30

Chapter 2

REVIREVIREVIREVIEW OF LITERATUREEW OF LITERATUREEW OF LITERATUREEW OF LITERATURE

31

Chapter 2Chapter 2Chapter 2Chapter 2

REVIEW OF LITERATURE

Prothrombin and thrombin was the subject of many protein chemical studies in the period

1960-1980 (Lundblad et al., 1976). In 1970s the amino acid sequences of human and

bovine prothrombin were determined (Butkowski et al., 1977). In l980s, the nucleotide

sequences of human and bovine prothrombin c-DNA were determined (Degen et al, 1983;

MacGillivray and Davie, 1984) and three years later the complete human gene sequence

(Degen and Davie, I987). During this period, thrombomodulin, the cofactor for protein C

activation by thrombin was characterized (Esmon and Owen. 1981). In 1989, the tertiary

structure of thrombin was elucidated (Bode et al., 1989) and this led to a number of groups

focusing on thrombin as a target for drug development (Tapparelli et al., I993).

Many studies in the period 1970-2000 demonstrated that thrombin had diverse effects on

cells (Chen and Buchanan, 1975; Coughlin, 2000). The molecular basis of many of these

effects was elucidated in 1991 with the identification of a proteolytically activated receptor

for thrombin (Vu et al., 1991). In the same year the two first reports on site-directed

mutagenesis of thrombin were published (Le Bonniec and Esmon, 1991; Wu et al., 1991).

Within 10 years, these were followed by over 40 reports on thrombin mutagenesis, providing

an extensive knowledge of the molecule: virtually every surface loop of thrombin had been

investigated. It was also demonstrated in 1990s that thrombin possess a sodium ion binding

site that is crucial for catalysis (Di Cera et al., 1995; Guinto et al., 1999). In 1998,

prothrombin-deficient mice were obtained by knockout techniques, but died within a few

days of birth from fatal hemorrhagic events (Sun et al., 1998). Overall the number and extent

of studies promote thrombin as one of the best-known proteases to date.

Numerous crystallographic structures of thrombin have been determined (over 150 in the

PDB). The initial structure of the D-Phe-Pro-Arg-CH2- thrombin complex provided great

insight into the structural basis for thrombin's specificity (Bode et al, 1989, 1992). The

binding of the D-Phe-Pro-Arg-CH2- group delineates the substrate-binding regions for

32

residues on the N-terminal side of the scissile bond. Access to the active site is partially

blocked by the 60-loop containing nine residues that represent an insertion into the

thrombin sequence compared to that of chymotrypsin.

The structures of the thrombin exosites were also elucidated in these initial studies. The

fibrinogen-recognition exosite and the heparin-binding site consist of positively charged

surface regions. Electrostatic calculations demonstrated that positive fields project from these

two exosites at the poles of the molecule, whereas the active site between the two poles has

a negative potential (Bode et al., 1992). The nature of interactions with the fibrinogen-

recognition exosite has been further refined in complexes with hirudin (Rydel et al., 1991),

fragments of fibrinogen (Martin et al., 1992; Stubbs et al., 1992) and of protease-activated

receptor 1 (Mathews et al., 1994). Active-site interactions have also been further explored in

structures with substrate analogs (Martin et al., 1992; Stubbs et al., 1992) and synthetic

inhibitors (Brandstetter et al., 1992). The structure of an unnatural complex between basic

pancreatic trypsin inhibitor and a mutated thrombin has also been determined (van de Locht

et al., 1997). Also, the structure of the thrombin-thrombomodulin complex has been

elucidated (Fuentes-Prior et al., 2000).

Two Na+ ion binding sites have been identified crystallographically by exchanging Na+ with

Rb+. One is intermolecular, found on the surface between two symmetry-related thrombin

molecules. Since it is not present in thrombin crystal structures having different crystal systems,

the other Na+ site is the functionally relevant one. The second site has octahedral coordination

with the carbonyl oxygen atoms of Arg221A and Lys224 and four conserved water molecules.

It is located near Asp189 of the S1 specificity site in an elongated solvent channel formed by

four antiparallel beta-strands between Cys182-Cys191 and Val213-Tyr228. This channel is

extending from the active site to the opposite surface of the enzyme. It was first noted in the

hirudin-thrombin structure. When Na+ binds to thrombin, a conformational change is induced

that renders the enzyme kinetically faster and more specific in the activation of fibrinogen

(Zhang and Tulinsky, 1997).

