaller alkyl substituent like methyl, resulting in submicromolar inhibitors.

32. Characterization and comparative evaluation of a structurally
unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.
Journal of Thrombosis and Haemostasis, 2:
1422–1428
First molecule to exhibit efficacy
in animal models of vascular
disease following oral
administration.

33. Characterization of a small molecule PAI1inhibitor, ZK4044
Thrombosis Research (2005) 115, 341–
350
⇒ZK4044 did not convert active PAI-1 to latent PAI-1,suggesting that the binding
of ZK4044 to PAI-1either directly or indirectly hinders the interaction of PAI-1 with
tPA and uPA. Similar interference of the interaction of PAI-1 and tPA by another
small molecule PAI-1 inhibitor, ARH029953XX, has been reported.
⇒ZK4044 probably binds to one of these two binding pockets near
⇒site on the surface of PAI-1 that is flanked by basic residues Arg76, Arg115
and Arg118 formed by a-helices D, E, F and h-strand s1A.
⇒ An adjacent binding pocket for small molecule inhibitors has been proposed
to be located in a space between h-strands s3A and s5A.