33

2.1 Thrombin substrates

The atomic level interactions between substrate and thrombin come mainly from the

crystal structures of thrombin bound to fibrinopeptide A (Stubbs et al., 1992; Martin et

al., 1992).

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues

7f-20f of the Aα-chain of human fibrinogen, has been co-crystallized with bovine

thrombin and is shown in Figure 2.1. Three crystallographically independent complexes

were located in the asymmetric unit by molecular replacement using the native bovine

thrombin structure as a model. Excellent electron density could be traced for the

decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of

thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at

the Arg-16f/Gly-17f bond, are not seen. The major specific interactions between the

peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr60A, Trp60D,

Leu99, Ile174, Trp215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a

hydrogen bond linking Phe-8f NH to Lys97 O; 3) a salt link between Glu-11f and

Arg173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly216; and

5) the insertion of Arg-16f into the specificity pocket (Martin et al., 1992).

Figure 2.1: The structure of residues 7-16 of the Aα -chain of human fibrinogen bound to

bovine thrombin (PDB ID: 1BBR). Some of the hydrogen bonding

interactions between thrombin and ligand ares displayed

34

Another study demonstrating the thrombin-substrate interactions was the crystal structure

of the factor XIII (FXIII) activation peptide, encompassing residues 28–37, complexed to

human α-thrombin (Sadasivan and Yee, 2000). The residues surrounding the thrombin

cleavage site in FXIII are of particular interest. The serine protease thrombin

proteolytically activates blood coagulation FXIII by cleavage at residue Arg37-Gly38.

FXIII in turn cross-links fibrin molecules and gives mechanical stability to the blood clot.

The 2.0 Å resolution x-ray crystal structure of human alpha-thrombin bound to the FXIII

(28-37) decapeptide has been determined as shown in Figure 2.2. This structure reveals

the detailed atomic level interactions between the FXIII activation peptide and thrombin

and provides the first high resolution view of this functionally important part of the FXIII

molecule. A comparison of this structure with the crystal structure of fibrinopeptide A

complexed with thrombin highlights several important determinants of thrombin

substrate interaction. First, the P1 and P2 residues must be compatible with the geometry

and chemistry of the S1 and S2 specificity sites in thrombin. Second, a glycine in the P5

position is necessary for the conserved substrate conformation seen in both FXIII (28-37)

and fibrinopeptide A. Finally, the hydrophobic residues, which occupy the aryl binding

site of thrombin, determine the substrate conformation further away from the catalytic

residues. In the case of FXIII (28-37), the aryl binding site is shared by hydrophobic

residues P4 (Val34) and P9 (Val29). A bulkier residue in either of these sites may alter

the substrate peptide conformation (Sadasivan and Yee, 2000).

Figure 2.2: Crystal structure of FXIII (28-37) activation peptide bound to human apha

thrombin (PDB ID: 1DE7).

35

The FXIII P9-P1 segment makes important contributions to binding and hydrolysis. A

series of peptides with substitutions at the P4 position (V, L, F, A, and I) have been

screened to find the relative activity. V34L mutation showed the highest binding

interactions compared to others. Solution NMR studies indicated that the P4-P2

interaction involving L34 and P36 was preserved in FXIII activation peptide V29F, V34L

and a helical turn involving F29 was still not visible (Trumbo and Maurer, 2002). Kinetic

studies of the role of individual substrate positions are highly worthwhile for

understanding the sources of thrombin specificity.

The presence of the V34L polymorphism increases the rate of FXIII activation by

thrombin. In synthetic peptides, a significant variation in rates of activation has been

demonstrated among different FXIII structures (Trumbo and Maurer, 2002). Using both

purified platelet derived and recombinant FXIII, Wartiovaara et al. (Wartiovaara et al.,

1999) demonstrated that thrombin cleaves the activation peptide from V34L FXIII more

rapidly than from wild type FXIII. The presence of the FXIII V34L polymorphism has a

significant effect on fibrin clot structure. Wartiovaara et al. have also shown that fibrin γ-

chain dimerization and α-polymerization are enhanced in the presence of FXIII V34L

(Wartiovaara et al., 1999). Ariens et al., using mass spectrometry, showed that the V34L

polymorphism does not alter the actual thrombin cleavage site. Analysis of FXIII subunit

proteolysis by SDS-PAGE and HPLC showed that FXIII V34L is cleaved more rapidly,

and at lower concentrations of thrombin, than FXIII 34V (Ariens et al., 2000). Ariens et

al. also demonstrated that the cross-linking activities of both FXIIIa L34 and V34 were

similar, but the increased sensitivity of the L34 isoform to thrombin activation resulted in

more rapid cross-linking of fibrin γ - and α -chains in the presence of the polymorphism.

Turbidometric measurements demonstrated that clots formed from plasma in the presence

of FXIII L34 had a shorter lag phase (1.08 versus 1.55 min) and a lower maximum

absorbency after 5 min, indicating the presence of thinner fibers and less porous (Ariens

et al., 2000) . Thinner fibers are more resistant to fibrinolysis in vitro and should

theoretically increase clinical thrombotic risk. Most clinical studies have actually

demonstrated decreased venous and arterial thrombotic risk in carriers of the FXIII V34L

36

polymorphism, however. A study by Lim et al. tries to give explanation for this paradox

(Lim et al., 2003). At low fibrinogen concentration, fibrin clots prepared from

homozygous L34 samples had decreased permeability compared to those from V34

homozygotes, similar to results from previous studies. However, at higher fibrinogen

concentrations clots prepared from L34 homozygotes had higher permeability than those

from V34 homozygotes. Therefore, the FXIII L34 polymorphism may protect against

thrombosis specifically in patients with high fibrinogen levels, who are known to be at

higher risk for thrombotic complications (Lim et al., 2003)

2.2 Thrombin inhibitors

More than 150 of thrombin-inhibitor complexes are known to date and are available in

protein data bank (PDB). These include many peptidic and non-peptidic thrombin

inhibitors. Thrombin inhibitors such as heparin and its analogs, which have been in

widespread therapeutic use for decades, are indirect thrombin inhibitors. They act as part

of an antithrombin complex and do not themselves interact directly with the thrombin

active site. This means that they can only inactivate soluble thrombin but cannot react

with fibrin-bound thrombin. Direct thrombin inhibitors are capable of inactivating both

soluble and fibrin-bound thrombin. This confers considerable therapeutic benefits since

these agents can inhibit the ongoing coagulation process within the clot itself, not just the

formation of new clot. Some direct thrombin inhibitors including peptides and

nonpeptides are discussed here.

Haematophagous animals have developed a rich reservoir of inhibitors for blood

coagulation proteases during evolution. Two known direct thrombin inhibitors, hirudin

and hirulog, are derived from a haematophagous animal. Hirudin and bivalirudin are

bivalent inhibitors that bind to the exosite I and active site of thrombin. Hirudin is a 65-

amino acids protein isolated from the salivary gland of medicinal leech Hirudo

medicinalis (Greinacher et al, 2008). It has a globular N-terminal domain and an acidic

C-terminal tail, both of which bind to sites in the thrombin molecule. This C-terminal tail

interacts with thrombin exosite-I through electrostatic and hydrophobic interactions. The

37

N-terminal domain binds to an apolar site near the active site of thrombin, obstructing its

accessibility. Hirudin is a unique thrombin inhibitor forming a tight stoichiometric

complex with the enzyme. It is the only inhibitor that blocks thrombin activity at very

low (picomolar) concentrations. Bivalirudin (Hirulog), a 20-mer polypeptide, is a

product of rational design by grafting the hirudin C-terminal tail to an active site binding

moiety D-Phe-Pro- Arg-Pro using four GIy residues as spacer (Gladwell et al., 2002).

Another tight-binding thrombin inhibitor, haemadin, of molecular mass ~5 kD was

isolated from the leech Haemadipsa silvestris. Although the amino acid sequence of this

inhibitor lacks homology with hirudin, the mechanisms of thrombin inhibition by these

inhibitors are the same. In the crystal structure, the N-terminal segment of haemadin

binds to the active site of thrombin, forming a parallel -strand with residues Ser214–

Gly216 of the proteinase. This mode of binding is similar to that observed in hirudin. In

contrast to hirudin, however, the markedly acidic C-terminal peptide of haemadin does

not bind the fibrinogen-recognition exosite, but interacts with the heparin-binding exosite

of thrombin (Richardson et al., 2000).

Another novel, fast- and tight-binding competitive inhibitor of thrombin, variegin, was

extracted from the salivary gland of the tropical bont tick, Amblyomma variegatum. A

middle region in this 32-mer binds to the thrombin active site. Thrombin cleaves variegin

within this region between Lys10–Met11. Remarkably, the active site function of

thrombin is inhibited by variegin long after the cleavage is completed. Structurally,

variegin is best compared with hirulog- 1 although the binding of s-variegin (32-mer,

synthetic fulllength variegin) to thrombin is about 15 times stronger than that of hirulog-

1. The active-site-binding residues in variegin reside within the sequence EPKMHKT,

which is also the site most different from hirulog-1. The s-variegin P1 (Lys) and P3 (Glu)

residues are both suboptimal for thrombin interactions relative to the corresponding

segment in hirulog-1 (DFPRPGGG). The stronger binding of s-variegin is thus most

likely a function of its P’ residues (MHKT tetrapeptide) (Koh et al., 2007).

38

The tsetse thrombin inhibitor (TTI) is also a potent and specific low molecular mass

(3,530 Da) anticoagulant peptide. It was identified from salivary gland extracts of

Glossina morsitans morsitans (Diptera: Glossinidae). This inhibitor is highly specific for

thrombin, showing no activity against a panel of 10 serine proteases, including trypsin

and chymotrypsin. In addition to its remarkable anticoagulant effect in vitro, TTI is also a

potent inhibitor of thrombin-induced platelet aggregation. It does not have any homology

with known anticoagulants. Of interest, preliminary N-terminal amino acid sequence data

from the purified protein did not show homology to any previously identified

anticoagulants or class of protease inhibitors. TTI is one of the most potent (Ki*5584 fM)

naturally occurring anticoagulants ever identified, with remarkable specificity for

thrombin (Cappello et al., 1996)

Another peptide inhibitor is PPACK which is a D-Phe-Pro-Arg chloromethylketone that

can irreversibly inhibit thrombin. The crystal structure of thrombin-PPACK complex is

given in Figure 2.3. PPACK binds to active site only. The exceptional specificity of

PPACK to thrombin can be explained by its interactions with a hydrophobic cage formed

by Ile174, Trp215, Leu99, His57, Tyr60A and Trp60D.

Figure 2.3: PPACK in the active site of thrombin (PDB ID: 1DWE)

Another class of thrombin inhibitors was obtained by preparing boronic acid analogs and

incorporating them into peptides. This Ac-(D) Phe-Pro-boroArg-OH is a potent,

competitive inhibitor of thrombin with a Ki value of 40pM. The crystal structure of Ac-

39

(D) Phe-Pro-boroArg-OH complexed with human alpha-thrombin presented in Figure

2.4 illustrates the boron atom covalently bonded to the side-chain oxygen of the active

site serine, Ser195. The boron adopts a nearly tetrahedral geometry, and the boronic acid

forms a set of interactions with the protein that mimic the tetrahedral transition state of

serine proteases. Contributions of the arginine side chain to inhibitor affinity were

examined by synthesis of the ornithine, lysine, homolysine, and amidine analogs of

DuP714. The basic groups interact with backbone carbonyl groups, water molecules, and

an aspartic acid side chain (Asp189) located in the thrombin S1 specificity pocket. The

variation in inhibition constant by 3 orders of magnitude appears to reflect differences in

the energetics of interactions made with thrombin and differences in ligand flexibility in

solution (Weber et al., 1995).

Figure 2.4: Crystal structure of thrombin with Ac-(D) Phe-Pro-boroArg-OH bound in the

active site (PDB ID: 1LHE)

The angiotensin-converting enzyme breakdown product of bradykinin, Arg-Pro-Pro-Gly-Phe

(RPPGF), is a stable metabolite of bradykinin. High concentrations of RPPGF inhibit thrombin-

induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel -

strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad.

RPPGF competitively inhibits -thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with

a Ki = 1.75 ± 0.03 mM. These studies indicate that RPPGF is a bifunctional inhibitor of

thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active

site of α-thrombin. RPPGF can be described as a substrate-directed thrombin inhibitor because

it is partially directed to the thrombin substrate PAR1 (Hasan et al., 2003).

40

Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-

isomers and unusual amino acids were prepared based upon the stable angiotensin

converting enzyme breakdown product of bradykinin – RPPGF. These peptides are direct

thrombin inhibitors prolonging the thrombin clotting time. FM19 achieved 100%

inhibition of threshold α- or γ-thrombin-induced platelet aggregation. The crystal

structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds

into the S1 pocket, its second residue Oic interacts with His57, Tyr60A and Trp60D, and

its C-terminal p-methyl Phe engages thrombin’s aryl binding site composed of Ile174,

Trp215, and Leu99. So FM19, a low affinity reversible direct thrombin inhibitor, might

be useful as an add-on agent to address an unmet need in platelet inhibition in acute

coronary syndromes in diabetics and others who with all current antiplatelet therapy still

have reactive platelets ( Nieman et al., 2008).

Argatroban is a univalent inhibitor and binds only to the active site of thrombin. It is the

first clinically used synthetic direct thrombin inhibitor (Bush et al., 1991). The crystal

structure of argatroban with human alpha thrombin is shown in Figure 2.5. It is an

arginine derivative small molecule (anhydrous MW=508.7). It is used as an anticoagulant

for prophylaxis or treatment of thrombosis in patients with heparin-induced

thrombocytopenia (Dhillon et al., 2009). Argatroban reversibly binds to the thrombin

active site and does not require the co-factor antithrombin III for antithrombotic activity

(Banner and Hadvary, 1991).

41

Figure 2.5: The binding of argatroban into the active site of thrombin. Some of the

hydrogen bonding interactions between thrombin and ligand is displayed

(PDB ID: 1DWC).

Another direct thrombin inhibitor undergoing clinal use is Dabigatron etexilate. It is used

for the prevention of venous thromboembolism (VTE) following orthopedic surgery. It

has stable, predictable pharmacokinetics and does not require routine monitoring. Its

chemical structure is shown in Figure 2.6. Dabigatran etexilate is the prodrug of the

active moiety dabigatran, an orally active agent that could replace enoxaparin in some

clinical indications. Issues relating to the use of dabigatran include its lack of antidote,

limited application in renal disease, and interaction with drugs such as amiodarone and

verapamil. Several trials investigating the use of dabigatran for other indications, such as

stroke prevention in atrial fibrillation and acute coronary syndromes, are underway.

Given its safety profile, efficacy, oral bioavailability and stable pharmacokinetic

properties, dabigatran may be a viable alternative to enoxaparin for thromboprophylaxis

in orthopaedic surgery (Verma, 2010).

42

Figure 2.6: Chemical structure of a direct thrombin inhibitor Dabigatran etexilate

Another orally active direct thrombin inhibitor is Ximelagatran. After absorption, it is

rapidly converted into its active form melagatran, a potent inhibitor of thrombin that

prevents both thrombin acivity and generation. Melagatron has a very poor oral absorption

due to the presence of a carboxylic acid, a secondary amine and an amide residue resulting

in a charged molecule at physiological pH. Concomitant food intake further reduces its

bioavailability. So, ximelagatran, a prodrug of melagatron was developed which has better

oral absorption due to better lipophilicity and uncharged nature at intestinal pH. The

structure of Ximelagatran is given in Figure 2.7. It was used for the treatment of venous

thromboembolism and prevents strokein atrial fibrillation. But FDA denied approval of

Ximelagatran in clinical use due to its hepatotoxicity (Lip, 2005).

Figure 2.7: Structure of an orally active direct thrombin inhibitor Ximelagatran.

43

AZD0837 is a new oral anticoagulant which is successfully progressed through phase II

studies. It is a selective and reversible direct thrombin inhibitor. AZD0837 rapidly

metabolized to AR-H067637. The half-life of AR-H067637 is 9–14 h in healthy subjects.

Phase II dose-guiding study investigated the safety, tolerability, and pharmacodynamic

efficacy of AZD0837 in the atrial fibrillation population. The results of this Phase II dose-

guiding study show that all tested doses of AZD0837 were generally well tolerated, and

suggest that AZD0837 at an exposure corresponding to the 300 mg of dose provides a

similar suppression of thrombogenesis at a potentially lower bleeding risk (Lip et al.,2009).

RWJ-671818, shown in Figure 2.8, was identified as a novel, low molecular weight,

orally active inhibitor of human alpha-thrombin (Ki = 1.3 nM) that is potentially useful

for the acute and chronic treatment of venous and arterial thrombosis. On the basis of

preclinical data, RWJ-671818 was advanced into human clinical trials and successfully

progressed through phase I studies (Lu et al., 2010).

Figure 2.8: Structure of RWJ-671818 with thrombin

Another active-site directed inhibitor of human α-thrombin is benzamide. The binding

mode of benzamide is same as that of Argatroban (Banner and Hadvary, 1991). The

crystal structure of benzamide with thrombin is given in Figure 2.9.

44

Figure 2.9: Benzamide (PDB ID: 1DWB) in the active site of thrombin

NAPAP is a peptidelike, benzamidine-based thrombin inhibitor. The starting point of the

search for noncovalent, nonpeptide thrombin inhibitors was the X-ray crystal structure of

the bovine thrombin complex formed with the NAPAP. It binds reversibly to the

thrombin active site by occupying the S3 pocket with its naphthyl group and the S2

pocket with the piperidine ring and by positioning its basic benzamidine moiety into S1

to form a salt bridge with Asp189. Additionally, the bridging glycine moiety of NAPAP,

in analogy to peptidic serine protease substrates, forms the canonical hydrogen-bonding

pattern with residues Trp215 and Gly216 at the rim of the specificity (S1) pocket. Despite

having significantly different chemical structures, NAPAP and Argatroban bind to

thrombin in a very similar “inhibitor binding mode” which is not that expected by direct

analogy with the binding of substrate (Sturzebecher et al.,1984). The crystal structure is

shown in Figure 2.10.

45

Figure 2.10: The structure of NAPAP complexed with thrombin (PDB ID: 1DWD)

On the basis of the X-ray crystal structure of NAPAP complexed with bovine thrombin, a

new structural class of nonpeptidic inhibitors was designed employing a 1,2,5-

trisubstituted benzimidazole as the central scaffold. Supported by a series of X-ray

structure analyses, the inhibitory activity of these compounds were optimized in a

number of iterative steps. Thrombin inhibition in the lower nanomolar range could be

achieved although the binding energy mainly results from nonpolar, hydrophobic

interactions. To reduce plasma protein binding and to improve in vivo potency, the

hydrophilicity of the inhibitors were increased by introducing a carboxylate group at a

position of the molecules, where it does not interfere with the thrombin binding site

interaction. From this series of inhibitors, BIBR 953 exhibited the most favorable in vivo

activity profile. Because of its highly polar, zwitterionic nature, oral absorption of BIBR

953 was insufficient. Therefore, a number of prodrugs were synthesized, from which

BIBR 1048 exhibited strong and long-lasting anticoagulant effects after oral

administration in different animal species. The chemical structure of BIBR953 and BIBR

46

1048 is given in Figure 2.11. On the basis of these encouraging results, BIBR 1048 was

chosen for clinical development (Norbert et al., 2002).

Figure 2.11: Chemical structures of BIBR953 (I) and BIBR 1048 (II)

The crystal structure of human alpha-thrombin in complex with a nonpeptidyl inhibitor

LY178550 has been solved to 2.07 Ǻ resolutions by the method of X-ray crystallography

as shown in Figure 2.12. The inhibitor binds to the active site in an L-shaped manner,

mimicking the bound conformation of the tripeptide arginal series of thrombin inhibitors.

The basic amidine of LY178550 forms a salt bridge with Asp 189 within the specificity

pocket, while the 4-benzylpiperidine side chain engages in a number of hydrophobic

interactions at the S2 and S3 binding sites. The inhibitor does not interact in any fashion

with the active site sequence Ser 214-Gly 216, as occurs with many of the inhibitors

studied previously. The indole N-H of the inhibitor forms a hydrogen bond to the

gamma-oxygen of the catalytic Ser195 (Chirgadze et al., 1997).

Figure 2.12: Crystal structure of thrombin in complex with LY178550 (PDB ID: 1D4P)

47

Thrombin inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure is discussed

here. The X-ray crystal structures of these complexes shown in Figure 2.13 reveal a

novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the

inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine

of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2,

and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to

point away from the active site, occupying a location between the S1 and S1' sites.

Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of

Trp60D contained in the thrombin S2 insertion loop defined by the sequence Tyr-Pro-

Pro-Trp. In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-

Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does

not engage in hydrogen bond formation with Gly216 of the thrombin active site

(Chirgadze et al., 2000).

Figure 2.13: Crystal structure of a benzo[b]thiophene derivative with thrombin (PDB ID:

1D3D)

The X-ray crystal structures of four beta-strand-templated active site inhibitors of

thrombin containing P1' groups was discussed here and given in Figure 2.14. Two of the

inhibitors have an alpha-ketoamide functionality at the scissile bond; the other two have a

nonhydrolyzable electrophilic group at the P1' position. The binding of lysine is

48

compared with that of arginine at the S1 specificity site, while that of D, L-phenylalanine

enantiomorphs is compared in the S3 region of thrombin. Four different P1' moieties bind

at the S1' subsite in three different ways. The binding constants vary between 2.0 µM and

70 pM. The bound structures are used to intercorrelate the various binding constants and

also lead to insightful inferences concerning binding at the S1' site of thrombin (St.

Charles et al., 1999)

Figure 2.14: Crystal structure of one of the four beta-strand-templated active site

inhibitors of thrombin (PDB ID: 1A46)

Another set of small molecule thrombin inhibitors are BMS-186282 and BMS-189090. The

crystallographic structures of their ternary complexes with human alpha-thrombin are given

in Figure 2.15. In both cases, the inhibitors, which adopt very similar bound

conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin

main chain in a manner like that reported for PPACK. They do, however, exhibit

differences in the binding of the alkyl guanidine moiety in the specificity pocket.

Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the

binding pocket. BMS-186282 only forms a hydrogen bond to the serine of the catalytic

triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are

highly selective for thrombin over trypsin and other serine proteases (Malley et al., 1996).

49

Figure 2.15: Crystal structure of (A): BMS-189090 (PDB ID: 1BMN) and (B): BMS-

186282 (PDB ID: 1BMM) in the active site of thrombin

In another study, two active site inhibitors RWJ50215 and RWJ 50353 of thrombin are

discussed. Structures of these inhibitors are given in Figure 2.16. Interactions between

the protein and the thiazole rings of the two inhibitors provide new valuable information

about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-

binding benzothiazole group linked to the PPACK motif. Interactions with the S1-S3

sites are similar to the PPACK structure. In RWJ-50215, a S1'-binding 2-ketothiazole

group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-

1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the

parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity

surrounded by His57, Tyr60A, Trp60D, and Lys60F. The ring nitrogen of the RWJ-

50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because

of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen

bond with the NZ atom of Lys60F (Matthews et al., 1996).

50

Figure 2.16: Structures of (A) RWJ-50215 (PDB ID: 1A4W) and (B) RWJ-50353 (PDB

ID: 1TBZ) in the active of thrombin.

Another three-dimensional structure of a complex consisting of human alpha-thrombin and

the active-site inhibitor RWJ-51438 is shown in Figure 2.17. The ketone carbonyl group of

the inhibitor is covalently linked to the OH- atom of Ser195, forming a tetrahedral

intermediate hemiketal structure; the benzothiazole ring N atom of RWJ-51438 forms a

hydrogen bond with His57. The carboxylate substituent on the benzothiazole group forms

salt bridges with Lys60F NZ and the NZ of the symmetry-related residues Lys236 and

Lys240, which introduces steric effects that perturb the 60A-60I insertion loop, especially at

residues Trp60D and Phe60H (Recacha et al., 2000).

Figure 2.17: Crystal structure of human alpha-thrombin and its active-site inhibitor RWJ-

51438 (PDB ID: 1DOJ)

51

Here, a novel class of noncovalent thrombin inhibitors has been designed, synthesized,

and tested in vitro. The main feature of these inhibitors is a 6,5-fused bicyclic core

structure that fills the S2 pocket of the active site of thrombin. The bicycle introduces

conformational constraint into the ligand and locks the Xaa-Pro amide bond into the

desired trans configuration. Among the known ring systems, the 7-thiaindolizidinones

(BTD) was selected as the basic template by molecular modeling. The influence of

several structural features was analyzed: the length of the argininal side chain, the

stereochemistry at C6, and the importance of making optimal use of the S3 pocket.

Finally, an X-ray crystal structure of inhibitor bound to thrombin was obtained at a

resolution of 2.3 Å as given in Figure 2.18. These designed thrombin inhibitors, which

were prepared by an efficient synthesis, showed high selectivity over trypsin and other

serine proteases (Wagner et al., 1998).

Figure 2.18: Crystal structure of a non-peptide thrombin inhibitor containing a bicyclic

core structure (PDB ID: 1BHX)

The crystal structures of a highly potent and selective low-molecular weight rigid

peptidyl aldehyde inhibitors complexed with thrombin have been discussed and showed

in Figure 2.19. Since the selectivity of two of the inhibitors was >1600 with respect to

trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also

determined. The selectivity appears to reside in the inability of a benzenesulfonamide

group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may

be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel

52

lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a

guanidinopiperidyl group at the P1 position that binds in the S1 specificity site.

Differences in the binding constants of these inhibitors are correlated with their

interactions with thrombin and trypsin. (Krishnan et al., 1998).

Figure 2.19: Crystal structure of thrombin in complex with rigid peptidyl aldehyde (PDB

ID: 1CA8)

Another series of retro-binding inhibitors of human α-thrombin was prepared to elucidate

structure–activity relationships (SAR) and optimize in vivo performance. The chemical

structure of the compound is given in Figure 2.20. Studies were focused on reducing

unnecessary functionality that was found to be a metabolic liability. Two compounds

were identified as orally active inhibitors of thrombin catalytic activity, and efficacious in

a thrombin-induced lethality model in mice (Edwin et al., 2002).

Figure 2.20: Chemical structure of a retro-binding inhibitor of human alpha thrombin

53

A recent study has designed a potent and selective inhibitor of thrombin with a

bis(phenyl)methane moiety binding into the S3 pocket and is shown in Figure 2.21.

These inhibitors bind with remarkable potency to the active site of thrombin. A

combination of X-ray crystallography and isothermal titration calorimetry provides an

explanation of the tight binding of the bis(phenyl)methane inhibitors. The first phenyl

moiety occupies the hydrophobic S3 pocket, resulting in a mainly entropic advantage of

binding. A rotation of a glutamate residue adjacent to the S3 binding pocket is observed.

The rotation of this glutamate into salt-bridging distance with a lysine moiety correlates

with an enhanced enthalpic contribution to binding for these highly potent thrombin

binders. This explanation for the magnitude of the attractive force is confirmed by data

retrieved by a relibase search of several thrombin–inhibitor complexes deposited in the

PDB exhibiting similar molecular features (Baum et al., 2009)

Figure 2.21: Structure of a bis (phenyl) methane inhibitor of thrombin.

Another class of potent thrombin inhibitors are 2-(2-Chloro-6-fluorophenyl) acetamides

having 2,2-difluoro-2-aryl/heteroaryl-ethylamine P3 and oxyguanidine P1 substituents.

The K(i) value of these is 0.9-33.9 nM. 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine

was the best P3 substituent, yielding the most potent inhibitor (K(i)=0.7 nM) and is

shown in Figure 2.22. Replacing the P3 heteroaryl group with a phenyl ring or replacing

the difluoro substitution with dimethyl or cyclopropyl groups in the linker reduced the

affinity for thrombin significantly. The aminopyridine P1 is also provided an increase in

potency (Lee et al., 2007).

54

Figure 2.22: Structure of a 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine

2.3 Computational design of thrombin inhibitors

Computational combinatorial ligand design (CCLD) is used to generate low-molecular-

weight active site-directed inhibitors of human thrombin inhibitors. CCLD generated a

series of molecules that showed same interaction patterns as those of known thrombin

inhibitors, i.e., hydrophobic moieties in S3 and S2, hydrogen bonds with the polar groups

of Gly-216, and benzamidine in S1. One of the successfully designed compounds is

involved in same interactions as in NAPAP-thrombin complex except for the hydrogen

bond with the CO of Gly-216. CCLD automatically converted one of the sp3 carbons of

the cyclopentane ring into sp2 nitrogen to obtain the secondary amide connection

between cyclopentane and benzamidine, which should facilitate synthesis. This

“NAPAP-like” compound was synthesized and tested in a binding assay and found to be

a relatively potent and selective inhibitor of thrombin. Its racemic mixture has an

inhibition constant (Ki) value of 1.7 µM for thrombin and is inactive against plasmin

(Caflisch et al., 1998).

55

In another study, a series of thrombin inhibitors were generated by using computer-

assisted multiparameter optimization process. The process was organized in design

cycles, starting with a set of randomly chosen molecules. Each cycle combined

combinatorial synthesis, multiparameter characterization of compounds in a variety of

bioassays, and algorithmic processing of the data to devise a set of compounds to be

synthesized in the next cycle. The identified lead compounds exhibited thrombin

inhibitory constants in the lower nanomolar range. They are by far the most selective

synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases

such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore,

these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic

stability, low serum protein binding, good solubility, high anticoagulant activity, and a

slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray

crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a

neutral moiety in the S1 pocket of thrombin (Riester et al., 2005).